Linkage Map Construction and Genetic Diversity Analysis based on Transcriptome-derived Markers in Brassica oleracea L.

Abstract

Molecular markers have been proven to be an essential and powerful tool for plant geneticists. The advent of molecular markers helps the geneticists in many genetic applications. Transcriptome sequencing, which is DNA sequencing of the mRNA pool of a given tissue, has enabled rapid marker discovery. In present study, two cabbage parental lines, C1184 and C1234, which is susceptible and resistant to black rot disease, respectively, were used for transcriptome sequencing. A total of 92,255 and 127,522 reads were generated, and clustered into 34,688 and 40,947 unigenes, respectively. 27 ESTs were found to have different expression between parental lines, and the most differentially expressed EST were known as Ankyrin repeat domain and RING finger-containing protein XBAT31 that could be considered as candidate gene for resistance to black rot. In addition, 2,405 SSR motifs derived from the unigenes of the black rot resistant parent C1234 were detected. Of these, trinucleotide motifs were the most abundant one (66.15%) observed among all the repeat motifs. Moreover, 1,167 SNPs were also identified between two parental lines. A total of 937 EST-based SSR and 97 EST-based dCAPS markers were designed and used for polymorphism analysis. These newly designed primers, together with the published SSRs and intron-based polymorphism (IBP) markers, were used to construct a genetic linkage map using an F2 segregation population of 97 individuals. The constructed genetic map composed of 265 loci (98 EST-based SSRs, 21 EST-based dCAPS, 91 public SSRs and 55 IBP markers) distributed on nine linkage groups spanning a total of 1,331.88 cM with an average distance of 5.03 cM between adjacent loci. The genetic map constructed in this study could serve as framework for identifying marker-trait associations through quantitative trait loci (QTL) mapping, map-based cloning and physical map alignment. On the other hand, molecular DNA markers were also useful for genetic diversity analysis. Of which, analysis of genetic diversity of crop plants is a key component for crop improvement program. In this study, 91 commercial B. oleracea cultivars belonging to six subspecies, which obtained from seed companies worldwide, have been characterized using 69 SSR markers. A total of 359 different alleles have been observed, with an average number of 5.20 alleles per locus. Polymorphism information content (PIC) values ranged from 0.06 to 0.73, with an average of 0.40. Kohlrabi cultivars showed the highest heterozygosity level among six subspecies

meanwhile kale cultivars exhibited the lowest one. Based on genetic similarity values, an UPGMA clustering dendrogram and principal coordinate analysis (PCoA) were generated to analyze genetic diversity. All of 91 cultivars were clearly separated into six different clusters with a propensity to cluster into its subspecies. Structure analysis exhibited six genetic groups, of which cabbage cultivars differentiated into two subgroups based on their head shapes, whereas cauliflower and kai-lan cultivars clustered together in the same group. In addition, 18 SSR markers were found to have 27 specific alleles that can be used to differentiate 22 cultivars. Genetic diversity and population structure analysis generated in this study presents new insight into the genetic structure and relationships among 91 B. oleracea cultivars and provides valuable information for future breeding of B. oleracea species.