Genome-wide Association Analysis Of Leaf Coloration And Study Of Regulatory Mechanism Of Anthocyanin Biosynthesis In Rice Leaf

Rice(Oryza sativa L.)is not only one of the most important food crops,but also a model plant for studying cereal crops and monocotyledons.With the improvement of living standards,people began to pursue higher nutritional value of rice.At the same time,rice plant breeders began to study how to improve the nutrients,such as anthocyanin contents of rice on the basis of high and stable yield.Anthocyanin is a kind of water-soluble polyphenolic compound with bioactive function.Dietary anthocyanins can help us to prevent or reduce the risk of chronic diseases such as cancer,cardiovascular disease,non-alcoholic fat hepatitis,diabetes and obesity.Black rice is a rare type of rice species which has anthocyanin specifically accumulated in pericarp.This rice is getting popular among consumers and breeders in recent years because of its high nutritive value and antioxidative properties.However,due to the scarce resources of black rice,and the research of anthocyanin synthesis pathway in rice was not deep enough,high quality black rice breeding and the development of black rice industry was impeded.In this research,a natural population of 533 rice accessions collected worldwide,30 wild rice accessions(one Oryza glaberrima,six O.nivara,twenty-three O.rufipogon)and 45 black rice accessions were used as materials,the mechanism of anthocyanin synthesis and regulation in rice leaves,and the domestication of related core genes were deeply analyzed,laid a foundation for the further analysis of anthocyanin synthesis and regulation pathway in rice grain and the formation mechanism of black rice.The results are as follow:The anthocyanin contents of leaf blade in tiller stage from 533 rice accessions collected worldwide(two years datas: Wuhan 2016 in summer and Lingshui 2016 in winter-2017 in spring),30 wild rice accessions(Wuhan 2016 in summer)and 45 black rice accessions(Wuhan 2017 in summer)were measured using high performance liquid chromatography(HPLC).The results show that only 67 varieties,which accounts for12.57% of 533 rice accessions have anthocyanin accumulated in leaf blade.Among 30 wild rice accessions,27 varieties shown to have anthocyanin accumulated,accounting for90%.Anthocyanin is widely accumulated in the leaves of wild rice,and most modern rice leaves have no anthocyanin accumulation,indicate that anthocyanin in rice leaves is an“ancestral trait”.There are 31 varieties that have anthocyanin accumulated in leaf blade,accounting for 68.89% of 45 black rice accessions.The proportion of anthocyanin in black rice material was significantly higher than that of normal rice varieties.Two gene regions were found to determine the accumulation of anthocyanin in leaves through the whole genome association analysis(GWAS).One gene region on chromosome 6,contains a known R2R3-MYB transcription factors OsC1,another gene region on chromosome 1,contains a b HLH transcription factors OsRb.Gene sequencing and haplotype analysis of OsC1 and OsRb were processed among 533 rice germplasm resources,at least 6 kinds of haplotypes were found in OsC1,OsC1 Hap 1 was a functional haplotype,Osc1 Hap 2-Osc1 Hap 6 were non-functional haplotypes.Osc1 Hap2 was the most important haplotype,with a frequency of up to 50% in the population.It mainly appeared in indica rice varieties,which may be the main factor causing no anthocyanin accumulation in indica rice leaves.While in the OsRb,at least 3 haplotypes were found,OsRb Hap 1 was a functional haplotype,Osrb Hap 2 and Osrb Hap 3 were non-functional haplotypes.Osrb Hap 2 had a size of about 6 kb reverse transcriptional transposon insertion the 5'UTR region,which resulted in a loss of transcription activity of OsRb gene,which may be the main factor leading to the accumulation of anthocyanin in japonica rice leaves.Gene sequencing and haplotype analysis of OsDFR of 533 rice core germplasms,at least two haplotypes were found.OsDFR Hap 1 was a functional haplotype,Osdfr Hap 2results in the loss of gene function due to a nonsense mutation of TCG?TAG in exon 2.Osdfr Hap 2 showed a frequency of 16.44% in the population,which not a main factor leading to the non-accumulation of anthocyanin in rice leaves.Population genetic analysis showed that OsC1 and OsRb non-functional alleles were selected in the process of rice domestication,which was the main reason for no accumulation of anthocyanin in the leaves of modern cultivated rice.OsDFR non-functional allele was not selected during the domestication of rice.The coding sequence(CDS)and the promoter region of functional OsC1,OsRb and OsDFR were separated from a black rice variety with purple leaf “Heishuai”.The CDS of OsC1,OsRb and OsDFR from Heishuai was driven by their own promoters respectively.Correspondingly,picked complementary receptor materials from 533 rice core germplasms with single gene mutation.The content of anthocyanin in the leaves of transgenic plants was analyzed.The result shows that POsRb::OsRb,POsC1::OsC1 and POsDFR::OsDFR from Heishuai can complement the phenotype of anthocyanin accumulation in leaf.When non-functional Osc1 and Osrb were complemented,the expression levels of specific structural genes in anthocyanin synthesis were increased significantly in transgene plants.Correlation analysis between the data of leaf tissue transcriptome and anthocyanin content in leaves of 268 rice varieties was conducted,found that expression levels of OsF3 H,OsDFR,OsANS,OsUFGT and OsRb were highly correlated with each others,and greatly positive correlated with leaf blade anthocyanin content.The result suggests that OsRb may regulate the expression of the late biosythesis genes(LBGs)of OsF3 H,OsDFR,OsANS and OsUFGT,and determine the content of anthocyanin in leaves.At the meanwhile,the correlation between the expression of all 233 MYB transcription regulation genes and the anthocyanin synthesis structure genes was analysed using the data of 268 rice leaf transcriptome,and an unreported MYB transcription factor OsP1 was found.The expression of the early biosythesis genes(EBGs)OsCHS,OsCHI and OsF3'H was highly correlated with that of OsP1 rather than OsC1,suggested that OsP1 may be involved in the expression regulation of OsCHS,OsCHI and OsF3'H.Yeast two hybrid assay revealed that either OsRb,OsB2,OsB1,or OsPAC1 interacts with OsC1,but neither of them show interaction with OsP1 in yeast.Dual-luciferase transient transcriptional activity assay was performed,confirmed that OsP1 protein had a transcriptional activation effect on the EBGs independent with b HLH,while OsC1 form a ternary MBW complex with OsRb/OsB2 and OsPAC1 to activate the promoter of the LBGs.Using leaf samples from 12 representative common rice accessions and 12 representative black rice accessions to detect and compare the relative expression levels of OsB2 and OsRb,indicated that OsB2 could complement the absence of OsRb in black rice.Investigation of the promoter of OsB2 in the collection of 523 rice accessions and 45 additional black rice accessions showed that the OsB2 allele with “gain-of-function”mutation existed exclusively in black rice accessions,which had a proportion of 1% in523 rice accessions.Taken together,OsRb rather than OsB1 and OsB2 is the really Pl locus in cultivated rice.Using a panal of 603 rice accessions,which consist of 70 black rice accessions and533 rice accessions,OsMYB3 was found to correlate with the accumulation of anthocyanin in pericarp through GWAS.OsMYB3 showed a high homology with OsC1 by the phylogenetic analysis of MYBs.OsMYB3+OsB2 showed a certain degree of activation to OsCHS and OsDFR through dual-luciferase transient transcriptional activity assay.Pericarp and endosperm tissue transcriptome analysis of 24 rice varieties indicated that OsMYB3 might involved the anthocyanin synthesis pathway in rice pericarp.