Genome-wide Association Study Of QTL Underlying Ferrous,zinc And Aluminum Toxicity Tolerance At Seedling Stage In Indica Rice Germplasms

Heavy metal toxicity directly affects rice quality and human health.Therefore,identifying genes tolerant to heavy metal toxicity is significantly important in rice breeding.We performed a genome wide association study?GWAS?to detect the trait-SNP associations for Fe,Zn and Al toxicity tolerance at seedling stage using 700K SNPs and 222 core indica accessions from IRRI.Transcriptomic analysis was also performed between tolerant and susceptible accesions.And BSA analysis was done by using a F₂ population derived from two accesions which had extremely opposite reaction to Al toxicity.This research preliminarily dissect the genetic mechnism at genome and transcriptome level,and provide valuable information for the improvement of rice varieties for heavy metal toxicity tolerance by MAS.The main results are summarized as follows:1.Population structure analysis of 222 accesionsThe SNP with minor allele frequency?MAF?less than 0.05 were removed,and finally 395,553SNP markers were selected for GWAS.Two subgroups of the 222 associations were found by PCA 3dimension plot.No obviously population structure was found in the remaining 211 accessions.The LD decay in the whole geneome was about 200 kb.2.Identification of QTL and candidate genes associated with heavy metal toxicity toleranceA total of 14 QTL for Fe toxicity tolerance and 16 QTL for Zn toxicity tolerance were identified,respectively.qSdw3a,qSdw3b,qSdw12 and qSFe5/qSZn5 were detected in the same genomic regions under the two stress conditions,indicating that there are probably common genes and mechanisms governing Fe and Zn toxicity tolerance in rice.Five most likely candidate genes of five QTL?qSh6/qSdw6,qSdw3a,qSdw3b,qSwc5,qSFe2?underlying aboveground traits under stress were inferred according to functional annotation.Three accessions?CC55,CC123 and CC155?showed strong Fe and Zn stress tolerance.A total of 15 and 4 QTL for Al toxicity tolerance were identified at seedling stage and germination stage,respecitvely.qSdw5 and qGRl5a was located in the same region.Six most likely candidate genes of six Al toxicity tolerance QTL?qSh7,qSdw2,qSdw3a,qSdw5,qSwc3 and qSwc8?were inferred according to functional annotation.Three accesions?CC105,CC163 and CC268?were strongly tolerent to Al toxicity,while CC180 was seriously susceptible to Al toxicity.3.Identification of new candidate genes for Al toxicity tolerance by BSA methodBulk sequence analysis was applied in a F₂ population derived from CC105?tolerant?and CC180?susceptible?under Al toxicity stress condition.The average sequence depth of parents and bulk samples was about 13X and 22X,respectively.A total of 568,239 SNP polymorphisms was used to analyse the SNP index in two bulk samples,and a candidate region of 0-2.0 Mb was found on chromosome 2.The?SNP-index that equal to 1 was found in three candidate genes?LOC_Os02g02580,LOC_Os02g03390 and LOC_Os02g03530?.Among them,LOC_Os02g02580belong to TBL protein fanmily which affeted in xylan O-acetylation of cell wall and associated with Al tolerance in Arabidopsis thaliana,and considered to be the most likely candidate gene.4.Haplotype analysis of genes related to heavy metal toxicity toleranceTotal of 12 reported genes and 38 candidate genes detected in this research were done haplotype analysis,finally 12 genes was found significant differences in correspongding traits among different haplotypes.The candidate gene LOC_Os06g17880 for ferrous toxicity tolerance and LOC_Os03g47360for Fe,Zn and Al toxicity identified in this research were found significant difference in their haplotypes.5.RNA-seq analysis of tolerant and susceptible accessionsRNA-seq analysis was performed in tolerant and susceptible accesions for Fe,Zn and Al toxicity respectively.105 differential expressed genes were identified in Fe,Zn and Al toxicity extreme accessions.GO terms analysis indicated that these differential expressed genes were mainly involved in electron transport,oxidoreductase activity,cell death and ATP binding.Four candidate genes?LOC_Os03g62170,LOC_Os05g04410,LOC_Os06g17880 and LOC_Os09g24700?detected in this research were also expressed differentialy in extreme accessions.These genes may be the new candidate genes associated with Fe and Zn toxicity tolerance.