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

Micronutrient deficiency is a significant causal factor associated with malnutrition in more than half of the human population.Micronutrient malnutrition,and particularly zinc(Zn)and iron(Fe)deficiency,is more prominent in children and women.Many strategies to reduce the prevalence of these deficiencies have been explored,including food fortification,supplementation,dietary diversification,and biofortification.Rice(Oryza sativa L.)is one of the main stable food crops and an important dietary source of essential micronutrients such as Zn,Fe,and selenium(Se).Approximately over 30% of the world’s population are Zn-deficient;thus,there is an urgent need to improve Zn concentration in rice.The importance of rice as a staple crop worldwide,combined with the long-term sustainability and costeffectiveness of biofortification compared with other approaches,makes biofortification of rice an appealing target for addressing these deficiencies.Biofortification programs are active in more than 40 countries and approximately 1.5 million farming households are already growing varieties of Znbiofortified rice.Our objective is to identify potential candidate genes responsible for grain Zn in rice.In the present study,a natural population of 503 rice accessions was evaluated for grain Zn concentration grown at two locations in two years,and the result demonstrated wide concentration variation among the accessions.The grain Zn concentration was in the range of 11.21 to 37.00 ppm with a mean of 20.87 ppm in Jiangxi province,2017(JX2017)and 15.88 to 41.25 ppm with a mean of 24.50 ppm in Hainan province,2018(HN2018).The genome-wide association study(GWAS)performed using mixed linear model(MLM)resulted in the identification of 69 QTL regions associated with grain Zn concentration.In total,1620 potential genes were identified,and the OsLCT1 gene encoding a low-affinity cation transporter was selected for further analysis.There were 9 haplotypes for OsLCT1,and Hap5 and Hap9 could be more favorable for breeding since it has a relatively high grain Zn concentration and lower grain cadmium(Cd)concentration.Moreover,correlation between expression level of OsLCT1 and grain Zn concentration were validated through RT-q PCR using three haplotype groups(Hap5,Hap6,and Hap9)containing more than 10 accessions.Furthermore,the CRISPR/Cas9 knockout lines of OsLCT1 also showed a significant reduction in grain Zn,Fe,and Cd concentration without any disturbance to plant architecture.In summary,we demonstrated the divergent role of OsLCT1 on grain Zn and Cd concentration,which could be useful for Zn biofortification breeding programs.The major Zn transporter family that plays an influx transporting role is believed to be the ZIP family(zinc-regulated transporter(ZRT)-,iron-regulated transporter(IRT)-like protein).We selected two uncharacterized ZIP family genes,the metal cation transporters OsZIP10(LOC＿Os06g37010)and OsZIP13(LOC＿Os02g10230).The phylogenetic analysis showed that OsZIP10 belongs to clade IV and OsZIP13 belongs to VIII.The CRISPR/Cas9 knockout lines of OsZIP10 showed reduction in grain Zn concentration while opposite trend was seen for OsZIP13.The OsZIP13 mutant showed increased in grain Zn concentration.Histochemical staining of the transgenic rice plants expressing the OsZIP10,and OsZIP13 promoter-driven GUS reporter gene showed strong signal in root for both OsZIP10 and OsZIP13.Quantification of grain Zn content of the OsZIP10 and OsZIP13 overexpression lines is in progress which could help us justify our results and support us in setting up our future research direction.