Category Archives: Agriculture

Arthropod pests were generally unaffected by local or landscape scale factors

Of importance to urban farmers, multiple on farm practices have been identified that may be implemented to increase CBC in urban farms . To our knowledge, this work has not yet been gathered and synthesized, making it difficult to translate research into practice. Here, we review and summarize relationships between local farm and surrounding landscape effects in UA on pest and natural enemy populations, as well as on the resulting levels of biological control . Our literature review focuses on four questions for UA systems: Which local on-farm practices and off-farm landscape factors affect insect and mite pest populations and their crop damage; natural enemy biodiversity ; ecosystem services through increased biological control; and which practices can be recommended to urban farmers to promote CBC? We searched for peer-reviewed literature, published before February 2019, that measured natural enemy and insect pest richness, abundance, and rates of predation and parasitism in UA systems. We further focused the review on intra-urban studies that measured differences in on-farm composition, practices, and surrounding off-farm landscape attributes to measures of insect abundance, richness, and community composition. We excluded studies that either focused on taxa that do not provide regulating ecosystem services relevant to CBC , or compared pest or natural enemy abundance, richness and composition between urban and rural green spaces, farms, or gardens. We did this because these measures do not explicitly focus on UA or local on farm predictors of arthropods. In some cases,dutch buckets we did include urban-to-rural studies if a subset of the samples met the intra-urban requirement. For these studies, we excluded the reported findings from rural or natural landscapes.

The review protocol followed the PRISMA systematic review framework and the methodologies described in Pullin 2006 . We searched three databases including Web of Science, the United States Department of Agriculture National Agricultural Library database , and the National Center for Biotechnology Information , using search terms that are common in the CBC literature: “Biological control,” “Herbivore,” “Pest,” “Parasitism,” “Natural enemies,” and “Parasitoid.” These terms were paired with “Urban agriculture” and “Urbanization.” Search terms were applied to titles, abstracts, and keywords. Our search protocol identified 675 peer-reviewed publications using this methodology. We removed all duplicates and reviewed the remaining articles for relevance. From these, we identified 15 articles that met our protocol criteria and were selected for review . For each publication, we collected information on authors, title, site location, site sample number, land type , sampling period, methodology, and taxa assessed. We then recorded the statistically significant effects of 16 explanatory variables common among studies for species richness, abundance , and levels of ecosystem services through biological controls . To further identify explanatory variables, and to align variables with reviewed literature, we categorize variables as “local factors” or “landscape factors.” Local factors were defined as biotic and abiotic features of the local agroecosystem , and landscape factors were defined as features of the surrounding landscape . For each explanatory variable, we counted the number of reportable results . Some explanatory variable measures, such as local or landscape factors combined into an index value, or measures that were not clearly defined were categorized as “landscape cover” or “structural diversity” .In four publications, herbivorous taxa were studied that are UA crop pests, but few local factors were presented that explained increases in pest abundance or richness. Moreover, studies often showed inconsistent results, and the only factor repeatedly associated with increased pest richness was increased perennial richness and abundance. Structural diversity of vegetation, host plant density, garden age and soil moisture were also identified as factors affecting herbivore richness and abundance, but these relationships were only measured as significant once.Local factors positively affected parasitoid and predator abundance and richness in thirteen of fifteen reviewed studies, with only 7% of the reported results showing negative effects on natural enemy abundance and richness.

Important local factors that positively affected natural enemy populations included increased floral abundance and richness, increased mulch and leaf litter cover, larger garden size, high plant species richness, more perennials, and increased structural diversity. Garden size was the only factor that differed between predator and parasitoid taxa, with larger gardens positively affecting parasitoid populations and smaller gardens positively affecting predator abundance.We reviewed UA literature to assess how local on-farm management practices and surrounding off-farm landscape features affect herbivorous insect pests, arthropod natural enemies, and measures of conservation biological control. This is a first attempt to synthesize the growing number of case studies in this field. We found that local and landscape factors differentially affect insect pests and their natural enemies, as well as ecosystem services received through biological control. Local on farm diversification and management most commonly affected natural enemy species richness, abundance, and ecosystem services with of reported results showing positive impacts. Relationships between measures of arthropod diversity and impervious urban land cover at the landscape scale are inconsistent, as they have both negative and positive effects on arthropod populations . Some reviewed studies found parasitoid abundance increased, but richness decreased with urban landscape cover , or that these relationships for predators are differential across taxa, region, and landscape scale . The differences across taxa, region, and surrounding urban landscape composition are all important considerations.However, insect pests were the least commonly measured taxa across these studies. Only two reviewed studies focused on intra-urban local and landscape herbivorous pest effects, and most studies did not assess relationships between insect pests and crop damage . Rates of parasitism were also unaffected by local and landscape factors, even though parasitoids are prevalent in urban gardens . Similarly, urban-to-rural studies report that parasitic Hymenoptera may be somewhat resistant to landscape-scale habitat fragmentation in larger non-garden habitat patches . However, in more urbanized landscapes with smaller habitat patches, landscape fragmentation has negative effects on Hymenoptera species diversity .

Our review identified gaps in UA CBC-related research, particularly on the topics of methodology and geographic breadth. The key methodological issues that we found in the literature include: lack of measured temporal effects; inconsistent sampling techniques across studies; coarse taxonomic identification and biodiversity metrics of focal taxa; and difficulty in accessing sufficient landscape data. Only three of the reviewed studies measured temporal effects , and the average sampling period was only 22 weeks. Clearly, more extensive year round sampling is needed to account for possible temporal changes between seasons. To this point,growing lettuce hydroponically local climate measures were rarely reported; only a third of studies measured temperature, and none measured wind speed or humidity. These local abiotic climate-related factors should be considered as climate change will increasingly impact urban arthropods in the coming decades. It is important to consider methods of insect sampling and units of ecosystem function in CBC research. Often the goal of UA studies is to better understand functionally important species distributions in fragmented landscapes with implications for agricultural ecosystem functioning. While measuring the richness and abundance of insects is an essential step to understand species distributions, it does not account for functional effects of biodiversity that are of use to UA practitioners. Nineteen of the studies used pan traps or sticky traps, standard but often superficial methods in insect population studies. These sampling methods can be too broad when investigating biological controls . It would be useful to measure the actual rates of prey consumption, for example, by using exclusion and sentinel prey in relation to natural enemy presence, or by rearing parasitized insects. Emerging technologies such as molecular gut content analysis of predators using DNA-based prey assays are an effective method to link predator to pest . While most studies included multiple methodologies, it would be useful to include more standardization in UA field sampling protocols for biodiversity and biological control to facilitate future meta-analysis. With regard to biodiversity metrics in UA CBC research, most of the reviewed studies offer only a coarse overview of species identification, especially for parasitic Hymenoptera, which are often only identified to super family. Though genus- and species-level identification are time-intensive and require skilled labor, species- or genus-level data is necessary to better investigate species-effects on CBC. This is particularly important because many of the parasitic Hymenoptera in the reviewed studies are aggregated as “beneficial,” but many are hyperparasitoids, or are potentially parasitoids of other natural enemy predators . More research is needed on UA pests because insect pests are the least measured taxa across studies, and most studies do not assess relationships between insect pests and crop damage and yield. Only two reviewed studies focused on intra-urban local and landscape herbivorous pest effects . Additional challenges exist in UA landscape studies, notably the availability of fine-scale landscape data. Urban ecologists have been limited in their access to geographical data at a scale smaller than 30 m.

Many studies use the US National Landscape Cover Database, which includes a measure of the impervious surface, but the scale is inappropriate for complex urban environments particularly, when considering effects on arthropods that respond to habitat heterogeneity at much smaller spatial scales . Ground-proofing landscape composition can necessitate consistent access to a private property which can be challenging. Alternative methodologies have been proposed such as aerial drones with high-resolution cameras, but limits to drone flight plans in residential areas or excluded flight space make flights difficult. We also found a strong bias towards UA studies in North America and Europe. It is unclear as to whether this bias is a relic of the database search itself or there is a distinct lack of literature available.Crop pests in urban landscapes can be challenging to control, and they can have a disproportionate impact on the smaller crop sizes common to urban farms. Herbivorous insect populations in urban areas can persist for more extended periods, have increased fecundity, and can even be larger . Under managed or neglected urban landscapes can exacerbate pest issues. Irregular irrigation, application of fertilizers or pesticides, and higher levels of air pollution can induce plant stress or vigor, creating favorable conditions and refuges for herbivorous pests that can emigrate to urban farms and gardens . Urban agriculture is often practiced without pesticides for health and environmental reasons despite these challenges. Instead, farmers find themselves relying on time and labor-intensive cultural and mechanical practices for pest management. Consequently, urban farmers have shown great interest in agroecological pest management , a proactive ecosystem services based approach that aims to reduce pest abundance and crop damage by increasing natural enemy populations through agroecological practices . For example, increasing vegetative complexity, implementing soil conservation practices, and introducing floral resources for nectarivorous natural enemies. These practices increase the amount of shelter, nectar, and pollenresources on urban farms, increasing natural enemy populations, resulting in increased biological control services . In rural agroecosystems, APM practices, landscape effects, and conservation biological control have been widely studied . Metaanalyses have found that on-farm management practices such as intercropping, crop rotations, and increased structural diversity increase natural enemies’ abundance, diversity, and ability to regulate pest populations . Increased landscape diversity surrounding rural agroecosystems has been shown to mediate arthropod diversity and abundance, with natural enemies showing a positive response to increased landscape complexity . The enemies hypothesis states that increased structural complexity should increase natural enemy abundance, diversity, and associated ecosystem services . This hypothesis has been investigated and questioned in agroecosystem management, with varying results at different spatial and temporal scales, most often in rural contexts. Ostensibly, diversification effects observed in rural agroecosystems should be observed in their urban counterparts. However, the effects of diversification on biological control services and APM in urban agroecosystems regarding this hypothesis are still being explored, especially in how natural enemies are affected by landscape factors such as fragmentation and isolation, common in urban landscapes . The extent of fragmentation effects on organisms in urban environments, and related ES, has been a persistent question, especially in urban agroecosystem management . Roads, parking lots, and buildings increase impervious surfaces, fragmenting and reducing green space connectivity and impacting the quality and area of suitable habitat . The reduction of available and appropriate habitats for urban flora and fauna decreases meta population connectivity and drives a decline in urban species diversity, selecting for more disturbance tolerant species and increasing the chance of localized extinctions .

Crop revenue declines vary by region and crop category

Water condition reports from the California Department of Water Resources this year indicated slightly higher precipitation in the Sacramento Valley compared to last year, and reservoirs were around 50% of historical average storage by this time of the year. However, the reservoirs in the San Joaquin Valley and Tulare Lake Basin are near all-time lows and precipitation was only 45% of the historical average. The drought impacts are especially severe because California’s population and share of permanent crops have both increased. Furthermore, the lack of water in 2015, together with impacts of additional groundwater pumping that occurred in 2014, may lower the water table levels enough to decrease pumping capacity in some areas. Every year, the state and federal water projects announce deliveries to water districts. Likewise, local water districts across the state inform their member farmers about expected water allocations. Growers, in turn, make planting decisions based on these expected water deliveries. In the early spring of 2015, ERA Economics conducted a survey of irrigation districts to assess the expected water deliveries for the irrigation season. The total water shortage is expected to be close to 8.8 million acre-feet statewide. Farmers and irrigation districts are able to partially offset some of the surface water shortage by pumping additional groundwater. Additional groundwater pumping in 2015 is expected to be 6.2 maf, resulting in a net water shortage of 2.6 maf. The map in Figure 2 shows a breakdown of net water shortages by basin. The region most affected by water availability is the Tulare Lake Basin,plastic pots large which includes parts of Fresno, Tulare, Kings, and Kern counties.

This area has the lowest precipitation and relies heavily on water imports from other basins and groundwater pumping, yet it provides more than 50% of all agricultural revenues in the Central Valley. The net water reduction of 2.6 maf in 2015 will be 60% higher than 2014 . We used the changes in irrigation water supplies and the Statewide Agricultural Production Model to estimate the crop mix and fallowing due to the 2015 drought. The SWAP model is an economic model, which simulates the response of farmers to changes in water supply. The model includes detailed information on crop acreages, values, and regional access to ground and surface water supplies. We linked the SWAP model to DWR’s groundwater-surface water simulation model, C2VSim. The C2VSim model estimates the groundwater levels and pumping capacities and feeds this information to the SWAP model. Declining groundwater levels increase the energy required to pump groundwater, and in turn the cost of using groundwater for irrigation. The SWAP model simulates the response by farmers to this increased cost of groundwater pumping. With net shortages of 2.6 maf for 2015, the SWAP model estimates fallowing nearly 564,000 acres statewide—562,000 of which would occur in the Central Valley. Other areas include the Central Coast, the South Coast and inland Southern California, which are less affected by decreased precipitation, have access to groundwater, or import water from other basins such as the Colorado River. Figure 3 shows a breakdown of estimated fallowing due to the 2015 drought. The bulk of the fallowing occurs in feed and grain crops, in addition to some field crops south of the delta. Fallowing of vegetables, orchards, and vineyards will be minimal, yet the Tulare Lake Basin may fallow some of these higher revenue per acre crops. We have conducted retrospective analyses of previous droughts and found that the SWAP model accurately estimates fallowing due to water shortages.

In addition, we can use multi-spectral satellite imagery to estimate the total idle land area using measures such as the Normalized Difference Vegetation Index . The NDVI estimates total idle land area, whereas SWAP estimates fallow land due to water shortage. In May 2014, predicted fallowing using SWAP was 410,000 acres in the Central Valley. We compared 2014 and 2011 NDVI measures and found there were an additional 450,000 acres of idle land in 2014 compared to 2011. Similar estimates by NASA Ames found around 500,000 acres of idle land. We note that SWAP estimates are directly based on water changes, whereas satellite based estimates measure all idle land. Patterns of idle land are consistent between SWAP and the vegetation index methods, and support the overall conclusion that the bulk of the fallowing occurs in the Tulare Lake Basin. During California’s periodic droughts, water shortages lead to fallowing and significant reductions in output from California agriculture. Adaptation methods include changing the typical crop mix by using water for crops with higher revenue per unit of water, substituting groundwater for surface water, water transfers, and additional use of technology. Estimated changes in water supplies were based on a survey of water districts conducted in the spring of 2015, public announcements of water deliveries from federal and state water projects, and information on groundwater tables from the California Department of Water Resources C2VSim model. We used the Statewide Agricultural Production Model to estimate the economic impacts of the 2015 drought. Our preliminary estimates of the impacts of the drought on cropping patterns were reported in Howitt et al. . We estimated an 8.8 million acre foot loss in surface water availability could be partially offset by increased groundwater pumping of 6.2 maf statewide. We used the SWAP model to estimate the additional fallowing of 564,000 acres, and a decrease in crop revenue of about $850 million. Additional losses for cattle and calves and dairies , due to reduced winter pasture and higher cost of forage crops, are also expected. The combination of these losses lowers revenue by about $1.2 billion, compared to a normal water year.

We estimate groundwater pumping costs could increase by $600 million in 2015 as a result of higher pumping volumes and lower groundwater levels.Figure 1 shows disaggregated losses. Based on data through late May, our SWAP model estimates that the Sacramento Valley, extending from Shasta County to the delta, will face revenue losses of over $200 million in feed and grain crops while maintaining most vegetable and orchard production. More recent data on water availability and planted acreage are likely to raise these estimates. The northern San Joaquin Valley, from south of the delta to just north of Fresno County, has relatively small losses and even revenue increases for some crops due to slightly higher crop prices for vegetables, orchards, and vines. Other areas, like the Central and South Coast, may also see slight revenue increases. As shown in Figure 1, we estimate that the Tulare Lake Basin will face severe revenue losses totaling about $620 million across all crop categories. Based on crop fallowing,planting pot seedlings about 8,550 direct full-time and part-time jobs could be lost, 7,670 in crops and nearly 980 in livestock and dairies. This estimate incorporates the fact that about two seasonal jobs equal one full-time equivalent job. Most employment losses occur in the Tulare Lake Basin. Changes in field crops and grains account for nearly 6,840 of the direct job losses. Impact analysis allows us to trace expenditure patterns in a regional economy caused by an economic event. When agricultural crop revenues are reduced due to water shortages, expenditures on agricultural-related sectors such as fertilizers, agrochemicals, or farm consulting services are also reduced. These indirect economic impacts cause the direct on-farm impacts to ripple through the economy. Moreover, those households that rely on agriculture and agriculture-related sectors for income spend less on consumer goods and services; these induced effects also ripple through the economy, reducing economic activity and jobs further. The sum of direct, indirect, and induced effects is often referred to as the total or multiplier effect of an economic event on the region’s economy. Howitt et al. uses this type of analysis to show the direct and total effects of the agricultural drought in the California economy. Multiplier effects on employment, sector output, and value added are examined. Employment represents full-time and part-time jobs; sector output refers to sales from agriculture and all other sectors in the economy; and value added is a measure of net gain in economic value after netting out any double counting across sectors. Value added includes salaries, self-employment income, other property income and indirect business taxes. Preliminary estimates in Howitt et al. show that indirect and induced effects from the 8,550 direct job losses and their spillover effects result in a loss of about 18,600 jobs in California in total. Likewise, direct agricultural revenue losses of about $1.8 billion generate a loss of $2.7 billion in state value of output across the whole economy. We estimate a loss of farm value added of $420 million and an overall loss of $1.25 billion in California value added. Despite the recurrence of droughts, California’s $45 billion agricultural economy has grown in recent decades, as it has shifted to commodities generating more revenue per acre and per acre-foot. This includes an increasing proportion of cropland devoted to tree and vine crops. Many of the expanding crop groups in California, including orchards, berries and vegetables, are more labor intensive than the more mechanized field crop categories. Impacts of the drought in the context of changes in overall farm employment merit careful examination.

For example, Howitt et al. estimated that the drought would cause a loss of about 7,500 direct farm jobs—almost 2% of about 400,000 farm jobs in California. Recently released annual data show that farm jobs in California rose by about 4,000 jobs, or about 1% from 2013 to 2014 . This result does not contradict losses due to drought and reinforces the point that labor-intensive agriculture has, in fact, been growing, and job growth would have been larger, but for the continuing drought.Figure 2 shows that while agricultural jobs grew in aggregate, that growth was in the fall and winter months of the 2013–2014 water year, in what is considered the non-irrigation season. The jobs associated with the main irrigated crop production period in the Central Valley, from April to September, showed no change in employment. Figure 3 shows that where irrigation availability declined most in the San Joaquin Valley, irrigation-season jobs fell in total—especially for contract workers. Figure 4 puts the 2014 employment data into perspective. Agricultural jobs have grown in California in each of the last five years from 2010 to 2014, especially in the non-irrigation season. Growth was faster in the two predrought years and 2014 was the second lowest. Much of the non-irrigation season employment occurs in coastal regions with year-round employment in berries and vegetables—crops that are minimally affected by the drought. Irrigation season employment, which is important in the Central Valley and is affected by drought, failed to grow in 2014—the only time in this five year period—after substantial growth in every year except 2011. These data are consistent with substantial drought-induced job losses for some of the most vulnerable workers in California in 2014 and again in 2015. The severe drought that California has been experiencing for several years has idled hundreds of thousands of acres of cropland, reduced crop yields, cut the cattle herd on pastures throughout the state, and reduced farm production value by billions of dollars. Nonetheless, despite the fact that California is the most important farm state in the United States—leading the nation in production of major commodities such as milk, tree nuts and many fruits and vegetables, and supplying about half of U.S. fresh produce—most consumers have seen no sign of the drought in the cost of food. This article lays out the facts and economic logic for why this is true. The previous articles in this issue have sketched the severity of this drought and what it has meant for irrigation water costs and availability, crop planting patterns, and production. This article takes the story through to food consumers.As the previous articles in this ARE Update issue document, the California drought is likely to reduce acreage by close to 10%. But, almost all that reduction in acreage is occurring in the Central Valley and among the major forage, grain and other field crops, for two reasons. First, in normal years, Central Valley agriculture relies on surface water deliveries from major government-owned or regulated projects.

All ag-MAR field sites were chosen based on SAGBI suitability prior to flooding

Glyphosate-resistant horseweed, or mare’s tail , was reported in 2005 and is one of the dominant weeds in and around raisin and tree fruit production areas of the San Joaquin Valley, as well as on roadsides and canal banks in the region . The level of glyphosate resistance in horse weed is relatively low, and resistant plants are usually injured to some degree following glyphosate applications, which suggests that resistance is not due to an altered target enzyme. Genetic comparisons of horseweed accessions from around the state suggest that there have been multiple, independent origins of resistance in this species, rather than the spread of resistance from a single-source population . Hairy fleabane populations resistant to glyphosate were reported in 2007 . Glyphosate resistance in hairy fleabane appears to be similar to resistance in horse weed in that selection has occurred in response to similar management strategies in perennial crops and surrounding areas ; multiple origins of resistance are suspected ; and growth stage and environmental conditions affect the level of resistance . The discovery by Moretti, Hanson et al. of hairy fleabane resistant to both glyphosate and paraquat raises questions about whether a common physiological mechanism is helping to confer resistance to these dissimilar herbicides, and research is ongoing to elucidate these factors. Junglerice resistant to glyphosate was first identified in 2008 in a Roundup Ready corn field in the Sacramento Valley ; since then, glyphosate-resistant junglerice has become widespread in orchards and field crops throughout California . Resistance appears to be due to mutations in the EPSPS target site , although some populations also appear to have enhanced EPSPS activity . Target-site mutations appear to be the most frequent mechanism among the accessions so far collected in California; however,blueberry pot size additional research is ongoing to determine whether the same is true with populations selected in orchards and in other regions of the Central Valley.

Several other common weeds in orchards and vineyards, including Palmer amaranth , three spike goose grass and witch grass , are suspected to have evolved resistance to glyphosate; preliminary research trials by UC researchers and California State University, Fresno, collaborators have been initiated to verify and characterize the putative resistant populations. Since the discovery of herbicide resistant weed bio-types in California, UC research and Cooperative Extension personnel, as well as university and non-university cooperators and students, have conducted locally relevant weed management research in the state. Research and extension efforts have included alternative chemical management techniques using various post emergence and pre-emergence herbicides along with mechanical control measures in an integrated approach. Applied research integrating agronomy, weed control, spray application technology and water management have been useful to regulatory agencies and have had positive impacts on water and air quality, wildlife habitat and water use . Information on the underlying mechanisms and genetic basis of resistance provides useful information to California weed managers in the longer term. This information is broadly applicable to the biology, physiology, evolution and control of weeds in other crops and regions at the local, national and international level. Although this paper has focused on the efforts of UC weed scientists and collaborators, the basic and applied scientific information developed in California supports similar research being conducted in other regions of the country and world. Like many other areas encompassed by the Endemic and Invasive Pests and Diseases Strategic Initiative, solutions to herbicide resistance are not simple and are affected by many biological, economic, regulatory and social factors. The diverse network of weed scientists and collaborators in a land-grant university system is well positioned to address these complex issues and respond to stakeholder concerns through applied and basic research, extension and outreach to affected agricultural industries, and education of future scientists and leaders. Ultimately, the goal of weed science research is to help growers maintain agricultural productivity and economic stability while increasing environmental sustainability.

Increasing groundwater use for agriculture and public utilities in the last century have put pressure on and diminished groundwater storage in California’s aquifers. The severe droughts that occurred over the last decade were exceptionally warm and dry, including some of the driest years since the late nineteenth century, further exacerbating the adverse effects of decreased ground water water resources . Years of decreased precipitation and increased groundwater extraction have rendered many of California’s groundwater basins and sub-basins to be in a state of groundwater overdraft, where out fluxes of groundwater through pumping or other natural processes greatly exceed influxes to groundwater storage. State agencies recently tasked with achieving groundwater sustainability by 2040, known as Groundwater Sustainability Agencies , have taken action to correct how we can locally manage groundwater resources in California in order to combat groundwater overdraft through the Sustainable Groundwater Management Act . Given how crucial groundwater is to California’s growing population and massive agricultural industry, it is imperative to practice sustainable management of this vital resource. The consequences of mismanagement of California’s groundwater resources are the driving force behind the implementation of methods that can help restore and increase groundwater storage in aquifer systems across the Central Valley. Rising in prevalence as a way of both maintaining and improving groundwater levels is managed aquifer recharge , a process that intentionally places more water into groundwater aquifers than would naturally occur using surface spreading or injection methods . The method of MAR used in this project, agricultural-managed aquifer recharge , spreads diverted surface water onto fallow agricultural fields to recharge groundwater supplies and store water for future use. The feasibility of MAR in agricultural settings depends on water availability, infrastructure, crop tolerance, and the suitability of soil to allow for deep percolation .

The suitability of soil in agricultural fields can be assessed using the Soil and Agricultural Groundwater Banking Index to determine if ag-MAR would be a viable and successful method of replenishing groundwater in a certain area, depending on the rate of deep percolation through the material, residence time of water in the root zone, soil chemistry, as well as topographic and other surface conditions . As long as conditions are viable, ag-MAR can be implemented annually, providing a reliable and sustainable source of stored groundwater to be used in times of drought when other water sources are deficient. In the coming years, GSAs may look towards ag-MAR as a powerful tool in sustainable groundwater management. However, ag-MAR is not only a tool to replenish our groundwater resources, as this application has multiple benefits to an environmental system,raspberry container size including nearby communities and wildlife. There are a limited number of studies that assess the multiple benefits of ag-MAR projects in addition to the apparent hydrologic response in a field’s underlying water table. Although ecological benefits may be harder to measure than hydrologic benefits, considering benefits to wildlife and the environment as design outcomes may have a positive influence on gaining stakeholders to implement more ag-MAR projects. MAR projects undoubtedly have the potential to bridge the gap between two distinct but connected fields of science. Ag-MAR projects are important, especially in California’s Central Valley, because they provide a sustainable way to manage and store groundwater while also being an ecological asset to migratory birds and other organisms that depend on wetland habitats . Across California, groundwater extraction accounts for 40% of the water supply for farms and cities . Colusa County, like many counties in the Central Valley, is highly dependent on pumping groundwater to support their agricultural production. Increased groundwater pumping has resulted in groundwater level declines of >20 ft over the last decade, which highlights the necessity of practicing sustainable groundwater management at the local level. Implementing ag-MAR in Colusa County gives support to farmers while recharging groundwater resources for local communities’ future water usage. The Nature Conservancy , in partnership with Colusa Groundwater Authority, has developed a multi-benefit recharge program to compensate farmers that volunteer their fields to be flooded during the fall season when water resources are limited but migratory bird and waterfowl habitat are in high demand during the fall migratory season . Desirable conditions in the fields that are conducive to both recharge and bird stopovers are those that best mimic a natural wetland habitat. The idea of the TNC project is to convert agricultural fields to temporary wetland habitats that have enough standing water and are free of orchards and other trees that would limit space in the fields and inhibit the bird’s eye view of the fields from above, as migrating birds are more inclined to stop in open flooded fields . Incentive programs like TNC’s on-farm multi-benefit recharge program or BirdReturns, are known to produce a large proportion of open water habitats in post-harvest rice fields during times of drought . BirdReturns and other incentive programs were responsible for providing, on average, 35% of the wetland habitat on the landscape during the 2013-2015 drought, with a few days even reaching up to 100% of the wetland habitat . Previous results of TNC’s incentive program have shown some of the largest average densities in shorebird presence in this agricultural region when wetland habitat was provided for migratory birds that are usually unable to stop in fallow rice fields . The timing of flooding during the fall also makes these ag-MAR sites valuable habitats for birds during migration season when habitats are in deficit, especially during drought .

Just as incentive programs provide a means of sustaining migratory bird populations during dry years, they also provide a way of restoring groundwater resources for use during drought, which further highlights the importance of multi-benefit recharge programs.The goal for this study was to develop two groundwater models using MODFLOW in order to simulate and understand the effects of conducting ag-MAR on selected field sites in Colusa County during the fall season. The first model developed was a large-scale regional model, called the parent model, which was built to derive a more refined, and local child model, which mainly focused on selected recharge sites. The development of the parent and child models allowed us to quantitatively and qualitatively assess the benefits of ag-MAR on the study area’s groundwater resources, the water supply of nearby communities, and as a wetland habitat for migratory birds. In addition to quantifying the effects to these beneficial users, with the results of our models we aimed to answer the following key research questions:1. Through the process of groundwater model development and analysis of results, what guidelines can we provide for optimizing the design of multi-benefit groundwater recharge projects like this in the future? 2. How does the timing, frequency, and amount of recharge affect the results of our model, and what are the benefits of changing each factor? Also, what would we need to monitor to measure these benefits? 3. In what ways can we use groundwater models in the context of understanding hydrologic and environmental impacts in multi-benefit recharge projects, and what answers can we derive from such models?The project area is located in Colusa County, California, in the northern Sacramento Valley region . Located in the Colusa groundwater subbasin of the Sacramento Valley groundwater basin, the project area is bounded by the foothills of the Coast Ranges in the west, and the Sacramento River and other surface water features in the east near the Sutter Buttes. The topography of the project area is mostly flat agricultural land, with higher topography in the southwestern area near the foothills of the Coast Ranges and in the east near the Sutter Buttes. With the exception of a few major urban centers and wildlife refuges, the land use in the area is predominantly agricultural. Agriculture in the area is supported with irrigation water supplied to growers via surface water features like canals and supply systems .Major surface water features in the study area include the Sacramento River, Butte Creek, Butte Slough, the West Borrow Ditch, the Colusa Trough, and the Glenn-Colusa Canal. The Sacramento River flows north to south along the eastern border of the parent model domain and serves as the principal stream in the Colusa Subbasin, significantly contributing to California’s water supply . Regionally, streams that drain the Coast Ranges and Sierra Nevada serve as tributaries to the Sacramento River .

Previous studies have shown that disruption of this gene leads to an albino phenotype

It has been reported that pre-assembled CRISPR/Cas9 ribonucleo proteins can be delivered into protoplasts to induce mutations, without the need for stable integration of CRISPR/Cas9 genes into the host-plant genome. Particle bombardment has also been used to deliver CRISPR/Cas9 ribonucleo proteins to wheat and maize cells, producing non-transgenic mutants. However, working with protoplasts, as well as utilizing biolistics, limits the potential for full-plant regeneration to some species and tissue types. Therefore, it is important to also develop alternative methods to produce non-transgenic CRISPR mutants of perennial crop plant species. In contrast to the limited success of plant regeneration from protoplasts, plant regeneration from leaf, hypocotyl, epicotyl, shoot, root, cotyledon, or callus explants has been well established for the majority of crop plant species, including many that are recalcitrant to regeneration from protoplasts. It has also been shown that proteins can be produced following transient expression of Agrobacterium T-DNA genes. In addition, Agrobacterium inoculation protocols have been developed for many perennial crop species. To circumvent the limited regeneration potential when using CRISPR ribonucleoproteins to produce non-transgenic mutants, we report a method for using Agrobacterium to transiently express the Cas9 and sgRNA genes in plant cells,drainage pot using tobacco as a model plant and PDS as a model target gene. We have also developed a high-throughput screening protocol utilizing next-generation sequencing in combination with high-resolution DNA melting analysis to efficiently identify mutants from a population of shoots regenerated in the absence of selection pressure.

We demonstrate that the combination of Agrobacteriummediated transient CRISPR/Cas9 expression with a highly efficient screening protocol makes it possible to efficiently obtain non-transgenic mutant plants, a method that should be applicable to heterozygous perennial crop species.Using tobacco as a model plant and an intron-containing GUS gene as a marker , we observed that transient expression of T-DNA genes in inoculated leaf discs peaked 3–4 days following Agrobacterium infection in the absence of kanamycin selection. Figure 1d shows the GUS activity in tobacco leaf discs 2–6 days post infection , and Fig. 1e shows the GUS activity in leaf discs after 5 additional days in timentin-containing media. The antibiotic timentin was used to suppress Agrobacterium growth following an initial 2–6 day co-incubation; thus, the GUS activities shown in Fig. 1e should result from stable integration of the GUS gene into the tobacco genome. The difference in GUS expression between explants in Fig. 1d and those in Fig. 1e is indicative of transient GUS expression, demonstrating that there are high levels of transient expression of the genes in the T-DNA region. The results in Fig. 1d, e indicated that a 3-day or 4-day co-incubation was optimal for Agrobacterium-mediated transient expression of the GUS gene. Three days of Agrobacterium co-incubation was subsequently used for transient expression of CRISPR/Cas9 genes. The sgRNA used in these experiments targets the beginning of the fourth exon of the endogenous tobacco phytoene desaturase gene , as shown in Figure 1c.We used this phenotype as a visual marker to identify tobacco mutants whose PDS gene had been edited following the expression of Cas9 and PDS-targeting sgRNA genes. We infected 415 tobacco leaf-disc explants in three independent experiments using 3 days of Agrobacterium co-incubation without any selection for transgenic cells or shoots .

A total of 197 shoots regenerated from infected explants exhibited the albino phenotype, indicating a mutation in PDS, demonstrating a mutation rate of 0.475 pds mutants per explant. However, due to the lack of chemical selection, the total number of shoots regenerated from each explant was very high, and therefore, the mutation rate per total regenerated shoots was quite low . These results indicate that pds mutant plants can be produced via Agrobacterium-mediated expression of Cas9 and sgRNA genes without using antibiotic selection. Ten independent pds mutant plants were randomly chosen for further analysis, and six are shown in Table 2. The specific genetic mutations in these plant lines were identified via high throughput sequencing, which demonstrated that all plants contained tetra-allelic mutations, meaning that all four alleles were mutagenized and no wild-type alleles could be detected. Microscopic analysis of plant tissues was unable to uncover the presence of green cells in albino pds mutants, suggesting that all PDS genes in all cells were mutated in these plants. The albinism resulting from the disrupted PDS gene enabled us to conveniently identify pds mutant shoots at early stages of shoot development in this study. However, the vast majority of desirable mutations for crop improvement are unlikely to display any visually identifiable phenotypes at the early stages of shoot development. When no selection pressure is applied during callus and shoot regeneration following Agrobacterium infection, the vast majority of regenerated shoots or plantlets should be non-mutant, as demonstrated above. Therefore, the ability to efficiently identify mutants lacking any visually identifiable phenotype from a population of regenerated shoots is essential for using the abovedescribed Agrobacterium-mediated transient mutagenesis system. Toward this end, we tested the effectiveness and efficiency of a two-step screening method using the newly produced pds mutants.

The first step, an initial identification of mutants, takes advantage of the high throughput nature of Illumina sequencing, and the second step, a fine identification of mutants, makes use of the high resolution of HRM analysis.Although our mutagenesis rate was relatively high per explant, without a visible phenotype, it would be diffificult to identify mutant plants due to the high number of regenerated shoots in the absence of chemical selection. Additionally, other CRISPR mutagenesis projects could have an even lower mutation rate than the one reported here. Therefore, we have developed a two-step method for high-throughput screening of shoots to identify the presence of targeted mutations. We first mixed leaf tissue from an albino pds mutant , pds-12, with leaf tissues from independently derived non-mutant shoots , regenerated from Agrobacterium-infected explants, at MT-to-WT ratios of 1:20, 1:41, and 1:83. We isolated genomic DNA from these pooled tissue samples and performed PCR reactions to amplify a 186-bp fragment that contained the sgRNA-target region on the fourth PDS exon . PCR products were sequenced on an Illumina platform to ~×60,000 to ×100,000 coverage. Next, we measured the amount of PCR product derived from the MT-to-WT ratios of 1:20, 1:41, and 1:83 mixed tissues and diluted it with ×6 the amount of PCR product derived from WT plant tissue. The diluted PCR product was also used for Illumina sequencing analysis. We observed that the PCR products derived from a 42- plant pooled tissue sample containing the pds-12 mutant showed a drastically elevated nucleotide variant frequency at positions 45–51 , which is consistent with verified mutations at these nucleotide positions . NVF is a measure of the frequency of abnormal nucleotides detected by DNA sequencing at a given position due to mutations or sequencing error. When we diluted the same PCR products with 6× WT PCR products , we observed significant reductions in NVF at positions 45–51 relative to the undiluted PCR product. The observed elevations and reductions of NVF before and after a 6× WT DNA dilution further verified the presence of mutations at nucleotide positions 45–51, as NVF resulting from sequencing error would be unaffected by dilution. As shown in Table 2, high-throughput sequencing uncovered four types of mutations in the pds-12 mutant line: a 1-bp deletion at position 51, a 5-bp deletion at positions 45–49, a 2-bp deletion at positions 49–50, and a 4-bp deletion at positions 46–49 . Similar results were observed using the 1MT: 20WT and 1MT: 83WT pooled tissue samples, with a more drastic elevation of NVF for the 1MT: 20WT samples and reduced elevated NVF for the 1MT: 83WT samples compared to the 1MT: 41WT samples . Using a single-blind approach ,growing raspberries containers we tested the accuracy of the mutant screening method based on elevations and reductions of NVF before and after a 6× WT DNA dilution as described above. We created eight 42-plant pools, five of which contained plant tissue from a single pds mutant plant line and three of which contained 100% WT plants. The five pds mutants used were pds-9, pds-10, pds- 11, pds-13, and pds-14. We confirmed that the screening method was reliable for identifying all 42-plant pools that contained pds mutant plants at a ratio of 1MT: 41WT , with 100% accuracy . Thus, the elevations and reductions of NVF before and after a 6× WT DNA dilution were excellent indicators of 42-plant pools that contained mutant plants.After identification of mutant-containing 42-plant pools, HRM analysis was used to identify individual mutant plant lines within each of the 42-plant pools .

To determine the sensitivity of HRM analysis, we performed HRM analysis on PCR products amplified from various DNA templates combined at different ratios. These template mixes were created using one-part pds-12 plant tissue combined with different parts independently regenerated non-mutant tissues in the following ratios: 1:1, 1:6, 1:19, and 1:29. Figure 3a shows that mutant-containing PCR products at ratios of 1:1, 1:6, and 1:19 could be distinguished from a wild-type DNA reference. The plant pool size we chose for subsequent HRM analysis was 7; thus, each 42-plant pool containing DNA from mutants could be divided into six pools of seven plants each. We also used a single-blind experiment approach to test the accuracy of HRM analysis to identify mutant plants. We created eight additional 7-plant pools, five containing a single pds mutant each , and the remaining three negative control pools containing only wild-type plants. The HRM analysis results are shown in Fig. 3b and demonstrate that all pooled samples containing pds mutant plants could be identified with 100% accuracy. Finally, upon the identification of mutant-containing 7-plant pools, individual mutant within each pool were identified via HRM based on a 1:1 mix between each putative mutant and a WT plant. Through this method, we successfully identified all pds mutant plants .To distinguish transgenic from non-transgenic mutants, we performed PCR on 29 randomly selected pds mutant lines using primers targeted to three regions in the TDNA fragment . Mutant plants were considered to be non-transgenic if they lacked a PCR product for all three primer sets . Approximately 17.2% of the tested pds mutant lines were determined to be non-transgenic following PCR analysis . As shown in Supplementary Figure 3, a non-transgenic plant , along with a transgenic plant , was cultured on MS media containing 100 mg/L kanamycin. The nontransgenic pds-7 plant died under kanamycin selection, while the transgenic pds-9 plant grew normally.Producing non-transgenic mutants of heterozygous perennial crop plants using CRISPR/Cas9 technology is highly desirable but challenging. We developed an effective method for producing and identifying CRISPR/Cas9- mediated non-transgenic mutant plants, which should be applicable to many perennial heterozygous crop plants. We have demonstrated that we can use Agrobacterium to transiently express CRISPR/Cas9 genes, and such expression can lead to the production of tetra-allelic, nontransgenic mutant plants. We have also demonstrated that the first step of our mutant identification, based on elevated and reduced nucleotide variance frequencies before and after a WT DNA dilution, using high throughput DNA sequencing analysis, is reliable and highly efficient. Furthermore, the second step of mutant identification, using HRM analysis, is simple and effective. With one sgRNA targeting the tobacco PDS gene, we achieved a 47.5% mutation rate using no selective pressure during callus or shoot regeneration. At least 17.2% of the pds mutant plants produced this way were non-transgenic, for an overall non-transgenic mutation rate of 8.2% . We expect that the rate of recovery for non-transgenic mutant plants following Agrobacterium-mediated transient expression of CRISPR/ Cas9 genes could be much higher than we reported here. One reason is that the albino phenotype caused by pds mutations used in this study can result in cell- or shoot growth disadvantages, which may have contributed to lower rates of mutant shoot production. Additionally, multiple sgRNA sequences may be used to target the same gene to increase the efficiency of mutant production. Protoplast-mediated delivery of CRISPR/Cas9 ribonucleoproteins offers advantages for creating non-transgenic mutant plants. However, regenerating plants from protoplasts can be difficult and has not been demonstrated to be possible for many important crop species. Low efficiency of plant regeneration from protoplasts has been reported in economically important crops such as avocado, grape, and apple. Furthermore, regeneration protocols have not been successfully demonstrated in many other plant species.

Self-compatibility is controlled by a single self-compatibility Sf dominant allele

Wide cultivation of almond, often under the more severe environments of Central Asia and the Mediterranean region, was possible because of the availability of a highly diverse gene pool, genetic recombination promoted by its self-incompatibility, and possibly, by interspecific hybridization and gene introgression involving other members of the Amygdalus subgenus. As a result, almond is an extremely variable species, with a high morphological and physiological diversity. This variability, measured with biochemical and molecular markers , has revealed that almond is the most genetically variable of the diploid Prunus cultivated species. In the Mediterranean Region, 2000 years of almond culture concentrated production to specific areas, where well-defined seedling ecotypes and local cultivars evolved. By the turn of the 20th century, most of these almond-producing countries had identified locally desirable cultivars that were often seedling selections of unknown origin. Thus, growers selected cultivars and landraces, which represented a rich genetic diversity. Most of these Mediterranean local cultivars have largely disappeared from cultivation in the last 50 years. Modern almond cultivation is based on a reduced number of cultivars grafted onto soiladapted clonal root stocks and cultivated under irrigated conditions when possible. Modern almond breeding started in the 1920s with the making of controlled crosses and seedling selections to meet changing agronomic and market demands. Currently,growing blueberries there are six active public breeding programs worldwide: the USA , Spain , Australia , and Israel .

Some private breeding programs exist also in the USA. In addition, there were various breeding initiatives in Russia, France, Greece, Italy, and Argentina. Different breeding objectives were developed according to regional agronomic, commercial, and market requirements. One of the main differences in the objectives is nut shell hardness. Two types of almonds are bred: soft-shelled and hard shelled . Common aims of Mediterranean breeding programs are self-compatibility and late-blooming, as most traditional almond cultivars are self-incompatible and early blooming.During the last 50 years, almond breeding for self-compatibility has mainly used two sources of Sf, local landraces originated in Italy and related species such as P. persica and P. webbii. Almond breeders have relied mainly on out crossing and, occasionally, on introgression from other Prunus species, for the development of new cultivars. Initially, in the USA and later in Russia and Mediterranean region , rapid genetic advances were achieved. In California, “Carmel” , as “Nonpareil” pollinizer, was the first cultivar release with extensive commercial impact. In Russia and the former Soviet Union, several late-flowering and frost-hardy cultivars were obtained in the 1950s with Primorskyi later used extensively for breeding in Europe. In the Mediterranean region, late flowering, productive, well-adapted, and resilient cultivars like Ferragnès or Masbovera were released with great success. The French self-compatible cultivar Lauranne showed a broad environmental adaptation, high production, and regular cropping. Although improved cultivars continued to be released, the amount of progress per generation diminishes since parents were continually drawn from the same gene pool. This situation has resulted in a potential loss of genetic variability in new breeding stocks and cultivars. Inbreeding depression in almond, expressed as low vigor, reduced flower number and fruit set, increased fruit abortion, lowered seed germination and seedling survival, increased leaf and wood abnormalities, and loss of disease resistance have been reported.

In addition, low self fruitfulness in self-compatible almond genotypes was suspected to be due to inbreeding. Regarding breeding for self-compatibility, male parents carrying the Sf allele and sharing the other S allele with the female parent are commonly used. In addition, crossing heterozygous self-compatible parents in breeding programs has been suggested to obtain homozygous self compatible genotypes to be used in further breeding. Such breeding strategies can narrow the genetic variability of crops when they lead to a reduced number of genotypes utilized as parents. Summarizing, modern almond breeding and production are dominated by a small number of widely distributed and related cultivars. This situation can lead to a potential increase of inbreeding depression and genetic vulnerability, i.e., susceptibility of most of the grown cultivars to biotic and abiotic stresses due to similarities in their genotypes. Therefore, it is needed to have up-to-date information of the relationships among genotypes used at breeding and production levels. Several almond populations have been analyzed with molecular markers in order to determine genetic variability and relatedness. However, these studies were performed with material from limited geographic areas and do not represent the current worldwide status of almond breeding stocks. Although genomic measures of inbreeding are more accurate than those obtained from pedigree data, pedigree-based analysis is a cost effective technique to estimate these parameters inbreeding populations and an alternative when genomic measures are unviable. Several reports have evaluated inbreeding based on pedigree data in breeding populations of fruit and nut tree crops. In almond, a pedigree analysis of 123 different genotypes from the USA, France, Spain, Israel, and Russia was reported. However, their work was mainly focused on North American genotypes and did not include many cultivars that have subsequently been released worldwide.

This study aimed to determine the genetic structure of current breeding stocks and breeding tendencies over the last 50 years using marker-verified pedigree data.Pedigree data of 220 almond genotypes were compiled from available bibliography and breeding records. From the 220 almond genotypes, 37 genotypes were no longer available as they were eliminated some time ago or were from discontinued breeding programs. To verify parental relationships of the rest of genotypes , we used SSRs, SNPs, and self incompatibility S-allele data from previous studies performed by the breeding programs taking part in this study . Marker data confirmed both parents of 71 genotypes and one parent of four genotypes and found three erroneous parentages. Two wrong parentages were found on the male parent of “Capella” and “Davey”, changing their pedigree to open-pollinated and a third incorrect parentage on “Yosemite” female parent, eliminating this genotype from the analysis. After the corrections made, pedigrees of 169 genotypes of known origin were analyzed . The origin of the genotypes were 59 from Spain, 56 from the USA, 16 from Russia, 11 from Israel, 10 from France, 7 from Australia, 7 from Greece, 2 from Argentina, and 2 from Italy. A pedigree data file was created. Each record in the file contained one cultivar or selection name, the female parent and the male parent, in that order. Once entered,square plant pots these data were available for inbreeding analyses such as determining the number of times a cultivar appeared in a pedigree as a male or female genitor. Genotypes of known origin were classified into two groups according to self compatibility: 104 self-incompatible and 65 self compatible.In summary, the inbreeding coefficient measures the probability that two alleles in a locus are identical by descent and so copies of the same allele from a previous generation. The pairwise relatedness measures the probability that two alleles at any locus are identical by descent between two different individuals. F and r range from 0 to 1, with values close to 0 indicating a low degree of inbreeding or relatedness and values close to 1 indicating a high degree of inbreeding or relatedness. The genetic contribution estimates the proportion of genome that comes from the same individual. Thus, a child will have 0.5 genome of either parent and a grandchild will have 0.25 genomes of his grandparents.To calculate F, r, and GC, parents of unknown origin were assumed to be unrelated and noninbred. The seed parent involved in all open pollinations was also assumed to be unrelated to the pollen parent. These assumptions, based on the fact that most almond cultivars are obligate out crossers because of their self-incompatibility, may lead to an underestimation of inbreeding. In the cases of genotypes of open-pollinated origin , numbers OP1, OP2, and OP3 were given to the pollen parent in order to be distinguishable for genetic studies. Also, all mutants were considered to have no genetic differences from the original cultivar, thus GC = 1. Since the differences between such mutants and the original cultivar are expected to be caused by a few mutations in the DNA, this simplification avoids the overestimation of inbreeding coefficients. Cultivars like Supernova and Guara were considered as “Tuono” clones. Regarding the different clones of the French paper-shell cultivar Princesse, used in both the USA and Russian breeding programs, we adopted the approach of Lansari et al.by analyzing both clones as the same cultivar. Historical reports suggest that the Hatch series “Nonpareil”, “I.X.L.”, and “Ne Plus Ultra” were seedling selections from an open-pollination progeny of the early-introduced cultivar Princesse.

This cultivar probably originated from the Languedoc region in France. Also, “Nikitskij” was selected in France in 1902. Because their specific origins remain uncertain, we analyzed these genotypes as nonrelated, which, however, could lead to an underestimation of inbreeding. Pedigree data were analyzed at four levels: worldwide, by country , by breeding program , and by genotypes carrying the Sf allele for self-compatibility.Our genetic study of almond breeding programs worldwide demonstrated that the most widely used cultivars were Nonpareil, Tuono, Cristomorto, and Mission. “Nonpareil” had a large influence in USA and Australian programs, where soft-shelled nuts are bred. This reference cultivar was present in all the breeding programs studied . The self-compatible “Tuono” and the late blooming “Cristomorto” were extensively used in the Mediterranean programs, where hard-shelled nuts are bred. “Mission” initially showed a considerable importance worldwide, but deeper analysis demonstrated that it was mainly influential in private American programs. Taking into account these results, we can establish two main breeding lines based on the use of three different founders: the European programs based mainly on “Tuono” and “Cristomorto” , and the North American–Australian programs based on “Nonpareil” . The French and Spanish breeding programs were based directly on “Tuono” and “Cristomorto”. In the French INRA program, the Italian cultivars Tuono and Cristomorto account for 60.0% of total GC and were present in the pedigree of all ten cultivars and selections evaluated. Also, the local French late-flowering and Monilinia-resistant cultivar Aï was a parent to both “Ferragnès” and “Ferraduel”. In the three Spanish breeding programs, the importance of “Tuono” and “Cristomorto” cultivars was very high, accounting to 46.2% of total GC. These two cultivars were present in the pedigree of 53 out of 59 cultivars and breeding selections from Spain. These results can be explained by the large influence of the French germplasm on the Spanish breeding programs, causing a high relationship between the programs of both countries . In the North American breeding programs, “Nonpareil” accounts for 43.7% of the total GC and was present in the pedigree of 48 out of 56 cultivars and breeding selections from the USA. In Australia, ‘Nonpareil’ accounts for 39.3% of the total GC and is present in the pedigree of 6 out of 7 cultivars and breeding selections. Also, “Lauranne” reaches an importance similar to ‘Nonpareil’, explaining the close relationship between the Australian and French programs . Even in other countries with noncontinuous breeding initiatives, such as Russia, Greece, or Argentina, the use of “Nonpareil” as a founder was common. Israel was the only country where these cultivars had a relatively low influence. This may be due to the extreme Israeli climatic conditions, forcing breeders to use locally adapted selections as parents. In Spain, the use of locally adapted cultivars such as Bertina at CITA as a donor for Polystigma ochraceum Sacc. resistance was successful but used only to a limited extent. Other examples of secondary founders include “Primorskyi”, used regularly as late-blooming and Fusicoccum-resistance donor in two of the Spanish breeding programs and “Eureka” and “Harriott” in the North American breeding programs.Pedigree analysis is a cost-effective and well-established way to monitoring inbreeding and relatedness among controlled breeding populations. However, the veracity of any analysis based on this kind of data relies on the accuracy of records collected across multiple institutions and by many breeders. In order to verify parental relationships of the genotypes under study, we used SSRs, SNPs, and self-incompatibility S-allele data from previous analysis carried out by the breeding programs taking part in this study. Our molecular marker analysis confirmed 146 parentage relationships and found three errors , which were corrected accordingly. Thus, the marker-based pedigree analysis performed showed only small parental changes and corroborates the consistency of the results reached by this study.However, several reports have demonstrated that large scale genomic analysis may provide more accurate results than pedigree analysis. This kind of genome-based pedigree analysis has already been performed in apple.

Similar marginal voids can also be seen in WT meristems treated with exogenous cytokinin

Although cytokinin responses are homogenously distributed in these meristems, the pWUS:eGFP-WUS pattern does not clearly show strong WUS expression in the peripheral regions where cytokinin induced stability might be expected. The results of cycloheximide and MG132 treatments do not help clarify this situation, as the alternating patterns of stability and instability cannot easily be explained in terms of the cytokinin signaling pathway alone. To do so requires assuming that the cytokinin phosphor-relay system has a previously undetected branch pathway, potentially regulating a protease with equally unusual phospho-dependent activity. However, this model is not much different than the observation that cytokinin influences WUS stability through both protein translation protein pathways, as both models require multiple steps with poorly known intermediates. Attempts to identify the possible intermediates using lists of cytokinin-targeted genes do not clearly help resolve this situation, as a meta-analysis found only five translation related genes, two of which modify mRNA, one that modified tRNA, and two that are involved with ribosomal RNA processing. The same list of cytokinin targets also contains six protease genes , while a single representative from the ubiquitin/proteosome pathway was down-regulated. In the absence of a clearly direct cytokinin-WUS connection, it is quite tempting to speculate that protein stability is a secondary effect of cytokinin responses. If so, stability may be a generic feature of cytokinin responses, which has the potential to affect all proteins simultaneously. Experiments with auxin on the other hand, suggest a much more direct link with WUS stability. Four hours of exogenous NAA treatment dramatically reduced pWUS:eGFP-WUS fluorescent levels,pot blueberries while comparable treatments with cytokinin took a minimum of 12 hours to show the slightest response in WUS expression.

The auxin–induced degradation was also readily blocked by cycloheximide treatments , indicating that the response requires protein translation. Still, exactly which proteins are translated, and how they affect WUS stability is not clear. Auxin induced degradation may have a functional significance for lateral anlagen though, as the concentration of auxin responses in distinct foci, would help rapidly reprogram the anlagen cells by degrading conflicting developmental proteins. This hypothesis is consistent with the large marginal voids of WUS and CLV3 expression found when cytokinin responses are ectopically induced with the pCLV3:GR-LhG4 x p6xOP:ARR1ΔDDK-GR system , which were often correlated to the presence of leaf primordia and sites of auxin accumulation.Although not quite as direct, other research has also shown that WUS transcript levels are indirectly linked to auxin transport. In addition callus tissue studies have found that induction of SAMs does not require cytokinin alone, but instead requires an appropriately high concentration of auxin or a balanced auxin/cytokinin ratio, clearly implying that auxin is a significant part of the process. Considering the overall organization of the SAM, this suggests a model where WUS helps stabilize the mutually exclusive pattern of auxin and cytokinin responses in the PZ and RM by activating the biosynthesis of both hormones and auxin transport genes within the CZ. The lack of hormone responses in the very cells that produce them is consistent with a similar pattern in root development, and given the often symplast-like environment in the SAM, a repressive mechanism may be necessary to prevent hormone response proteins from spreading into the CZ and suppressing biosynthesis. The fields of protein stability and instability brought about by the hormone responses also appears to define the number of WUS producing cells, and eventually, the concentration of WUS molecules that reach the CZ, forming an indirect, but stable set of feedback loops that share WUS as an anchor.

The CLV3 pathway may represent another feedback loop within this framework, as it is also activated by WUS in the CZ, similar to the postulated activation of hormone biosynthesis genes. Although the intermediate steps are not clear, CLV3 appears to suppress cytokinin-induced proliferation, as seen by the hypersensitive response of clv3-2 mutant to exogenous cytokinin . By doing so, it may potentially function as a third feedback loop, negatively regulating WUS transcription though a mechanism that is slightly more direct than either hormone pathway alone. It would thus be of great interest to learn what proteins regulate WUS transcription in the RM, as the ahk2/3/4 RNA in-situ clearly shows that cytokinin responses are not involved.Meristems used for nuclear/cytoplasmic measurements were stained with FM4-64 and DAPI to help delimit cell walls and nuclei, respectively. Cell volume was assumed to be cubic rectangular, and estimated by calculating depth as an average of length and width measurements. Nuclei were assumed to be spherical, and their volumes were calculated directly from their maximum cross-sectional diameter. The relative concentration of fluorophores in each subcellular compartment was then estimated by sampling a representative volume to calculate the average concentration in “fluorphore units/µM3 ” units, based on the voxel dimensions in the optical section. This figure was then multiplied by the volume of each subcellar compartment to estimate the relative amount of fluorophore units present in each. At least 4 cells from each of three tissue layers were sampled in each meristem, and a minimum of 5 meristems were sampled for each treatment, for a total of 180 measurements. In May 2016, an environmental scientist at the Indian Institute of Science, T.V. Ramachandra, made headlines with his assertion that Bengaluru 1 — the center of India’s information technology boom and one of its fastest growing cities—would be “dead” in five years . The claim struck a nerve, and led to a series of English medium news articles and social media posts debating Ramachandra’s statement . As evidence of its continuing resonance, the discussion again surfaced in August 2017, with headlines proclaiming Bengaluru’s demise in three years . Although Ramachandra’s contentious argument was based on his study about environmental degradation in the city , he expressed the effects of this decay in terms of food and urban life.

He is quoted in Deccan Herald as saying, “what’s the point [of] earning better when the food that you eat is adulterated? As a result of unplanned urbanisation, Bengaluru is going to be an unliveable and dead city in the next five years” . With this bleak prediction, Ramachandra challenged narratives proclaiming the economic advantages of rapid urban development—higher wages and a burgeoning middle class—by suggesting that money means little when one’s food is inedible and the urban environment is uninhabitable. Ramachandra’s assertion and the intense debate that it inspired are indicative of the broader insecurities and aspirations that anchor this dissertation. I trace the shifting food supply chain that connects Bengaluru with its agrarian periphery in order to understand the changing relationships among food producers and consumers in the context of rapid urbanization. With analmost 47% urban growth rate between the 2001 and 2011 , Bengaluru and its outlying communities have changed drastically since the start of the city’s information technology boom in the early 1990s. As the city expands and its landscapes and livelihoods shift, urban residents express regret at the loss of the calm and idyllic “Garden City,” replaced by unhealthy ecologies and human communities. But, they also proudly proclaim Bengaluru to be a “cosmopolitan” city, with restaurants and bars befitting a globally connected technology hub. This ambiguous relationship with Bengaluru’s shifting food scene extends to the edges of the city, where farmers whose fields sit alongside factories and housing developments are in an economically and environmentally precarious position, and who recognize that they are likely the last generation to farm their land. They are also aware, however, that their proximity to the city opens up new opportunities, including higher prices for their commodities,square plastic plant pots access to private schooling for their children, and the potential to sell their land at a great profit to real estate developers. Food, a daily need packed with political and cultural meaning, is a particularly productive site of analysis to discover how processes of production, exchange, and consumption intersect with the insecurities and aspirations of an expanding cityscape. This project analyzes the relationship between food supply chains and the city. Tracing food networks illuminates shifting desires and consumption patterns as well as changing relationships among urban and agrarian communities. Examining how the expanding city affects food supply chains sheds light on how changing livelihood structures, infrastructures, and class relationships intersect with issues such as food safety and access. How food is produced, exchanged, and consumed can tell us about the relationship between bodies and changing cityscapes. Advocates of the second intervention that I consider in detail in this dissertation understand their project as a challenge to corporate assertions about the efficacy of “direct” supply networks. Increasingly, urban middle and upper class residents of Bengaluru are growing their own food for home consumption. For these individuals, gardening offers two interventions: one, a way to ensure food quality and safety in the context of increasingly untrustworthy food sources; and two, a site to contest the social and environmental ills of urban development. In asserting the value of food cultivation among urban professionals, Bengaluru’s middle and upper class gardeners generate new understandings and practices of contemporary urban life. However, these projects remain anchored in existing class and caste inequalities that make cultivation a leisure activity for some and a livelihood strategy for others.

The stakes for these projects are high—while they are guided by the concerns and desires of a particular class segment , they have repercussions for the entire food system and thus affect everyone. It is therefore critical that we understand these projects, as ideological forms and concrete practices that promise to shape Bengaluru’s food ecologies going forward. What is lost by focusing on some problems and ignoring others? What solutions emerge to answer these problems? Whom do these solutions benefit, both intentionally and otherwise? It is critical to consider these questions because the rate of urbanization worldwide has generated multi-fold challenges for ensuring food security, sustainability, and safety. Yet the effects of urbanization on food systems remain understudied . Feeding the world’s growing cities is of pressing concern as farmers abandon agriculture in the face of mounting economic insecurity and climate-related instability. This dynamic is particularly pressing in India, where UN-Habitat’s 2016 World Cities Report predicts that the urban population is expected to grow by an additional 300 million residents by 2050. In this context, a primary concern is feeding the burgeoning urban population while also ensuring food security for rural communities. Hunger and malnutrition remain critical struggles in India. The results of the 4th National Family Health Survey show that among children under five years old, 38.4% are stunted , 21% are wasted , 7.5 are severely wasted, and 35.7% are underweight . These numbers are closely linked with poverty: the percentage of underweight children decreases as the “wealth quintile” increases, from 49% among the lowest to 20% in the highest. Among adults, “deficiencies in the diet of both women and men are observed among those with little or no schooling, those in rural areas, those in poorer households, and those belonging to scheduled tribes and scheduled castes” . As these numbers suggest, many in India struggle with food insecurity, and this struggle is especially acute among marginal communities. My interviews with residents of a slum community in Bengaluru suggested that hunger was a daily reality. For these families, the rations provided by India’s Public Distribution System were not sufficient to cover their families’ dietary needs. They supplemented their rations in several ways—with food from their family members who remained farmers in their ancestral village, for example, or vegetables that were damaged and therefore cheaper—but they struggled to have enough to eat. This dissertation does not explore the lived realities of food insecurity among the urban and rural poor. This is not due to the declining significance of hunger, but rather because in India today, problems other than food insecurity motivate many interventions into the food system. Despite the continued challenges of hunger and malnutrition, the majority of my interlocutors— ranging from government officials to NGO leaders—who were involved in making and advising policy decisions were relatively unconcerned with food insecurity. This is at least in part due to my empirical focus on fruits and vegetables, which have not been part of India’s strategy for ensuring national food security.

Cell identity in the SAM is thus largely an issue of location rather than its developmental history

The astute student however, will note that this interpretation is not quite universal, as some researchers further postulate the existence of a fourth “Organizing Center” inserted between the CZ and RM tissues. Although not shown in Figure 2.0, the OC is equivalent to the rounded apex of L3, pushing the remaining part of the RM somewhat deeper into the stem. Until better genetic evidence is available though, only CZ, PZ, and RM will be used for the remainder of this dissertation. While the numbering system shown in Figure 12 does provide a useful set of spatial coordinates, it is also somewhat misleading as it implies that the SAM is static structure, unchanging over time. This could not be further from the truth. Instead, it must be remembered that the SAM is a site of plant growth, and as a result its cells are in a state of constant flux as they divide, grow, and differentiate. For example, the repeated perpendicular divisions that occur in L1 and L2 actually cause these layers to expand sideways, where the displaced cells eventually bend around the curve of the apical dome and become part of the cylindrical stem surface. The motion is reminiscent of the path taken by water droplets in an umbrella-shaped fountain, though the individual plant cells move considerably slower. If growth by lateral displacement is followed to its logical extremes, it is important to note that all of the founding cells will be pushed off to the sides over time, while new ones take their place in the middle. No single cell in the SAM is a permanent resident. The overall shape and size of an SAM is perhaps more analogous to a standing wave,growing pot where stability is the illusion caused by a dynamic equilibrium. Maintaining that wave is of course a difficult challenge, as the inputs to that equilibrium must be precisely matched to its outputs at all times.

Failure todo so would quickly rob the plant of its ability to grow, with obvious consequences for survival. Exactly how this balance is maintained is not fully understood, but the motion of the cells makes at least one part of the process perfectly clear: the cells must change their identity as they are moved from one place to another. Those that start in the CZ for example, switch to PZ gene expression patterns as they move further away from the middle, and may later adopt leaf and flower identities as they are incorporated into mature organs.The ability of a cell to determine its location within the SAM structure is thus of paramount importance, yet it must do so in the absence of any stationary reference point. So far as currently understood, each cell solves this problem in exactly the same way a person would do so: it talks to its neighbors. Based on what the individual cell sees and what its neighbors report seeing, it is possible to work out exactly where the cell is located in the overall plant structure. Of course in actual plant tissues such communication occurs largely through to the exchange of proteins, hormones, and RNA molecules, though increasingly evidence suggests that mechanical forces in the cell wall may also contribute some information [2]. Some molecules can travel further distances than others, some are modified en-route in order to become functional, and still others move from cell to cell in precise patterns, much like the knight in a game of chess. When these molecules are produced in different areas of the plant, the surrounding cells can estimate their relative locations to each other simply by reading the chemical bar-code in their local milieu, and then develop accordingly. At the present time, only a few such routes of chemical communication have been identified, two of which are plant hormones: auxin and cytokinin. Auxin is best known for increasing the volume of cells, though it also has roles in apical dominance and tropism growth patterns. Cytokinin meanwhile is known for stimulating cell division, in addition to other roles in senescence and pathogen responses.

Together the function of the two hormones would appear to complement each other very well in terms of overall growth, yet within the SAM they appear to mix about as well as oil and water. Cells that respond to auxin often don’t respond to cytokinin, and vice versa. Why this should be so is not well understood, but studies of root vasculature development suggest that their mutual exclusion is actually used to generate spontaneous patterns that help guide plant development. In callus tissue, the two hormones are often found to have response patterns arranged in a polka-dot like arrays, where each hormone “dot” is surrounded by a circular field belonging to the other. The SAM is organized around a single such dot, where cytokinin responses occur in the RM, and auxin responses occur in the PZ which often occur in discrete foci corresponding to new lateral organs. The CZ cells in contrast, do not appear to be sensitive to either hormone, but instead express both auxin and cytokinin biosynthesis genes . The production of cytokinin in the L1 and L2 is also consistent with the distribution of bioactive cytokinin concentrations observed with immunological techniques and with GFP reporter systems. This suggests a stable arrangement of three mutual exclusion zones within the SAM, which closely correspond to the known CZ, PZ, and RM tissues. Root apical meristems in contrast, appear to be based on the reciprocal arrangement, as roots have an auxin response dot in the middle surrounded by cytokinin responses in the overarching root cap, concentrated in the root cap columella cells.Another potential communication system that has been extensively studied involves a potential feedback loop between the CZ and RM cells, thought to be carried out by WUS and CLV3. WUS is a homeodomain transcription factor produced exclusively within the RM, but is capable of moving 2-5 cell diameters away from its center of origin.

WUS has also been shown to activate transcription of CLAVATA3 in the overlying CZ cells by directly binding to the CLV3 promoter. CLV3 in turn, is thought to be a small secreted oligopeptide that is modified with a few arabinose sugars. The mature glycoprotein then travels through the apoplast to reach leucine-rich receptor kinases in the RM, such as CLV1 or BARELY ANY MERISTEM1, thereby triggering a signaling cascade that ultimately suppresses WUS transcription. Many of the intermediate biochemical steps however, have not yet been fully identified, which makes it difficult to fully reject the feedback loop null hypothesis. There is also evidence of a more complex set of feedback loops, as WUS has been found to regulate components of the cytokinin signal transduction pathway , and exogenous cytokinin are able to stimulate WUS transcription. Altered cytokinin signalling pathways have also been shown to affect CLV3 expression patterns. WUSCHEL-LIKE HOMEOBOX5 , which is closely related to WUS, is known to participate in auxin pathways within the root, while the generation of SAMs from callus or root tissue has repeatedly been shown to require a pre-incubation on auxin rich media, where it may actually stimulate auxin transport . Micro RNA molecules may also be involved, as a variant of AUXIN RESPONSE FACTOR 10 that was resistant to miR160a was able to increase WUS and CLV3 expression patterns. Clearly, there is a lot going on. To help clarify how such cross-talk contributes to SAM structure, the research presented in this dissertation explores two closely related subjects. The first is the regulation of CLV3, which was studied by resolving the promoter structure of this gene in chapter 3. The results suggest that CLV3 is regulated in part by auxin responses,square pot while activation and/or repression is likely to be controlled complicated set of cis-motifs in the 3’ enhancer region. The presence of these 3’ motifs in a known transposon also suggests a novel origin of the WUS/CLV3 feedback loop. Chapter 4 meanwhile, explores the possibility that WUS and cytokinin responses form a second feedback loop necessary for SAM structure. This was done by narrowing down the possible cellular and biochemical routes by which cytokinin could affect WUS transcription, translation, and protein movement. The results however, suggest that the two pathways are atlargely independent of each other, though cytokinin responses may increase WUS stability in the RM. Unexpectedly, the data also found that the absence of cytokinin responses in the CZ is a critical part of SAM structure. The cytokinin response-free cells were also found to have an enhanced protein degradation mechanism, which may help shape the WUS protein gradient. Interestingly, WUS proteins were found to be rapidly degraded following auxin treatments, suggesting a model in which the SAM structure is defined by cytokinin-induced stability in the RM, and auxin-induced protein degradation in the surrounding CZ and PZ cells.The WUS-CLV3 feedback loop has long been an attractive model to explain how SAM structure is maintained in a dynamically changing cellular environment. Simply by combining activation of CLV3 with the repression of WUS, computer simulations have repeatedly shown that this is sufficient to maintain constant population of cells with CZ and RM identity. However, despite the simplicity of this model, the molecular mechanisms that carry out the feedback loop have instead revealed a number of potential complications. On the forward path for example, WUS is known to be a bi-functional transcription factor, activating and repressing several hundred different target genes.

Currently it is not currently known exactly how WUS switches from activator to repressor, but it has been shown to directly bind to DNA motifs in AGAMOUS and CLV3 regulatory regions, where it activates their transcription. Additional binding sites on repressed targets such as KANADI1, YABBY3, ASYMMETRIC LEAVES2 have also been identifie. Complicating this model of is the observation that CLV3 activation requires both WUS and SHOOTMERISTEMLESS in leaf tissues, suggesting that the presence of WUS alone is not sufficient. In addition WUS has also found to directly interact with the GRAS domain transcription factor HAMl, as well as the potent transcriptional repressor TOPLESS. TPL itself further has been shown to assemble a protein complex with Sin3 ASSOCIATED PROTEIN and HISTONE DEACETYLASE 19 [49, 50], suggesting a potential link between WUS and chromatin modification. In order to discriminate between the two models, this study began by attempting to identify the cis-regulatory environment around the CLV3 locus. The CLV3 expression pattern was firstcarefully recorded with a GFP reporter, which in contrast to previously published RNA in-situ’s, found layer-specific differences in CLV3 transcriptional output. The regulatory regions of CLV3 were then annotated by mapping predicted transcription factor binding sites and computationally significant cis-motifs, which were further resolved with phylogenetic footprinting. This analysis found that CLV3 has a very simple 5’ promoter, containing an auxin responsive element, suggestive of ubiquitous expression. The 3’ enhancer in contrast, contained at least 3 large cis-regulatory modules, two of which were found within a naturally occurring transposon, while the 3rd included several known WUS binding sites. On the basis of promoter deletion experiments, all three cis-regulatory modules were found to be required for CLV3 activation. The existence of the transposon in turn, has several implications for the evolution of the WUS-CLV3 feedback loop and Brassicaceae plant anatomy. Previous reports of the CLV3 expression pattern have consistently found it localized to the apex of the SAM, where it is often used as an indicator of CZ cell identity. Within this region, the expression pattern is somewhat variable, as previous RNA in-situ revealed a narrow inverted cone-shape, while GFP and GUS reporters often produce more indistinct rounded shape 3-4 cell layers deep. In contrast, the present study found a slightly more complex pattern when viewed as a longitudinal section. In perfectly centered sections, the pCLV3:mGFP5-ER reporter often appears in an inverted cone shape, but the expression zone is noticeably broader than the previous RNA in-situ results . As the section plane is displaced from the central axis and becomes more tangential, a conspicuous gap is frequently visible, where the L2 cells have less fluorescence than those immediately above and below. This suggests a bi-partite expression pattern where a flat, circular domain occurs specifically in the L1, and a second spherical domain occurs underneath in the L3 cells . In order to identify the CLV3 regulatory structure, this work began by annotating all known regulatory motifs on an 8kb genomic sequence centered on At2g27250.

Suppose that the polygyny threshold in equation were satisfied by an equality

In order to produce analytically tractable results, we simplify by assuming throughout that there are only two types of males, rich and poor, with rich males being a fraction u of the population. All rich are identical, as are all poor. The rich males are indexed by r and the poor by p. We now demonstrate two theoretical results with the potential to resolve the polygyny paradox. First, diminishing returns to additional wives arising from causes other than necessity to share a husband’s rival material wealth will reduce the number of wives acquired by each rich male. Second, because of this fact, a highly unequal wealth distribution with few extraordinarily rich men may produce little polygyny, while a less unequal wealth distribution with a larger fraction of rich men may produce a greater extent of polygyny. Two rich men, for example, can be expected to have more wives in total than one very rich man whose wealth equals their combined wealth. For this same reason, the Gini coefficient—see table 2 for a definition—is not a sufficient statistic for the analysis of the relationship between polygyny and wealth inequality. We take up each of these results, in turn, before assessing if our empirical estimates are consistent with this explanation.If we assume that male demand is limiting, then equation determines the number of wives each rich man will have. It is clear from inspection of equation that a greater extent of diminishing fitness returns to additional wives produces a lower male demand for additional wives. This is demonstrated mathematically in the electronic supplementary material. Determining the effect of greater diminishing returns to additional wives when female supply is limiting is more challenging. As noted above, if female supply is limiting,1 litre square plant pots the value of n* implied by the polygyny threshold inequality in equation has no closed form solution.

To address this challenge, we proceed as follows.Then a reduction in d, holding all other terms constant, would reduce the right-hand side of the equation—the fitness of each of the n wives of a rich man—while having no effect on the left-hand side—the fitness of a singleton wife. Thus, holding all else equal, an offsetting decrease in n would be required to restore the equality. This is demonstrated in the electronic supplementary material by differentiating equation with respect to d. This means that a man who was just barely rich enough so that an unpaired woman would choose to marry him as wife number under the initial d, would, under the lower d, be unable to secure the unpaired woman’s partnership. As such, an increase in the extent of diminishing returns to additional wives will reduce both male demand for, and female supply to, polygynous marriage. Our results imply that if d , 1, a larger quantity of moderately rich men can be expected to have more wives in total than a smaller quantity of even richer men, holding constant the total wealth held by the rich across these cases. This first finding will interact with our second finding, discussed below, concerning the effects of the population density of the rich class of men on the frequency of polygyny and the level of wealth inequality. In the electronic supplementary material, we present an alternative approach to account for diminishing marginal returns to increasing number of wives and find that our insights do not depend on the specific way in which diminishing fitness returns to increasing number of wives are modelled.To address prediction P1, we present empirical estimates of m and d . These values are estimated using a multi-level regression model fit to our individual level data; methodological details are provided in the electronic supplementary material. In all but four of the populations in our sample, the estimated d coefficient is reliably less than 1. This result provides cross-cultural empirical support for the first of the two conditions needed to generate a transition to a greater degree of monogamy with increasing wealth inequality.

Note two further results also shown in figure 5. First, our estimates for m are quite low, particularly across the agricultural economies. Second, our estimates of d 2 m are positive in almost all populations, including those that are concurrently polygynous and those that are serially monogamous. The consistently small values of m across all of our samples, even the monogamous ones, was unexpected. However, these low values reflect changes in male fitness per wife. Because of biological limits to the rate of reproduction in human females, significant increases to wealth are constrained to have less than proportional effects on fitness per wife. The effects observed here are more likely to reflect the ability of males with more than a threshold level of resources per wife to minimize offspring mortality, rather than to significantly enhance their own fertility. Though not discussed in detail here, our data suggest that male wealth impacts male fitness primarily by increasing the rate of wife acquisition rather than by increasing reproductive success per wife . Our second point addresses the possible concern that our estimates of d may be low, in part, because we use times married as our measure of polygyny. While it is true that men can accumulate a greater maximal number of marriage years through concurrent polygyny than serial monogamy, figure 5a demonstrates that the use of times married is an appropriate measure of polygyny for our purposes. Across almost all populations, the elasticity of fitness with respect to times married, d 2 m, is positive and reliably non-zero. Because these estimates measure the population-specific effects of cumulative number of wives on reproductive success, they demonstrate that an increased number of marriages leads to increased reproductive success in both types of marriage systems—concurrently polygynous and serially monogamous.We have established that there exists a strong cross-cultural pattern of decreasing—but reliably non-zero—fitness returns to increasing number of wives for reasons beyond rival wealth sharing.

We now turn our attention to testing if the transition to agriculture is associated with a decreasing fraction of wealthy males. In our theoretical model, we assume a discrete two-class wealth distribution, but empirical wealth data typically have continuous distributions. To deal with this issue, we consider two proxy measures for per cent rich in our empirical data: the minimum percentage of men that account for a fraction f of the total wealth and the frequency of men with more than c wealth, where c is the empirical midpoint in each population between the average wealth of males with one wife and the average wealth of males with two wives. More details about these metrics are included in the electronic supplementary material. Table 3 provides population-level posterior estimates of the completed wealth and completed polygyny measures, with the mean estimates by subsistence type shown in the bottom panel. To address prediction P2, we calculate empirical estimates of the fraction of rich men by production system . We find that agricultural populations have a significantly reduced frequency of wealthy individuals relative to horticultural populations. All four panels show reliable differences in mean per cent rich between the horticultural and agricultural subsistence modes. This lesser fraction of wealthy individuals suggests a decreased number of men both able and willing to take second wives. This in turn leads to reduced levels of per cent female polygyny in contexts where large wealth differentials are not able to underwrite large differentials in wives due to the existence of diminishing fitness returns to such additional wives. A limitation of this last result is that it is based on data from only four agricultural populations, three of them concentrated in a restricted region and time period . Moreover, a more informative dataset would come not from agricultural populations in the time period between the 1700s and 2000s, but rather from the agricultural populations in which monogamy actually began to emerge denovo. In our main analysis, we use estimates derived from the individual-level records available in the populations shown in table 1; in the electronic supplementary material,macetas por mayor we present comparable analyses that include 14 additional wealth distributions from historical agricultural populations. The results of this supplementary analysis are consistent with our arguments here—and in fact show stronger and more reliable effects in the direction predicted by P2. These supplementary data, however, are based on sometimes contested reconstructions of the historical wealth distributions pieced together by archaeologists and economic historians;they must be appreciated within the constraints associated with such forms of data.Using individual-level data from 29 populations, we show evidence of a general cross-cultural pattern of decreasing marginal fitness returns to increasing number of marriages. Further, using these same 29 datasets , we demonstrate the existence of an increasingly skewed distribution of material wealth in class-based agricultural societies . Both of these empirical findings are consistent with our model-based explanation for the decline of polygyny in societies engaged in agricultural production.We use cross-cultural data and a new mutual mate choice model to propose a resolution to the polygyny paradox. Following Oh et al., we extend the standard polygyny threshold model to a mutual mate choice model that accounts for both female supply to, and male demand for, polygynous matchings, in the light of the importance of, and inequality in, rival and non-rival forms of wealth.

The empirical results presented in figures 5 and 6 demonstrate two phenomena that are jointly sufficient to generate a transition to more frequent monogamy among populations with a co-occurring transition to a more unequal, highly stratified, class-based social structure. In such populations, fewer men can cross the wealth threshold required to obtain a second wife, and those who do may be fabulously wealthy, but—because of diminishing marginal fitness returns to increasing number of marriages—do not acquire wives in full proportion to their capacity to support them with rival wealth. Together, these effects reduce the population-level fraction of wives in polygynous marriages. Our model demonstrates that a low population-level frequency of polygyny will be an equilibrium outcome among fitness maximizing males and females in a society characterized by a large class of wealth-poor peasants and a small class of exceptionally wealthy elite. Our mutual mate choice model thus provides an empirically plausible resolution to the polygyny paradox and the transition to monogamy which co-occurred with the rise of highly unequal agricultural populations. We, however, cannot yet explain the causes of the unexpectedly substantial decreasing marginal fitness returns to increasing number of marriages. A purely statistical explanation of our results could be that we have missed some important rival form of wealth, which if accounted for would result in a larger estimate for m and hence a reduced estimate of the degree of diminishing returns to additional wives for reasons other than the sharing of rival wealth. Another possibility, already mentioned, is that in some of our datasets the very wealthy could be deliberately limiting their reproductive success , which would also drive m downwards. In addition to these possible statistical effects, there are a number of other plausible causes of the diminishing returns to additional wives observed in our populations. One possibility is that a male’s time and attention are rival inputs to his own fitness. This situation is likely to arise when paternal investment is essential to offspring survival and well-being. A male’s time can also be rival in other fitness relevant ways. For example, it may be difficult for a single wealthy man to effectively mate guard a large number of wives. With a wealth ratio of mr ¼ 2 and a per cent rich of u ¼ 0.5, a single rich man will have to monopolize his two wives in the face of challenges from a single unmarried man on average; however, with a wealth ratio of mr ¼ 10 and a per cent rich of u ¼ 0.1, a single rich man will have to defend his 10 wives from nine unmarried men on average. As the wealth ratio grows even more skewed, this situation could become increasingly difficult to manage . A related possibility is that a growing number of unmarried men could socially censure wealthy polygynous males, imposing costs on them that reduce male demand for and/or female supply to polygynous marriage. A third possibility is that sexually transmitted infection burden could diminish returns to polygyny, if polygyny enhances infection rates.

All cultivated citrus species or citrus relatives are susceptible to the disease

The model also predicted that limited areas in California are climatically favorable for HLB establishment, but the probability of establishment was predicted to be much lower compared to Florida. To increase the confidence regarding the model projection, a Principle Component Analysis was performed to investigate the climate similarity of regions with proven HLB presence with the California climate. PCA analysis showed that the climate in areas around Los Angeles overlapped with the climate of regions where HLB is currently present. On a global scale, HLB predictions from MaxEnt combined with expert knowledge could be informative for countries such as Australia, New Zealand, and European countries, where HLB has not been reported thus far. Oxytetracycline has been used extensively for over 60 years in agriculture for both plants and in animal health. The tetracyclines as a chemically diverse family of compounds have multiple activities across many organisms. Within this large family of compounds, a select subgroup has discreet and potent activity against α-proteobacteria, including CLas, while demonstrating inactivity against human bacterial pathogens labeling them as “non-antibiotic tetracyclines” a designation accepted by the FDA. Our efforts in tetracycline semi-synthesis have produced three separate series of compounds found effective against the surrogate strain Liberibacter crescens and with increased potency, while in a study of over 35 derivatives distinct structure-versus-activity parameters have emerged guiding the further design and synthesis of compounds active against Liberibacter species. One series of positional derivatives was three orders of magnitude more active than oxytetracycline,macetero de 7 litros while the others were at least two orders of magnitude more active in this surrogate strain.

The most potent compounds derived from the screening were examined in further studies of trans-bark uptake in young citrus trees, showing that proper formulation demonstrated transport throughout the plant and into the canopy. Further studies in HLB-infected citrus trees by bark or foliar application decreased PCRbased bacterial levels after one month of treatment while shoots collected from HLB-infected Valencia orange shoots showed significant repression of L10 and 16S mRNA levels. These preliminary results demonstrate that chemically-modified tetracycline derivatives are active specifically against HLB, lowering infection burden in whole plants, and are considerably more active than oxytetracycline. The case for novel and inexpensive compounds specifically designed for citrus plants harboring α-proteobacteria infections will be presented along with its potential to replace oxytetracycline for agricultural use. Besides the availability of financial and technological resources, and citrus growers’ awareness about the damages that mismanagement of the Huanglongbing can cause to their and neighbors’ groves, the decision of applying the Asian citrus psyllid control and symptomatic trees eradication by citrus growers depends on an economic analysis of the benefits and costs of any measure adoption in the short , medium , and long-term . Although basic information about the temporal progress of HLB and damage in plants of different age, scion/rootstock combination, and under different HLB management still scarce, a macro of Excel was developed to estimate and quantify the future impact of HLB in groves adopting different kinds of disease management. In this study, that macro was adapted to simulate the impact on production of citrus groves under different scenarios of HLB management , grove ages at the beginning of the epidemic , and initial incidences of HLB-symptomatic trees .

Considering that the assumptions and models were corrected, for high expected yield of healthy groves and moderate annual rate of HLB incidence in scenarios with ACP control , it can be concluded that groves younger than 6 years old at the time of HLB detection will have higher expected productivity in any term and initial disease incidence only if managed with eradication and reset of symptomatic plants and ACP control. Older groves with lower initial disease incidence may have a medium-term productive survival , only controlling ACP enough to not allow the secondary spread of the disease. However, the impact on fruit quality of symptomatic trees kept in the field was not considered in this study. The Asian citrus psyllid, Diaphorina citri Kuwayama, transmits Candidatus Liberibacter asiaticus, causal agent of the deadly bacterial disease called Huanglongbing or citrus greening. D. citri and Ca. L. asiaticus are well established in Florida, and have been reported from other states including Louisiana, Texas, and California; they pose a real threat to the entire US citrus industry. In order to discover known and unknown D. citri viruses which might be useful for virus-based biological control strategies, we constructed small RNA cDNA and RNA-seq libraries and used next generation Illumina-based sequencing for selected, worldwide D. citri populations from Taiwan, China, Brazil, and the US Through deep sequencing analysis and de novo contig assembly, larger contigs were obtained. BLASTX and tBLASTx searches against the viral database available in GenBank suggested distinct viruses shared protein sequence similarity with Reoviruses, and with Picorna-like viruses from the genus Iflavirus. Subsequent RT-PCR and Sanger sequencing confirmed the presence of these viruses in some but not all populations of D. citri investigated in this study. Here, we are reporting the first Picorna-like virus discovered in D. citri. However, our analysis suggested that, although encoded proteins show low but significant similarity to viruses belonging to the genus Iflavirus, the D. citri virus is not an Iflavirus and appears to be representatives of a new genus.

We were able to build 70% of the genome of this new virus through bioinformatics analysis. The genome sequences were completed by filling up the gaps using RT-PCRs and specific designed primers. Both 5` and 3` UTR full-sequences were obtained using RACE strategies. Our intent is to engineer this virus and assess its effects on D. citri. Sustainable long-term measures to combat HLB via breeding or genetic engineering methods are hampered by the fact that no true genetic resistance has been found in citrus germplasm.However, the degree of HLB susceptibility or tolerance varies among citrus species. There have been suggestions toward the identification of innate ‘Ca. Liberibacter asiaticus’ resistance-associated molecular mechanisms in citrus plants for application in breeding or genetic engineering crop development programs. Furthermore, a recent study showed that a continuous heat treatment of 40°C to 42°C for a minimum of 48 hours was sufficient to significantly reduce Las titer or eliminate Las entirely in HLB-affected citrus seedlings. Plant exposure to one form of stress has been shown to serendipitously induce resistance to other forms of stress. In this study, we conducted proteomics analysis of heat-treated HLB-affected lemon plants, detected proteins that were markedly up-regulated only in plants that were simultaneously exposed to heat and Las. This suggests that heat treatment induces proteins in Las-infected citrus plants that could play an active role in the suppression of Las growth. Hence, this research demonstrates that: the application of a proteomics approach to elucidate the molecular mechanisms involved in heat-induced Las-resistance in citrus plants and the use of the information from proteomics analysis to develop genetically-modified Las-resistant citrus plants by altering host gene expression to mimic heat-induced conditions. A nationwide survey was conducted to come up with a comprehensive data on the extent of HLB incidence and strains, and its insect vector on Philippine citrus. A total of 120 citrus farms in the various citrus growing areas in the country were surveyed. Typical symptoms of HLB were found in varying degrees of severity in 57% of the farms surveyed. The typical HLB symptoms assessed include sectoral yellowing, mottling,macetas 30 litros small leaves with zinc deficiency symptoms, interveinal chlorosis, vein corking, lopsided fruit, inverted fruit coloration, and aborted seeds. Disease samples were collected and confirmed through starch Iodine test and Polymerase Chain Reaction . There was a good agreement between the visual assessment of symptoms and identification by iodine starch test and PCR. Besides HLB, the occurrence of Asian Citrus Psylla was also noted through visual search and stem tap technique. ACP was noted in 12% of the farms surveyed. In some farms, however, no ACP was found although HLB symptoms were observed and disease spread is evident due to the presence of newly infected trees within the orchards. Disease samples were also graft-inoculated onto differential hosts to determine the HLB strains present in Philippine citrus. Based on disease index on differential hosts, Strain I and II are the predominant strains of citrus HLB found in the Philippines.The Asian citrus psyllid is responsible for transmitting greening or Huanglongbing to citrus. It is considered the most debilitating disease of citrus worldwide. Currently, citrus growers rely on insecticides to control ACP. ACP is a dominantly visual insect and, as such, it is important to understand its visual behavior. Previous studies have shown that young trees planted on a bed covered with metalized polyethylene mulch reduced ACP populations and, consequently, incidence of HLB. However, the actual mechanism whereby the metalized mulch protects trees from ACP is not known. One hypothesis is that radiation of ultraviolet light from the ground confuses the approaching psyllid and disrupts flight. To test this hypothesis, we developed laboratory experiments to evaluate ACP response to UV light. We tested both light emitting diodes and monochromatic colored visual targets produced with narrow band pass filters. ACP were attracted to UV more strongly than to blue and red, but less strongly to yellow and green. UV LEDs emitting a wavelength of 375 nm were found to be the most attractive to ACP amongst a range of UV LEDs . There was no difference between male and female ACP in terms of their attraction towards UV. We hypothesize that metalized mulch, which reflects UV, could be disorienting to the ACP as UV light is in nature primarily found in the sky.

These studies improve our understanding of ACP visual behavior and provide the basis for future studies.Early warning surveillance is crucial in areas that are not yet infected with Las or that have been newly planted with healthy trees. The success of HLB control in these areas relies on the detection of disease and instigation of mitigation procedures as early as possible in the epidemic. Control measures that are instigated too late may not be effective and will incur far greater control costs and disease-induced yield losses than epidemics that are caught early. Surveying and scouting is however expensive and surveillance programs necessarily cover large geographic areas, stretching fiscal and manpower resources. Understanding how to target resources across vast spatial areas is not trivial given the complex spatial distribution of plantings, vector dispersal patterns, interactions between residential and commercial citrus areas and unknown points of disease entry. Using state-of-the-art epidemic simulation methods coupled with geographic information systems , we incorporate this information and use it to simulate realistic spread patterns. By combining the epidemic models with stochastic optimisation algorithms, we are able to identify optimal ‘smart-surveillance’ programs that maximize the probability to achieve early warning based on the predictions of spread. We identify a number of general rule-of-thumbs to help inform optimal surveillance design. In particular, we illustrate that the optimal spatial distribution of sampling resources at the landscape scale depends on the sensitivity and specificity of sampling at the scale of individual trees and plantations . Thus, as new sampling, diagnostic, and detection technologies become available, the methods we are developing can help identify deployment strategies that get the most out of available technologies. The method can be tailored to specific areas and regions, each of which have unique environments and face different situations in terms of, for example, vector densities and inoculum pressures. Psyllid host searching behavior is complex and sophisticated. It can be influenced by host species, growth stage, and physiological condition, psyllid gender and mating status, behavioral plasticity, usurpation by phytopathogens of host aromas, and psyllid-induced emission of foliar volatiles. ACP relies on visual cues to locate its host plants, but evidence suggests that olfactory cues mediate visual response. Scent lures could potentially enhance psyllid response to the bright yellow and green background colors of the sticky traps used in ACP monitoring programs. However, because host foliage aromas are complex, dynamic, and species-specific, developing an effective scent lure is challenging. Here we explore some aspects of lure composition and deployment which may increase trap yield: Scent lure efficacy may be influenced by setting. In residential areas, a generalized scent lure comprised of volatiles common to several host species may work well, while a scent lure used in groves might need to mimic the scent of the grove trees to be effective. Alternatively, a lure that mimics ‘super hosts’, such as orange jasmine, may work well in a variety of situations.

Of the fruits evaluated the fewest isolates were obtained from grapes

Given that both methods capture genetic relatedness among accessions, a significant relationship between these two methods is expected.Principal component analysis revealed significant population structure defined by geography and use, but strong signals of genetic structure also exist at the level of pedigree relatedness. Previous work determined that 75% of the accessions evaluated here are related to at least one other accession by a first-degree relationship, and over half of the accessions are interrelated and form a single, complex pedigree network.Both the strong population-level and pedigree-level signals of genetic structure in our sample present challenges in genetic mapping as these are significant confounding factors when performing GWAS. Moreover, the rapid LD decay previously described for this and other diverse populations of V. vinifera suggests that millions of SNPs are required for well-powered GWAS in grapes.Despite our relatively low marker density and the challenges presented by strong genetic structure, we performed GWAS for all 33 phenotypes. For most traits, we found no convincing GWAS signals . However, we reasoned that we may find SNPs associated with key traits that experienced strong selection during domestication and breeding because selection results in extended LD surrounding the targeted loci, thereby requiring a lower SNP density than that required to map-unselected traits. We hypothesized that, by combining association mapping with selective sweep mapping ,macetas con drenaje we may identify loci associated with traits targeted during grape domestication and breeding. A key transition in grapevine domestication was the switch from dioecy to hermaphroditism: all wild Vitis species, including the ancestor of V. vinifera, are dioecious, and nearly all V. vinifera are hermaphroditic.

Hermaphroditism was likely the first, and arguably the most important, transition from wild vines to cultivated grapes: it enables self-pollination and subsequent clonal propagation of elite cultivars without the need for pollinators.Dioecy is found at low frequency in our sample: only 50 of the 550 accessions with flower sex data were labeled as dioecious. Despite this low frequency, we identified SNPs significantly associated with flower sex on chromosome 2 . The most significantly associated SNP overlaps with the 1.5 Mb region repeatedly identified via linkage mapping.This SNP is also found within the fine-mapped 143 kb region believed to harbor the causal flower sex locus.We therefore demonstrate that, even with only 50 accessions carrying the ancestral dioecy phenotype, we successfully map the flower sex locus at relatively high resolution using GWAS relative to traditional linkage mapping approaches. A genome-wide Fst scan comparing dioecious to hermaphroditic accessions also revealed that the SNP most strongly associated with flower sex had the highest Fst value genomewide, consistent with the effect of selection for hermaphroditism at this locus . If grape domestication resulted in a rapid increase in the frequency of the hermaphroditism allele, one would expect extended haplotype homozygosity, and thus extremely high xpEHH values, in and around the flower sex locus. While none of the xpEHH values at the flower sex locus fall within the top 1% most extreme values genome-wide, we do observe a suggestive peak with xpEHH values within the top 2.6% of genome-wide xpEHH values . xpEHH values in the bottom 1% of the genome-wide distribution are found directly adjacent to the flower sex locus identified here. We have no explanation for why a potential signature of selection could exist for dioecy in such close proximity to the flower sex locus. There are SNPs with extreme xpEHH and Fst values, indicating potential selection for hermaphroditism, at the distal end of chromosome 1 ranging from positions 366 to 467 kb . This genomic region overlaps with the region previously associated with flower sex in a bi-parental mapping population using the same Vitis9KSNP microarray employed for this study.

However, thisregion is several Mb from the locus highlighted in Figure 6a that color within a diffuse peak on chromosome 2 between 10 and has been repeatedly associated with flower sex. We hypothesize 17 Mb . Although the genomic region containing that this distal signal of selection is due to inaccurate localization significant GWAS hits for color overlaps the VvmybA1 gene, the of the array’s SNPs in the reference genome since, when this trait most significantly associated SNP found here is 3.6 Mb from the is mapped using genotyping-by-sequencing in the same bi- known causal mutation. Our inability to map the known color parental population, the flower sex colocalizes with the known locus with precision is consistent with results from rice and flower sex locus according to the reference genome.It is unclear Arabidopsis where markers with the strongest association why such mismapping occurs with the Vitis9KSNP array data, but signals were not found directly at known causative loci. Moreover, unexpected hybridization of non-targeted paralogous regions this result is unsurprising given the relatively low marker density may possibly contribute to these observations. of the SNP array employed here. Skin color in grapes is largely controlled by a single locus on While the diffuse association signal for grape color spanning chromosome 2, where a retrotransposon insertion in the MYBA1 nearly 7 Mb indicates that we have poor mapping resolution for gene results in a loss in pigmentation by disrupting anthocyanin this phenotype, it also suggests the presence of long-range LD biosynthesis.Although rare, white-skinned grapes have been potentially caused by selection. Saccharomyces cerevisiae is an important experimental model organism in addition to its commercial significance as the predominant yeast species during wine fermentation. Modern strains of S. cerevisiae are thought to have arisen in Asia given the diversity of strains and reproductive isolation observed in a study of S. cerevisiae isolates from human-associated and non-human-associated environments in China .

Distinct linages were observed for isolates from primeval and secondary forests . However this study considered few isolates from wine environments and found fewer isolates of S. cerevisiae from fruit sources and more from tree bark, rotting wood and soil samples than from fruit samples. The authors concluded that grape and orchard isolates were similar to those of the wine European lineage. Our goal was to evaluate in greater depth the diversity of natural vineyard isolates from two wine regions in China. Several studies have reported on the genetic diversity of S. cerevisiae strains in different wine-producing regions. These studies revealed that geographic region , climatic conditions , vintage , grape varieties and must characteristics , inoculation of starter yeasts , and SO2 addition affected the diversity of S. cerevisiae observed. In many cases genetically distinct strains of S. cerevisiae were isolated from the same fermentation during wine fermentation . The diversity of S. cerevisiae strains present in fermentations has been shown to play an important role in the characteristics of the final product . Numerous molecular methods have been developed to study the ecology and population dynamics of S. cerevisiae strains . Interdelta sequencing typing uses the variation of the number and position of the delta element, a repeated sequence that flanks the Ty1/Ty2 retrotransposon , that allows interpreting strain similarities and evolutionary or adaptive distance . A succession of different S. cerevisiae strains are established during native as well as inoculated fermentations that could have positive or negative effects on the course of fermentation and wine quality . Vezinhet et al. analyzed the evolution of S. cerevisiae strains isolated from spontaneous fermentations during six consecutive years. These authors concluded that the wide distribution of some strains in the studied areas and their presence over years,macetas 7 litros constitute evidence for the occurrence of specific indigenous strains representative of an enological region. China is an important wine-producing country and while some studies have investigated indigenous yeast species and population dynamics during wine fermentation within local viticulture regions ; few studies have focused on the breadth of the diversity of S. cerevisiae wine genetic resources of China. A study of human- and non-human-associated strains of China found novel distinct lineages only distantly related to the wine strain linages . The genetic diversity and relatedness of indigenous wine S. cerevisiae resources have not been extensively compared with that of wine strains isolated from other geographic regions. Ningxia and Xinjiang provinces, where the strains in this study were isolated, are two of the oldest wine producing regions in China. Shanshan, Xinjiang in northwestern China belongs to a temperate continental climate, with an average temperature of 12˚C. It is situated 92°22′E, 42°87′N with an average altitude of 3986 m.

Qing Tongxia, Ningxia in north central China also belongs to a temperate zone with an arid and semi-arid climate. It is situated 105°21′ to 105°21′ E, 37°36′ to 38°15′N with an average altitude of 1118 m. A comparison of the genetic diversity of S. cerevisiae resources in different viticulture regions of China with isolates from other diverse geographical regions is of importance to the study of global S. cerevisiae ecology. In the present study, interdelta sequence typing with improved primers was used as genetic marker for the distinction of S. cerevisiae strains. Dendrograms were constructed based on similarity among different patterns of bands and the genetic relationships of all strains were evaluated. The strains used in the study were either isolated from fermentations of different grape varieties in the Ningxia and Xinjiang Provinces in China or obtained from the Department of Viticulture and Enology Culture Collection at the University of California, Davis. The aims of the present work were to evaluate the genetic diversity and relatedness amongSaccharomyces strains of different geographic origin, to establish a strain collection to preserve the S. cerevisiae genetic resources of China, and to identify strains useful for further development for commercial wine production in China. Fifty-four isolates collected from fifteen spontaneous fermentations of grapes grown in China and one commonly used commercial yeast, Lavin RC212, were used in this study and obtained from the collections of the College of Enology, Northwest A&F University, Yangling, Shaanxi, China. This set of strains was selected on the basis of interdelta sequence profiles from a total of 349 isolates collected from fifteen spontaneous fermentations of grapes grown in Shanshan, Xinjiang and Qing Tongxia, Ningxia. Fifty-nine yeast colonies were isolated from six spontaneous fermentations of different commonly used grape varieties: Red Globe, Small-berry Thompson Seedless, Big-berry Thompson Seedless, Merlot, Mixed red and Mixed white in Xinjiang. Two hundred ninety isolates were obtained from nine spontaneous fermentations of the grape varieties Cabernet Gernischt, Cabernet Sauvignon, Cinsault, Merlot, Pinot Noir, Riesling, Sauvignon Blanc, Semillon, and Yan73 in Ningxia . The grape must fermentations were allowed to proceed spontaneously at 25~28˚C for 7~11 days until dry. Fermentations were sampled at early, mid and the final stage of fermentation, and serial ten-fold dilutions were inoculated onto WLN and incubated for five days at 28˚C.These yeasts were differentiated and classified according to colony morphology and color. S. cerevisiae isolates were purified and then maintained in 20% glycerol at -80 °C until further analysis, resulting in the selected set of 349 isolates for the interdelta sequence analysis. The composition of the different grape musts is reported in Supplemental Table 1 for Ningxia and in Supplemental Table 2 for the fermentations from Xinjiang. The fifty-four yeast strains selected from this larger population of isolates represented the major strain clusters of interdelta sequence profiles identified in the earlier preliminary study. The origins of the 54 isolates used in this study are shown in Table 1. Identification of S. cerevisiae was confirmed by PCR-RFLP of the 5.8S-ITS rDNA using restriction enzymes HaeIII, HpaII, and ScrFI as described by Li et al. . Strains were maintained in frozen stocks at -80˚C before use. Note that a similar strain numbering system was independently used by Wang et al. in their study but the strains are unrelated. We retained our numbering system since that is the designation given to the strains in the Northwest A&F University strain collection. Other strains were obtained from the Wine Yeast and Bacteria Collection of the Department of Viticulture and Enology at the University of California, Davis. The data from all fifty-two Saccharomyces isolates listed in Table 1 of Liu et al. were included in this study as the method of interdelta sequence analysis was identical. These yeast strains were collected from California, France, Italy, Northern Europe, and Spain .The interdelta sequence patterns obtained after gel electrophoresis were used for the construction of a presence/absence matrix, taking into account the total number of different bands observed. The interdelta sequence patterns were obtained following electrophoresis.

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