DNA Fingerprinting And Heterotic Group Classification By Using KASP Markers In Cabbage(Brassica Oleracea L.var.capitata)

Cabbage(Brassica oleracea L.var.capitata)is widely cultivated in our country,playing an important role on the annual vegetable supply,and its heterosis is strong.In recent years,the number of cabbage varieties and the cultivation area of cabbage has been steadily increasing.However,the homonyms and synonyms in cabbage occurs frequently,even some off-grade seeds have been traded in the market,which has caused enormous economic loss.Therefore,to construct the DNA fingerprinting and identify varieties rapidly and accurately are of great importance in identifying counterfeit seeds and solving disputes over property rights.Cabbage is a typical cross-pollination crop.Its major breeding method is to make hybrid combinations with inbred lines.And how to combine the parents plays a crucial role in cabbage breeding.At present,the main methods of parent selection were based on the breeder’s experience,morphological characters or geographic origins.Therefore,it is urgent need to classify the heterotic group by molecular markers,which can provide scientific guidance for heterosis utilization and improve the efficiency of combination development in cabbage breeding.In this study,the SNP markers were developed by whole genome re-sequencing data from 50inbred lines,and the KASP method was used to genotype the major varieties and inbred lines of cabbage.The DNA fingerprinting of the major cabbage varieties was constructed and the cabbage inbred line heterosis group was classified based on genotyping data.The results are as follows:1.Development of whole genome KASP markers in cabbageFifty inbred lines were re-sequenced by using the Illumina HiSeq 2500 Sequencing Platform,and the Pooled Mapping algorithm was used to analyze SNP type based on resequencing data.A total of2.54×10~6 SNP loci were obtained by aligning resequencing data to the reference genome of cabbage(02-12).The SNP loci were screened with four criteria:(i)Polymorphism between 40%and 60%,(ii)failure detection in less than 20 accessions,(iii)evenly distribution on each chromosome,(iv)no other variations in the flanking 50 bp for each site.Finally,five hundred SNP markers were selected with high PIC value,low missing rates and even distribution on each chromosome,with 55.6 loci per chromosome.Then KASP primers were designed based on these loci,and 442 of them were successfully transverted into KASP markers,account for 88.4%of all the 500 SNP markers.Nine markers,randomly selected from 442 KASP markers,were used to genotype ninety-six cabbage inbred lines by using the KASP platform,and the genotyping results of nine markers showed that these KASP markers could be used for further experiments.2.DNA fingerprinting of major cabbage varieties based on KASP makersThe KASP markers,developed throughout the whole genome of cabbage,were used to genotype59 major cabbage varieties.In 442 KASP markers,twenty-five KASP markers were un-genotyped in more than five materials and were removed from further analysis.Based on the genotyping result,fifty pairs of core primers were selected with high PIC value,no loci missing and even distribution on each chromosome.Fingerprinting database of 59 major varieties was established by 50 core markers,and the core SNP markers were validated by artificially-mixed population.3.Heterotic group classification of cabbage inbred line by using KASP makersAccording to the difference of the cultivation season,all of the cabbage inbred lines were divided into three types:spring cabbage,autumn cabbage,and winter cabbage.All the inbred lines contained 77spring cabbage inbred lines,72 autumn cabbage inbred lines,and 97 winter cabbage inbred lines.Based on the genotypic data,genetic similarity among different inbred lines were analyzed,and then the heterotic groups were classified along with their relationships.Spring cabbage inbred lines were classified into seven heterotic groups,and six heterotic groups for autumn cabbage,five heterotic groups for winter cabbage.In order to verify the accuracy of the heterotic groups classification,the parents of 50 developed elite combinations were analyzed.Except the combination‘Jiali-Green×Jiali-Gray’,the parents of remaining 49 developed elite combinations were all from different heterotic groups or subgroups,which indicated that the group classification was reasonable.Therefore,we suggest that the parents of combinations should be selected from different heterotic groups or subgroups,which will guide the parental selection in cabbage breeding and improve the breeding efficiency.