QTL Fine Mapping And Mining Of Candidate Genes For Berry Color Of Grape

Grape(Vitis L.)is one of the most widely cultivated and economically valuable fruit trees in the world.Berry color is the important economic traits of grape,which is mainly determined by the content and composition of anthocyanin in grape berry skin.MYBA1 and MYBA2 transcription factors(TFs)play important roles in the regulation of anthocyanin biosynthesis in grape berries.The mutations of these two TFs well explained the origin of white-skin grapes.However,for the red-skin grapes,the variety of berry color is very abundant,with many intermediate coloring types from pink to purplish black.This indicates that the berry color of grape is a typical quantitative trait.Thus,it is difficult to fully explain the molecular mechanism of berry color variation based on the existing researches.In order to better explore the regulatory mechanism of grape berry color,the hybrid population of V.vinifera ‘Red Globe' and ‘Muscat Hamburg' was used as the material.The berry color of the population was systematically evaluated.We also analyzed the relationship between Vv MYBA genotypes and the berry color of the population.A highdensity genetic map was constructed based on the whole-genome resequencing(WGS)of the population,and QTL mapping for grape berry color was carried out based on this genetic map.After combining the results of QTLs and transcriptome analysis,several candidate genes were selected that may be related to anthocyanin accumulation and berry color determination.And preliminary functional verification of individual candidate genes was conducted.The main results of our research are as follows:1.The berry coloration of the population showed obvious trait separation from yellowgreen to purple-black.The berry color of the red-skin progeny was normally distributed.The total anthocyanin content in the berry skin varied from 1.43 mg/kg(FW)to 1869.99 mg/kg(FW)in the population.A total of 12 kinds of anthocyanins were detected in the berry skin within the population.The pelargonidin-based anthocyanins were not detected in this population,and trihydroxylated(delphinidin-based,petunidin-based and malvidinbased)anthocyanins were generally higher than dihydroxylated(cyanidin-based and peonidin-based)anthocyanins.In this population,methylated anthocyanins accounted for a large proportion of anthocyanins,in more than half of offspring,methylated anthocyanins accounted for more than 80% of total anthocyanins.The acylated anthocyanins were mainly acylated by coumarine in the berry skin of hybrid population.The content and composition of anthocyanins in grape berry skin showed important effects on the berry color.There was a significant positive correlation between the berry color and the total anthocyanin.The proportion of dihydroxylated and trihydroxylated anthocyanins in grape berry skin also had an important effect on the grape berry color.The higher the proportion of trihydroxylated anthocyanins,the darker the grape berry color.2.The genotypes of Vv MYBA1 and Vv MYBA2 were analyzed.The white-skin offspring of the population were homozygote of non-functional Vv MYBA gene alleles,and the number of functional Vv MYBA allele was significantly positively related to berry color.However,there were also some progeny whose berry color inconsistent with the number of functional Vv MYBA alleles in this populatin.This study confirmed that the Vv MYBA genotype was closely related to the grape berry color,but it also indicated that the additive effect of Vv MYBA gene could not fully explain the grape berry color variation.3.A high-density genetic map of grape was constructed based on 27,454 high quality SNP markers developed by whole genome resequencing of hybrid population.The total genetic distance of the genetic map was 1442.638 c M,and the mean genetic distance among the 1554 bin markers was 0.928 c M.Based on the high-density genetic map and the data of population berry color traits,10 QTLs related to the regulation of grape berry color and anthocyanin biosynthesis were identified.In addition to the major QTL col-2-1 that contained the Vv MYBA gene cluster on chr2 was detected,nine micro effect QTLs were identified on chr2(col-2-2 and col-2-3),chr4(col-4-1 and col-4-2),chr6(col-6-1),chr11(col-11-1 and col-11-2)and chr17(col-17-1 and col-17-2).This indicates that grape berry color is controlled by a main QTL and multiple micro effect QTLs.4.In combination with the annotation of genes in QTL regions and the transcriptome analysis of the parents,19 candidate genes related to grape berry color were screened.Most of these candidate genes were TF genes.8 MYB(include Vv MYBA1 and Vv MYBA2)TFs,3 b HLH TFs,1 GRAS,NAC,LIM,b ZIP and S1fa-like TFs were selected,which may play important roles in the regulation of anthocyanin accumulation in grape berry skin.The expression pattern of candidate gene Vv MYB86(VIT?17s0000g09080)located at the col-17-1 loci was contrary to the anthocyanin accumulation process of the grape berry,and the typical C1 and C2 inhibitory motif were detected in its amino acid sequence.Heterologous transformation in tobacco showed that the Vv MYB86 TF could significantly inhibit the accumulation of anthocyanin in transgenic tobacco petals.This suggests that Vv MYB86 may negatively regulate the synthesis of anthocyanins.To conclude,based on the systematic evaluation of berry color trait and the highdensity genetic map of grape,a number of new QTLs related to grape berry color were identified in this study.Combining with transcriptome analysis,multiple candidate genes for grape berry color regulation were screened in the mapped QTL regions,and the function of individual candidate gene was preliminarily verified.It is significant for further researches on grape berry color regulation.