Analysis On Genetic Evolution Relationship Of Germplasm Resources And QTLs For The Main Traits Of Fleshy Taproot In Radish

Radish（Raphanus sativus L.）as an important vegetable crop in China,has an annual planting area of 1.3 million hectares and a total output of 45 million tons,which plays an indispensable role in China’s vegetable industry structure and agricultural industry economy.Fleshy taproot as the main organ,the quality trait and yield trait as the important targets of radish breeding.Normal growth and morphogenesis of radish are the basis of good appearance quality and yield formation.The morphological traits of radish taproot composed with numerous elements and most of them are quantitative characters,each of which has diverse genetic variation.Traditional and single technique is hardly to make breakthrough of the genetic theory of these complex traits,which has become bottleneck problem to restrict radish genetic improvement and new cultivars development.In addition,the radish taproot formation is a complex process underground.If only through phenotype observation or by means of previous experiences,it is hardly to improve radish morphological characters effectively in the short term.Therefore,it is important and urgent to explore genetic background of radish germplasm resources in depth for establishment of the representative and diverse natural population,and artificial populations for further study,and to uncover the main QTLs or key genes underlying the major traits of radish taproot by combining multiple omics technologies for efficiently molecular assisting selection and genomic wide design breeding.In this study,we studied the genetic diversity and phylogenetic relationship of a large collection of Raphanus germplasm resources,identified main QTLs and candidate genes related to major traits of radish fleshly taproots,and preliminary analyzed the function of the key genes.Through combination of SSR-Seq,resequencing,GWAS and QTL mapping,the main results are as follows:1.Analysis on genetic diversity and evolution relationship of Rahpanus germplasm resources.Raphanus has undergone a lengthy evolutionary process and has rich diversity.In order to deeply understand the phylogenetic relationship and genetic diversity between and within species,through SSR-sequencing and multi-analysis of 939 wild,semi-wild and cultivated accessions,we discovered that the European wild radish（EWR）has a higher genetic diversity.Frequent intraspecific genetic exchanges occurred in the whole cultivated radish population,and there was considerable genetic differentiation within the European cultivated radish（ECR）population,which could drive radish cultivated variety diversity formation.Among the ECR subpopulations,European primitive cultivated radishes（EPCR）with higher genetic diversity are most closely related to the EWR population and exhibit a gene flow with rat-tail radishes（RTR）and black radishes（BR）/oil radishes（OR）.Among Asian cultivated radishes（ACR）,Chinese big radishes（CBR）with a relatively high diversity are furthest from the EWR population,and most Japanese/Korean big radishes（JKBR）are close to CBR accessions,except for a few old Japanese landraces that are closer to the EPCR.RTR is sister to the clade of CBR（including JWR）,which suggests that the RTR may share the most recent common ancestry with CBR and JWR population.In addition,Japanese wild radishes（JWR）,（namely,R.sativus forma raphanistroides）,which have a strong gene exchange with the JKBR,OR and RTR population.American wild radishes（AWR）have a gene flow with European small radishes（ESR）,suggesting that the AWR developed from natural hybridization between the EWR and the ESR,and Japanese wild radish may be the closest evolutionary ancestor of cultivated radish in Asia.Overall,this demonstrates that Europe was the origin center of the radish,and that Europe,South Asia and East Asia appear to have been three independent domestication centers.The EPCR,AWR and JWR,as semi-wild populations,might have played indispensable transitional roles in radish evolution.Our study provides new points into the origin,evolution and genetic diversity of radish and facilitates the collection,conservation,utilization and exploitation of radish germplasm resources,and it laid a foundation for subsequent screening of natural populations of radish germplasm resources for genome-wide association analysis.2.Genome-wide association analysis of main traits related to radish fleshy taproot based on natural population.We measured the fleshy taproot weight（FTW）and fleshy taproot length of cultivated radish in 2017（426 accessions）and 2018（512 accessions）,respectively,and we also detected the composition and content of glucisinolate in 288 radish roots.Based on the genotyping data of these materials,there was found no significant loci associated with fleshy taproot weight in 2017,and 20 QTLs in 2018.In addition,18 and 13 loci were identified for taproot length in 2017 and 2018,respectively,and there was existed overlap loci in two years.In addition,three aliphatic glucosinolates and three indole glucosinolates were identified in288 radish germplasms root.The total contents ranged from 6.94 umol/g·DW to 126.41umol/g·DW,with an average of 32.43umol/g·DW.A significant locus（Chr6:5567062-5606944）was identified and contained 11genes.3.High density linkage map construction and QTL mapping for main traits of radish fleshy taproots based on RILs population from cultivated varieties.The radish advanced inbreed lines‘CX16Q-1-6-2’（P1）,which had a long cylindrical fleshy taproot with white peel and white flesh.The advanced inbred line CX16Q-25-2（P2）,which had nearly round fleshy taproot with purple red peel and white flesh.Through hybridization of these two lines,we got a F₂ segregation population of 1200 individual plants and a RIL population of 188 lines（F₆ generation）.Through resequencing of the RIL populations,we constructed a high-density linkage map,which included 4447 bin markers distributing in 9 linkage groups,the total length was 1513.345c M,and the average map distance was 0.34c M.The Lengths of the nine linkage groups were between 114.633c M to 208.515c M.The length（114.633c M）and the bin markers（321）of LG8 was the lowest,and the length（208.515c M）and bin markers（642）of LG1 was the highest.The maximum distance between markers was 2.129c M.Based on the linkage map of the RIL populations,the QTL mapping were performed for total plant weight,root weight and fleshy taproot weight were located at one locus on chromosome 2 and 7,respectively.On chromosome 2,the interval sizes was 0.78Mb,0.77Mb and1.14Mb,on chromosome 7,the interval sizes was 1.37Mb,1.19Mb and 1.09Mb.Two loci of each trait explained the total phenotypic variation was 42.92%,42.19%and 40.54%,respectively.QTLs for TW were discovered and the QTL located on chromosome 2 had the region size of 770Kb,which was overlapped with a QTL in GWAS analysis and included 78genes.By gene function annotation,sequence analysis and transcriptome analysis,we screened out RsDAR4 gene related to fleshy taproot weight for further function study.As regard to TL,QTLs were localized on chromosome 2,4,7 and 9 from mapping result of RIL population.The QTL（Chr9:14739571～38275973）was overlapped with the locus（Chr9:36646145～36647914）found in 2017 GWAS analysis.One QTL of TD was mapped on chromosome 2 and the interval size was 3.22Mb,explaining the phenotypic variation was18.08%.As regard to FTSI,QTLs were localized on chromosome2,4,7 and 9 from mapping result of RIL population,which explained total phenotypic variation was 51.27%.4、Linkage map construction of RILs population and QTL mapping for main traits of radish root based on a RIL population from cultivated and wild radish interspecific hybridization.We used advanced wild radish inbreed line‘QT15Q-4P2’and cultivated radish inbred line‘QT15Q-4P1’to construct a F₂ population and a RIL population（F₅ generation）,which contained 1200 individual plants and 125 lines for QTLs mapping of total taproot weight（TTW）and taproot diameter（TD）.By resequencing the wild RIL populations,we constructed a high-density linkage map,which included 2746 bin markers distributing in 9linkage groups,and a total length was 1327.523c M.The maximum length between markers in linkage group 1 and linkage group 2 were 90.139c M and 37.303c M,respectively.This may be due to that the relationship between cultivated radish and wild radish is relatively distant,the large fragment chromosome loss in the offspring,or due to the limited population size caused severe partial segregation.Other seven linkage groups were basically normal,the length of LG5（164.295c M）was the longest and the bin markers was the second（348 bin markers）.The length of LG7（112.306c M）and the bin markers number were the smallest.Based on the linkage map of wild RIL population,we mapped TTW on chromosome 7and the region was ranged from 30.09Mb to 31.44Mb,the size of the interval was 1.35Mb,which explained the phenotypic variance was 12.56%.According to the BSA-seq,the taproot enlargement/TTW trait was located on chromosome 7 and the region was ranged from30.09Mb to 31.44Mb,and the two positioning methods coincide with each other.The results showed that the interspecific cross between cultivated radish and wild radish,the taproot enlarged could let the TTW increased in segregation individuals.And at the same time,the expanded taproot with less lateral roots and fibrous root,and the taproot with less lignification.Through gene function annotation and transcriptome analysis,that the genes Rs0x7c034258（A4）,Rs0x7c034371（SUS1）and Rs0x7c034395（SPS1）were noteworthy to further study.In conclusion,on the basis of clarifying the genetic diversity and genetic relationship between and within species of radish,we identified the important QTLs contributing to FTL,FTW and RW of radish.By integration and application of natural population and artificial population,GWAS,QTL mapping,and BSA-Seq discovered the RsDAR4 could be the key gene for FTW.The outputs of this study will provide a new theoretical basis for genetic mechanism of radish taproot morphogenesis,but also provide theoretical guidance for scientific genetic improvement of target traits of radish root.