Our Thai rice population belongs to the indica group . While its size is relatively small compared human studies, GWAS with similar population size has been effective in Arabidopsis and rice. Indeed, diversified composition of our population, lack of strong sub-population structure, and its homozygosity facilitated GWAS.Altogether, GWAS using this Thai rice population lev eraged more than 110,000 SNPs to identify 448 SNPs associated with salt tolerance, which were located in 200 loci in the rice genome. As presented in Table 3, 73% of candidate genes from association mapping associated with salt stress were located within salinity tolerance QTLs identified in bi-parental segregating populations. Functional annotation of the 200 identified genes revealed a number of plausible candidates. The gene annotations we employed relay on the presence of a protein domain or of a homolog with a known functionin rice and other crop species such as maize or sorghum, as well as Arabidopsis. Two chromosomes contained the highest number of reported salt QTLs overlapping with 7 of our candidate loci: chromosome 1, which included 16 QTLs and chromosome 2, which included 10 QTLs. The nature of the candidate genes indi cates that different molecular and cellular strategies have evolved to favor survival during salt stress. Several genes belong to the receptor kinase family , encoding signaling factors during environmental stresses. LOC_Os01g18850, one of candidate genes detected by GWA mapping of UFG trait, encodes SQUAMOSA promoter binding protein-like transcription factor , a plant specific TF, whose function was suggested to affect a broad range growth and development processes, including flower development and 19 SPL genes were identified in rice. The role of SPL gene in salt stress response has been studied by Mao et al. . The 31 SPL genes were identified in maize and the expression profiles of SPLs revealed that most SPL genes were induced under salt stress condition.

This enzyme, common to bacteria,grow table hydroponic plants and humans, shares a common catalytic center, a heme with an iron coordinated to the thiolate of a conserved cysteine. They oxidize disparate substrates through activation of molecular oxygen. The plant P450 gene superfamily plays crucial roles in plant metabolic processes. Narusaka et al. analyzed the expression of 49 Arabidopsis P450 genes under various stresses, including salt stress, and found that 29 P450- genes were induced by various stresses. In the CYP709B subfamily of P450, a cyp709b3 Arabidopsis mutant showed sensitivity to salt stress during germination and high salt-damage at the seedling stage. In rice, Tamiru et al. reported that a P450 gene, OsDSS1 located on chromosome 3 was involved in growth and drought stress responses. Compared to WT, the dss1 rice mutant exhibited improved recovery after germination under drought stress. Additionally, ectopic expression of the P450 gene PtCYP714A3 from Populus trichocarpa was studied in rice. Transgenic rice expressing PtCYP714A3 was semi dwarf with improved tolerance to salt and osmotic stress, resulting in higher survival rates than WT. Interestingly, several novel candidate loci with 144 significant SNPs identified from this GWA mapping were found on chromosome 10, in which no salt QTL was reported. This represents the highest density of significant SNPs found in the same LD block . Interestingly, seven of these SNP-associated genes encoded F-box domain containing proteins . Previous studies have reported the role of F-box proteins in regulating various abiotic stress responses in Arabidop sis, wheat and rice. A conserved N-terminal F-box domain , is a component of the multi-subunit of ubiquitin E3 ligase, an enzyme in the last step of the ubiquitination pathway. The rice gen ome harbors more than 600 F-box genes whose divergence is consistent with adaptive roles and regulation of 25 of these genes responds to salinity stress. Rice seed lings over expressing F-box protein gene, MAIF1 reduced inhibition of root growth and tolerance under salt stress compared with WT.

Salt induced the expression of OsMsr9, a novel rice putative F-box containing protein, especially in the panicle. Over expression of OsMsr9 increased root length, shoot length and survival rate under salt stress. Moreover, SNP with the lowest p-value found on chromosome 12 of GWA mapping of UFG was located in LOC_Os12g36630, which was annotated as a universal stress protein domain containing protein. In fact, USP genes are widely distributed across many organisms including plant, which encode a protein containing the 140–160 highly conserved residues of the Universal Stress Protein A domain . These genes were reported as environmental stress-responsive genes and played role in the ability of plant to respond to the stresses. To date, there are no report on the role of USP genes in salt-treated rice. However, in the study on OsUsp1 in rice under oxygen deficiency condition, it was found that OsUsp1 expression was strongly induced within 1 h of submergence and it played a role in ethylene-mediated stress adaptation in rice. Furthermore, the role of the USP protein in enhancing oxidative stress has been reported in the plant model Arabidopsis. They found that the over-expression of AtUSP conferred a strong tolerance to oxidative stress, primarily via its chaperone function.Fresh and minimally processed fruit and vegetables contribute significantly to the burden of food borne illness in the U.S.. While introduction of pathogens can occur at any step from production through consumption, preharvest contamination of leafy greens has been identified in a number of recent outbreaks. Recurrent outbreaks and product recalls associated with leafy greens and the isolation of Salmonella or Escherichia coli O157:H7 from implicated product suggest that specific plant bacteria interactions or environmental conditions might favor the introduction and persistence of these pathogens in the production environment. To evaluate the risks of preharvest contamination of leafy greens, the ability of E. coli O157:H7 to colonize or survive on plants has been assessed under both laboratory and field conditions.

On plants in growth chambers, E. coli O157:H7 was found to grow and survive under specific conditions on lettuce seedlings and mature plants. Growth of E. coli O157:H7 was generally associated with the phyllosphere,growing strawberries in pots or the aerial portions of plants. Endophytic growth has also been demonstrated, but mainly for plants in hydroponic solution. Young lettuce leaves were found to sustain higher populations of the pathogen than older leaves. Although controlled conditions in the laboratory provide an opportunity to study virulent strains of human pathogens, such investigations do not fully mimic the biotic conditions plants are exposed to in the field. Shortly after application of E. coli O157:H7 onto lettuce in production regions, culturable populations of the pathogen rapidly declined, regardless of the season or location . However, E. coli O157:H7 can apparently survive, at least in low quantities, on field-grown plants, and viable cells were found on a fraction of lettuce plants for several weeks after inoculation. Studies of E. coli O157:H7 survival on plants in the field have generally relied on colony-based assessments coupled with enrichment methods to detect the presence/absence of the pathogen. Such approaches do not permit measurements of total cell amounts or the detection of individual cells that are viable but injured and no longer recovered on standard laboratory growth medium. These sub-lethally injured cells may still be infectious or capable of resuming growth under favorable conditions. The latter possibility is relevant to leaf surface habitats, which are characterized as stressful environments for microorganisms and are subject to fluctuating temperatures and moisture levels as well as other environmental insults. Therefore, culture-independent methods, such as fluorescence based cell detection or genetic approaches, might be complementary for measurements of pathogen presence and survival on plants. In particular, combining membrane-impermeable, DNA intercalating compounds, such as propidium monoazide , with quantitative real-time PCR might offer novel opportunities to sensitively and selectively detect pathogens among a background of other microorganisms in environmental samples. PMA efficiently enters cells with compromised but not intact membranes, and, upon photo activation, PMA is irreversibly cross linked to DNA and prevents PCR amplification of target sequences. In this study, we developed a PMA real-time PCR assay to quantify both viable and total E. coli O157:H7 recovered from lettuce plants in the laboratory and field; these results were compared with quantities of this organism determined by culture methods. We also used this approach to design plant inoculation and incubation conditions in the laboratory that more closely mimic field environments and could be used to study the plant associated behaviors of virulent outbreak-associated strains of the pathogen.

Accurate measurements of pathogen amounts and viability are important for risk assessments and control mechanisms aimed at preventing outbreaks of food borne illness. This study was the first to apply a culture-independent method to quantify the number of E. coli O157:H7 cells on plants inoculated with the pathogen in the field. We also compared the utility of the method for quantifying virulent and avirulent pathogen survival on laboratory-grown lettuce. The first step in the development of culture-independent methods for bacterial enumeration is to ensure that the technique is optimized for the specific organism and environmental conditions of interest. While numerous assessment methods are available, we selected PMA combined with real-time PCR because this technique offered the possibility of rapid enumeration of viable cells in a variety of environments. For E. coli O157:H7 ATCC 700728, we first identified appropriate real-time PCR primers, PMA concentrations, and PMA incubation times with a goal of selective and sensitive detection of viable E. coli O157:H7 cells in the presence of high levels of dead E. coli O157:H7 cells. Among the results was the confirmation that PMA-mediated PCR-inhibition is incomplete when the product size is less than 200 bp. Although our tests on PMA real-time PCR revealed some potential short-comings with the method, most notably the interference of high quantities of inactivated cells and the relatively high numbers of cells required for detection, these parameters were within the range found in other PCR-based studies. E. coli O157:H7 strains ATCC 700728 and EC4045 were inoculated onto leaves of potted lettuce plants and incubated in a growth chamber under temperature and low humidity conditions that favored the decline of cell numbers in trends similar to those recorded in field trials performed in the Salinas Valley. This approach differs from most other laboratory studies that examined plants exposed to high moisture and temperatures supportive of pathogen growth. Within 2 days after inoculation on lettuce plants, the number of E. coli O157:H7 ATCC 700728 cells able to form a colony on TSA declined 1000-fold when the pathogen was initially applied at a level of 6 log CFU per plant or per leaf using an aerosolized spray and declined .105 -fold when either strain was applied directly in 2-ml drops. The numbers of culturable cells remained similar for the subsequent 5 days, suggesting that the initial events after contact with the plant are crucial for determining the survival of the organism on lettuce. In contrast with plate counts, real-time PCR targeting of the E. coli O157:H7 ATCC 700278 and EC4045 inoculants showed that the genomic DNA of the organisms remained on the lettuce in quantities nearly equivalent to the inoculum concentrations over the duration of the 7-day experiment. This finding confirmed that both strains adhered to the leaf surfaces and the recovery method was sufficient to remove the majority of the cell inoculants from the plant. Moreover, the results indicated the presence of high numbers of intact E coli O157:H7 cells on lettuce even when very few were able to form colonies on TSA. Exposure of E. coli O157:H7 suspensions to PMA prior to real time PCR quantification facilitated the detection of cells with an intact cell membrane, and these cells were likely viable. Application of this method to E. coli ATCC 700728 and EC4045 recovered from growth chamber lettuce indicated that a significant fraction of the pathogen inoculants were viable, even though colony-based assessments indicated otherwise. This finding was particularly evident for E. coli O157:H7 inoculated onto leaves by direct drop inoculation. While an average of 5 log CFU per leaf were viable as measured by PMA real-time PCR, less than 1 log CFU per leaf were enumerated by culturing. These results are supported by previous studies reporting that E. coli O157:H7 enters into a viable but not culturable state on lettuce leaves incubated at cold temperatures. Similar outcomes were reported for other E. coli including E. coli O104:H4 in water or after exposure to toxic concentrations of copper.