Construction Of High-Density Genetic Map And Identification Of Red Root Skin Trait In Radish(Raphanus Sativus L.)

High density genetic map is a crucial tool for improve the reference genome assembly,quantitative trait locus(QTLs)mapping associated with important agronomic trait,and comparative genome research.Radish(Raphanus sativus L.)is an important annual or biennial root vegetable crop belonging to the cruciferous family.The fleshy taproot of radish has high nutrition and medical value,and different radish varieties existed significant differences between quality traits.Using the traditional molecular markers,the previously constructed radish genetic map has low marker density and less functional markers,which greatly limite the QTL mapping of important quality traits.In this study,using the radish advance inbred lines‘NAU-LB'with long cylindrical and white skin and `NAU-YH' with short cylindrical and red skin as well as their 137 F2 individuals,a high density genetic map was constructed by whole genome resequencing and then the QTL mapping for 11 quality traits were carried out.The genetic analysis of F2,BC1P1 and BC1P2 indicated that the root skin color is controlled by a single gene.The identification of alternative splicing,single nucleotide polymorphism(SNP),insertion/deletion(InDel)and simple sequence repeats(SSR)were conducted based on three transcriptomes data.Mapping of the candidate gene was conducted for radish root skin trait in radish by the BSA-seq and fine mapping approach.The main results obtained were as follows:1.The whole-genome resequencing of the two parents and 137 F2 individuals were performed.In total,5.63Gb,7.46Gb and 404.44Gb raw reads were obtained from‘NAU-LB',‘NAU-YH'and 137 F2 individuals,respectively.The data of the two parents were aligned to the radish refenrence genome by BWA software.Base on GATK software,a total of 2,569,469 SNPs were developed,of which 821,217 SNPs were homozygous with "aa×bb" type.Genotyping for the F2 population and genetic linkage analysis was performed.A high-density genetic map was constructed with 2852 recombination bin markers.The total genetic distance of the bin map was 1306.8 cM,with an average distance of 0.46 cM between adjacent bin markers.Comparison of the bin map and the reference genome revealed a high level of collinearity among pseudo-chromosomes.The appropriate recombination of LG1 and LG7 has an important effect on the correction of radish genome assembly.A collinear analysis showed that the radish R8 and R9 have high level of synteny with Brasscia.rapa chromosomes A08 and B.oleracea chromosomes C9,respectively.The other chromosomes of radish have a low collinearity with B.rapa and B.oleracea,indicating that chromosome rearrangement occurs between genomes,which would provides a theoretical basis for revealing the specific genome structure in radish.The recombination rate along each chromosome was estimated by comparing genetic and physical length.Highly uneven distribution of recombination was observed among chromosomes.A total of 504 recombination hot regions were identified,which are enriched near gene promoters and terminators.The recombination rate positively correlated with SNPs,gene density and GC content.Functional annotation indicated that genes within recombination hot regions were mainly involved in metabolic process.The QTL analysis was performed on the phenotype of 11 traits(such as skin color,root weight,soluble protein)using the multiple QTL model of MapQTL software.A total of 18 QTLs were detected,and distributed in LG1,LG2,LG4,LG5 and LG8.The LOD values were ranged from 2.55-6.92.The phenotypic variation of a single QTL were distributed in the range of 8.1%-21.2%,the number of QTL for each chromosome was ranged from 1 to 5.The maximum number of QTL was detected on LG2,up to 7 QTL.2.The population of F2,BC1P1 and BC1P2 were constructed by crossing between'NAU-LB'(white root skin)and 'NAU-YH'(red root skin).The genetic analysis of the three populations confirmed that the root skin color was controlled by a single gene.The R locus was anchored to the 9.0Mb-11.6Mb on chromosome 7 by BSA-seq analysis.In this study,three radish transcriptome datasets were collected and aligned to the reference radishgenome.A total of 56,530 alternative splicing(AS)events were identified from three radish genotypes with intron retention(IR)being the most frequent AS type,which accounted for 59.4%of the total expressed genes in radish.In all,22,412 SNPs and 9436 InDels were identified,respectively.The ratio of SNPs with nonsynonymous/synonymous mutations was 1.05:1.A total of 43,680 potential SSRs were identified in 31,604 assembled unigenes.Moreover,35 SNPs and 200 InDels were randomly selected and validated by Sanger sequencing,83.9%of the SNPs and 70%of the InDels exhibited polymorphism among the three genotypes.In addition,the 15 SNPs and 125 InDels were found to be unevenly distributed on 9 linkage groups.Fine mapping was performed using 3 markers and 112 white individuals.The target gene was limited between InDel6 and InDel11,and the physical distance was 74kb,was consistent with the result of QTL mapping for red skin by a high-density genetic map.A gene belonging to the R2R3-MYB transcription factor family member within the candidate interval was identified.Its coding sequence shared highest homology of AtMYB90 gene.The full DNA sequence of RsMYB90 was 1315bp.Quantitative and semi-quantitative PCR results showed that the RsMYB90 was high expressed in root of 'NAU-YH',indicating the RsMYB90 might be an important functional factor to control the red root skin in radish.The research results have laid an important foundation for dissection the molecular mechanism of red root skin,and provided theoretical basis for the selection of high anthocyanin content in radish varieties.