Genome-wide Association Study And Critical Gene Identification Of Quality Characters In Radish(Raphanus Sativus L.)Taproot

The improvement of nutritional quality characters is an important objective in vegetable genetic breeding.The most nutritional quality characters belong to the quantitative traits controlled by multiple genes,and are affected by various environment factors.It results in the long improvement period and slow regeneration of excellent varieties,seriously hindering the process of vegetable quality breeding.Radish(Raphanus sativus L.)is an important root vegetable crop in Brassicaceae family,its taproot contains high contents of dry matter(DMC),total soluble solids(TSSC),soluble protein(SPC),vitamin C(VcC),soluble sugar(SSC),glucosinolates and anthocyanins,which significantly affect radish quality.To date,there are a few studies on the above traits,including germplasm selection and physiological fields and molecular genetic mechanism.However,key genes related to the formation of nutritional quality characters remain unclear,which has limited the improvement and breeding process of the high-quality radish cultivar.Therefore,how to analyze the formation mechanism of nutritional quality characters and accelerate the quality breeding process has become one of the important research subjects in radish genetic breeding.Currently,genome-wide association analysis(GWAS)technology can be an efficient and accurate method to solve this problem,realizing the genetic analysis of the complex traits and the identification of candidate genes.In this study,179 radish accessions were used to construct a natural population with abundant genetic variation.Based on the SNP data obtained by resequencing and phenotypes of target traits,GWAS was performed using mixed linear model(MLM)model to identify the associated SNPs and the candidate genes.Furthermore,the biological function of key gene involved in the formation of radish red-skin color was analyzed by transient and stable expression,dual luciferase and yeast one-hybrid(Y1H)assays.This result will provide new insights into the gene regulatory network of radish red-skin color formation.Additionally,the potential function of the basic leucine zipper transcription factor(bZIP TF)family were characterized in anthocyanin biosynthesis at the whole genome level.The main achievements were as follows:(1)GWAS analysis of nutritional quality characters in radish taprootIn this study,a total of ten important nutritional quality characters were investigated and collected from 179 cultivated radishes across two years,including DMC,TSSC,SPC,VcC and SSC,and four aliphatic glucosinolate components,including glucoraphenin(GRE),gluconapin(GNA),glucoerucin(GER),glucoraphasatin(GRH),and skin color in radish taproot.Except red-skin color,the frequency distribution of these traits showed normal distributions,indicating that they belonged to typical quantitative traits.A total of 179 radish accessions was resequencing,generating 6,145,767 raw SNPs.After filtered with the coverage depth? 3,minor allele frequency?0.05,missing rate<0.2,a total of 1,222,458 high-quality SNPs was obtained.Phylogenetic analysis showed that the black genotypes from Germany and other genotypes from Asia were significantly separated,and 177 radish genotypes from Asian were further divided into four categories,including majority of white skin radish,red skin radish,landraces(e.g.green skin radish),and other germplasm including the genotypes from Japan,consistent with the results of population structure and PC A analysis.In GWAS analysis using MLM model,a total of 54 SNPs was found to be significantly associated with DMC,TSSC,SPC,VCC and SSC,and 136 candidate genes were identified in their corresponding genetic regions(±10 kb).Furthermore,three important trait-related genes were identified by RT-qPCR,for example,RsEXPB3 for DMC,RsPIN5 for VcC,and RsSUS2 for SSC.These results provided important information for further study on the formation mechanism of nutritional quality characters in radish taproot.In the GWAS analysis for four aliphatic glucosinolates(GRE,GNA,GER and GRH)in radish roots,it was found that the non-synonymous SNP located in RsGSL-OH1 gene was significantly associated with the GNA content.Further analysis showed that there were two haplotypes in 179 radish genotypes,including high GNA content type(RsGSL-OH1HGNA)and low GNA content type(RsGSL-OH1LGNA)and RsGSL-OH1HGNA genotype contained the above non-synonymous SNP,which was positively correlated with GNA content.Additionally,two highly homologous genes RsGSL-OH2 and RsGSL-OH3 were found in the genetic region of the non-synonymous SNP.Moreover,for GRH content,Rs2OGD genes were further proved to be positively correlated with GRH content by RT-qPCR approach.Interestingly,RsGSL-OH1,RsGSL-OH2,RsGSL-OH3 and Rs2OGD belong to 2-oxoglutarate and Fe(?)-dependent oxygenase(2OGD)family,indicating that 2OGDs might contribute to the diversity of aliphatic glucosinolates by participating in the side chain modification of aliphatic glucosinolates.(2)GWAS analysis and functional verification of candidate genes for radish red-skin colorWhen a GWAS approach was performed for red-skin color of radish taproot,a myb domain protein 90 gene(designated as RsPAP2)was identified,which was highly homologous to,a Production of anthocyanin pigment 2 gene(PAP2)related to anthocyanin biosynthesis in Arabidopsis.RT-qPCR analysis showed that the expression level of RsPAP2 in the rich-anthocyanin tissues(root skin,stem,sepal,petal and anther)of red-skin radish was significantly higher than that of white-skin radish.Genetic analysis showed that the over-expression of RsPAP2 in Arabidopsis and radish plants led to increase of total anthocyanin content,accompanied by up-regulation of the structural genes(e.g.C4H,UFGT and F3H)involved in anthocyanin production.Moreover,RsPAP2 was found to independently activate the promoters of RsUFGT and RsTT8,as shown by dual-luciferase and Y1H assay,indicating RsPAP2 had the ability to regulate some anthocyanin-related genes.Additionally,the co-infiltration with and could strongly activated the promoters of more structural genes(RsANS,RsCHI,RsPAL and RsUFGT),indicating that RsTT8 was an important interaction factor of RsPAP2,which contributed to coordinated regulation of the anthocyanin biosynthesis process in radish roots,and RsPAP2 could also activate the transcription of RsTT8 gene.(3)Genome-wide characterization of bZIP gene family identifies potential members involved in anthocyanin biosynthesis in radishThe bZIP TFs,as one of the largest and most diverse transcription factor families,play crucial roles in anthocyanin accumulation as well as various abiotic stress responses.Anthocyanins are most abundant in red-skin radish,which exhibit strong antioxidant activity that offers benefits for human health.Here,a total of 135 bZIP-encoding genes were identified from radish genome.Synteny analysis showed that 104 radish and 63 Arabidopsis bZIP genes were orthologous.Transcriptome analysis revealed that 10 RsbZIP genes exhibited high-expression levels in radish taproot(RPKM>10).Specifically,RsbZIP010 exhibited down-regulated expression under cadmium,chromium and lead stresses,whereas RsbZIP031 and RsbZIP059 showed significant down-regulation under heat and salt stresses,respectively,indicating that they might be served as critical regulators in radish taproot under abiotic stresses.RT-qPCR analysis indicated that the expression of RsbZIP011 and RsbZIP102 were significantly up-regulated in red root skin,stem,vein,sepal and petal of radish,which was consistent with anthocyanin content,suggesting that they could participate in anthocyanin biosynthesis pathway of radish.Furthermore,the promoter sequences of several anthocyanin-related genes such as CHS,ANS and UFGT were found to contain G-box or ACE-box elements that could be recognized by bZIP family members.These results will facilitate further investigation on the regulation mechanism of RsbZIP genes in anthocyanin biosynthesis and response to abiotic stress in radish.