Identification Of Genetic Loci For Development Of Leaf Hairy In Rice Through Genome-wide Association Study

As the staple food source for more than one half of the world's population,rice(Oryza Sativa L.)is one of the most important food crops in the world.Leaf hair,derived from epidermal cells of leaves,stretches out of the hairy protrusions.It functions with resistance to biotic and abiotic stresses in rice,such as reduction in heat and water loss of leaves,improvement of tolerance to freeze,and prevention from ultraviolet radiation.Therefore,it is of great importance to identify genetic loci related to leaf hair development and subsequently isolate corresponding genes in rice for foundamental research and molecular breeding.So far,few genes associated with leaf trichomes has been cloned via map-based cloning strategy.In recent years,genome-wide association study(GWAS)has become a booming method to research on complex traits and has been applied in studying agronomically important traits of crops.Here,we selected 149 rice varieties with abundant phenotypic and genetic variations to detect the genetic loci related to development of leaf trichomes with GWAS.A total of 61,128 single nucleotide polymorphism(SNP)markers were selected.Using a mixed linear model(MLM)with-log10P>4.5(FDR<0.01)level,we found 12 SNPs significantly associated with leaf hair.Upstream and downstream 120 kb to the highest SNP loci(240 kb)was considered as interval for candidate genes,among which the candidate gene LOC_Os06g44750 was previously reported to be involved in the formation of rice leaf hair.DNA sequencing showed variations in coding region of the gene HL6 which cause amino acid changes among 149 rice varieties.Real time RT-PCR demonstrated that transcript amounts of HL6 in hairy leaf varieties,and,were significantly higher than those of varieties with less leaf hair.We then constructed the overexpression vector and CRISPR/Cas9 vector for HL6 to confirm its biological function.Haplotype analysis on the coding region of HL6 gene in 149 rice cultivars indicated that there existed at least 9 haplotypes,mong which two haplotypes were dominant.Our study not only verifies that the GWAS method can be utilized to rapidly identify candidate genes related to leaf hair,but also provides genetic basis for dissect molecular mechanism for leaf hair formation.