Finetuning of metabolomic traits such as amylose content in rice and sugar content in wild strawberry recently were made possible via CRISPR-Cas9 gene-editing technology. Similar approaches can be taken in cultivated strawberry for flavor improvement, but not before the biosynthetic genes responsible for metabolites production and their regulatory elements are identified. Our pipeline has proven to be effective in identification of novel causal mutations for flavor genes responsible for natural variation in volatile content and can be further applied to various metabolomic and morphological aspects of strawberry fruit such as anthocyanin biosynthesis , sugar content and fruit firmness. These findings also will help breeders to select for genomic variants underlying volatiles important to flavor. New markers can be designed from regulatory regions of key aroma volatiles, including multiple medium-chain volatiles shown to improve strawberry flavor and consumer liking , methyl thioacetate contributing to overripe flavor and methyl anthranilate imparting grape flavor . In the present study, a new functional HRM marker for mesifurane was developed and tested in multiple populations . These favorable alleles of volatiles can be pyramided to improve overall fruit flavor via marker assisted selection. Specific esters are shared with apple , certain lactones are shared with peach and various terpenes are shared with citrus . Syntenic regions and orthologous genes could be exploited for flavor improvement in those species. Additional insights were gained for the strawberry gene regulatory landscape, SV diversity, complex interplays among cis- and trans- regulatory elements, and subgenome dominance. Previously, Hardigan et al. and Pincot et al. showed a large genetic diversity existing in breeding populations of Fragaria × ananassa, plastic grow pots challenging previous assumptions that cultivated strawberry lacked nucleotide variation owing to the nature of its interspecific origin and short history of domestication .

Our work corroborated their findings and showed that even highly domesticated populations harbor substantial expression regulatory elements and structural variants. Over half of the expressed genes in fruit harbored at least one eQTL, and 22 731 eGenes had impactful cis-eQTL. The distribution of trans-eQTL is not random, but rather is concentrated at a few hotspots controlled by putative master regulators . The aggregation of trans-eQTL also was observed in plant species such as Lactuca sativa and Zea mays . Furthermore, we observed a substantial number of trans-eQTL among homoeologous chromosomes, similar to observations in other allopolyploid plant species . In cotton, physical interactions among chromatins from different subgenomes have been identified via Hi-C sequencing , supporting a potential regulatory mechanism among homoeologous chromosomes. However, owing to the high similarity among four subgenomes and limited length of Illumina reads, false alignment to incorrect homoeologous chromosomes could arise, leading to ‘ghost’ trans-eQTL signals. Future studies are needed to scrutinize the homoeologous trans-eQTL and investigate the mechanism behind this genome-wide phenomenon. Higher numbers of trans-eQTL in the Fragaria vesca-like subgenome are consistent with its dominance in octoploid strawberry . By contrast, the highly mixed Fragaria viridis- and Fragaria nipponica- like subgenomes contained much smaller numbers of trans-eQTL. The characterization of naturally-occurring allelic variants underlying volatile abundance has direct breeding applications. First, this will facilitate the selection of desirable alleles via DNA markers. Second, understanding the causal mutations in alleles can guide precision breeding approaches such as gene editing to modify the alleles themselves and/or their level of expression. From a broader perspective, multi-omics resources such as this one will have value for breeding a wide array of fruit traits.

Enhancing consumer satisfaction in fruit ultimately will depend on the improvement of the many traits that together enhance the overall eating experience.In a fruit tree orchard system, individual trees are composed of two genetically different genotypes, one being the rootstock which includes the mass of the tree below the soil surface to a graft union about midway up the trunk. Rootstocks can be selected for pest resistance or tolerance towards adverse soil conditions, and they can also influence vigor and cropping . The second portion of the tree is referred to as the scion and accounts for most of the above-ground mass, usually chosen for fruit production traits . Over the last 40+ years, the University of California has had a peach rootstock development program that has identified several promising size controlling rootstocks which allow for the establishment of new commercially viable orchard systems . New dwarfing rootstocks for peach must be graft compatible, reduce vigor, and not diminish marketable fruit production by reducing fruit size or quality . Previous peach rootstock trials monitored vigor control and grafting compatibility in conventional planting systems however, yield parameters such as fruit size and quantity have not been as thoroughly evaluated using these rootstocks in pedestrian orchard systems . Fruit size is paramount in peach production as larger fruit, free of cosmetic imperfections, have a higher market demand and therefore higher market value . It has been reported that peach fruit produced on trees with size-controlling rootstocks can tend to be smaller in size than fruit on trees with more vigorous rootstocks .Vascular tissue known as xylem is responsible for the movement of water and nutrients in all trees. In trees, every year a new ring of xylem forms surrounding the previous year’s growth and water conduction in the xylem often occurs only in this outermost annual ring . It has been reported that size-controlling peach rootstocks contain a higher proportion of narrow diameter xylem vessels and fewer larger vessels when compared to more vigorous rootstocks in addition to having an increased axial diameter .

Both characteristics create a reduction of hydraulic conductance in the size-controlling peach rootstocks compared to traditional, vigorous rootstocks. Reduced hydraulic conductance, as demonstrated by and , can cause reductions in stem water potential during mid-day hours that can lead to a reduction in vegetative growth .An mean peach fruit’s fresh weight is composed of over 80% water . Thus, it is reasonable to assume a reduced hydraulic conductance created by size controlling rootstocks could hinder fruit size. However, the relationship between fruit growth and water availability is dynamic and depends on the developmental stage of the fruit, the severity of water limitations, and the component of growth being considered . It has been reported that mild water stress applied during the intermediate developmental period of slow fruit growth has no effect on crop yields but significantly reduces vegetative growth in peach . Fruit developmental stages may differ in time of initiation and duration among peach varieties, an example of this would be an early vs. late harvested cultivar as demonstrated by . Fruit growth occurs in stages from fruit set to harvest, in all cultivars, and during the final growth phase of peach fruit is when 65% of a fruit’s dry weight and 80% of a fruit’s fresh weight are accumulated . Available water varies throughout the growing season, including diurnal fluctuations brought on by daily temperature fluctuations , day-to-day changes brought on by a shift in evapotranspiration , and possible seasonal changes brought on by the formation of new xylem . Water conduction in the tree is largely dependent on newly formed xylem each spring and the new xylem cells are smaller in size-controlling rootstocks. It is thought that the spring flush of vegetative growth is limited in trees on size controlling rootstocks compared to growth on vigorous rootstocks because of temporary reductions in root hydraulic conductance caused by smaller xylem vessels. A question that arises from these findings, does the reduction of water conductance in dwarfed peach trees also limit fruit growth?In peach production, fruit size is often manipulated with the use of a management practice known as fruit thinning. With fruit thinning, shortly after fruit set, a portion of immature fruit is removed from the tree to reduce carbohydrate competition among those remaining. It is widely recognized that fruit size is largely influenced by crop load, with larger fruit size obtained as the crop load is reduced . Quality of fruit may also be affected by crop load, low-cropped trees have been shown to produce larger and firmer fruit than those from heavily cropped trees . Although minor in comparison tocarbohydrate demand, big plastic pots fruit size may also be diminished by inducing higher water stress with larger crop loads. An experiment by found that larger crop loads were responsible for reducing midday stem water potential in nectarines.

MacFayden et al., concluded that an increased crop load also increased the fruit water deficit which may reduce fruit growth in peach. According to another study by , rootstocks also influenced the crop load’s effect on fruit size, and more vigorous rootstocks had larger fruits at specific crop loads. The fore mentioned findings relay the importance of better understanding the relationship between fruit size and crop load among vigorous and reduced-vigor rootstocks.While crop load per tree is controlled by thinning, crop load per area is most influenced by planting density. The reduced vigor and overall size of trees on size-controlling rootstocks facilitates the establishment of high-density plantings . The primary principle in establishing an appropriate planting density for an orchard using trees on size controlling rootstocks is that total tree dry matter production and crop yield are related to total light interception . This principle holds for essentially all crops . However, although higher light interception often leads to higher yields, yield may also vary significantly with other environmental stressors such as available water, nutrients, temperature, and amount of time the fruit has for growth . Orchard systems with increased planting densities have also been shown to reach maximum yield capacity earlier than conventional plantings since the trees are able to fill out their allotted space more quickly . In a small trial using the ‘Summer Bright” nectarine cultivar, trees that were pruned to a standard height of 12 to 13 feet or limited to heights of 8 or 9 feet produced similar sized fruit and crop yields. The reasoning for this was that, despite the height difference, both tree shapes had equal planar volume and therefore intercepted similar amounts of photosynthetically active radiation .The goal of this study was to address three production characteristics and their relationship with four different orchard systems. 1) Fruit size: can peach orchard systems using trees on size controlling rootstocks produce fruit of equal size compared to orchard systems with trees on vigorous rootstocks? 2) Fruit count: if crop load per area is similar among size-controlling and vigorous systems is fruit size also similar? 3) PAR interception and yield: is there a difference in the relationship of fruit production vs light interception among orchard systems with vigorous rootstocks and those with size-controlling rootstocks? A better understanding of production capabilities will allow researchers and growers to better estimate the potential of an orchard system on size-controlling rootstocks as a commercially viable option.In April 2015, an orchard system trial was established at the University of California Kearney Agricultural Center, Parlier, CA. The research block consisted of two peach [Prunus persica Batsch] scion cultivars, June Flame and August Flame grafted onto three different rootstock genotypes: HBOK 27 , P-30-135 , and Nemaguard . Controller 6 was used in two of the four training systems . The C-6 V was a high-density planting system with an in-row spacing of 1.2m and trained to the KAC-V perpendicular V pruning system . The C-6 Quad system was pruned to a Quad V where four main scaffolds are selected in each tree and pruned to resemble an open vase, the system also had a larger in-row spacing of 2.4 m . The Controller 9 Quad system was identical to the C-6 Quad system with the only difference being the rootstock. Between-row spacing was 4.6m in all systems using size controlling rootstocks. Nemaguard was used as the commercial standard rootstock with a planting density of 2.4m in-row spacing and 5.5m between-row spacing . Shortly after harvest, orchard systems using size-controlling rootstocks were topped to a height of 2.5m while systems using the Nemaguard rootstock were topped at 3.5m. The four systems were divided into three replications for each of the two scion cultivars making a total of eight unique orchard systems. Each replication consisted of four rows of trees with the northern and southern most rows used as guard rows, the first and last two trees in each data row were also considered guard trees making nine trees in each of the two inner rows the sample size per replication . In total, each cultivar was represented by approximately 54 data trees .