Genome Wide Association Study And Identification Of Candidate Genes Responsible For Grain Selenium Content In Rice (Oryza Sativa L)

Rice has been cultivated as the main food crop globally and contributes 50% of human food consumption.A lot of dietary nutrients are sublocalised in the rice grain.Selenium(Se)is associated with various health benefits,such as enhancing immunity,delaying the advancement of AIDS in patients infected with HIV,decreasing cancer incidence and retaining male fertility.Se enrichment in food is considered an effective approach of providing Se to humans for cancer prevention.Se and S exist in Group VIA elements and exhibit similar chemical properties and share similar biochemical pathways for uptake through roots,translocation pathway between organs,and metabolism activities within the plant.Se-metabolism is energy-enzyme dependent mechanism that takes place in the plant cytosol and plastids of leaf cells.Selenate enters the cytosol of leaf cells via SULTR transporters localized in the plasma membranes.Selenate is then transported into the plastid by the chloroplast membrane-localized SULTR3;1 transporter.Genes encoding enzymes involved in the primary S/Se assimilation pathway are upregulated when their growth is restricted by S supply or when Se supply is increased.In this study,501 rice accessions were explored from a diverse panel to identify genes regulating selenium(Se)content in rice grain.Genome-wide association study approach was used for identification of valuable quantitative trait loci(QTL)and respective candidate genes responsible for Se concentration in rice grains.These rice accessions were cultivated in two different years and locations i.e.,2017 Jiangxi(JX)and 2018 Hainan(HN),respectively.Significant genetic variation for grain Se content was revealed between indica and japonica subspecies across the two environments.Average Se concentration was marginally higher in indica as compared to japonica subspecies across the two environments.Clear variations regarding frequency distributions of accession’s phenotype from both environments predict strong environmental effect on Se concentrations in the rice grain.ANOVA analysis indicated that all factors had significant effect on Se concentration in rice grain,however,environment effect was much greater than both genotype and environment × genotype interaction effect.Overall,fourty five QTLs were identified on all chromosomes except chromosome 2.21 candidate genes were identified within these QTL regions and the following three genes were selected for haplotype analysis.LOC＿Os01g071930 encoding a sulfate transporter was identified to be the most favourable candidate gene located 158 kb downstream of the peak SNP of q Se1-2,because selenium is chemically analogous to sulfate and can be transported by sulfate transporter.LOC＿Os06g0695800encoding an ABC transporter was detected to be located at 119 kb upstream of the peak SNP of q Se＿ind＿6-1.Similarly,LOC＿Os04g0642000 encoding another ABC transporter was detected to be located at 68 kb upstream of the peak SNP of q Se4-1 which was identified in the composite population in HN.Expression level of LOC＿Os01g071930 among different haplotypes was compared through RTq PCR analysis.Hap2 was shown to have higher expression than Hap 1 and Hap 3 and also higher Se content at least in one environment.Besides GWAS,the reverse genetic approaches were integrated to evaluate the effect of four sulfate uptake,translocation or metabolism-related genes on selenium content in rice grain.The genes were as follows;LOC＿Os10g28440 encoding sulfate transporter 3.1,LOC＿Os10g28630 encoding homocysteine S-methyltransferase 2(HMT2),LOC＿Os04g08350 encoding cysteine synthase C1,and LOC＿Os04g52900 encoding an ABC transporter.Knockout line of these four genes was successfully developed through CRISPR/Cas9-mediated genome editing in a japonica rice variety Zhonghua 11(ZH11).The concentration of Se in grain was significantly reduced in the knockout lines of all these genes as compared to the wild type control ZH 11.Furthermore,the expression patterns of these genes in different tissues were examined through RT-q PCR analysis.LOC＿Os04g52900 and LOC＿Os10g28440 were highly expressed in the leaf while LOC＿Os10g28630 and LOC＿Os04g08350was highly expressed in the mature root and leaf.Expression pattern of LOC＿Os10g28440(encoding sulfate transporter 3.1)was validated through histochemical staining of the transgenic rice plants expressing its promoter-driven GUS reporter gene.Transgenic rice plants overexpressing the LOC＿Os10g28440 gene was developed and evaluation of the grain Se content is underway.This study will provide some candidate genes for breeding Se-enriched rice variety.