High-throughput development of simple sequence repeat markers for genetic diversity research in Crambe abyssinica

Weicong Qi, Feng Lin, Yuhe Liu, Bangquan Huang, Jihua Cheng, Wei Zhang, Han Zhao

The allohexaploid Crambe abyssinica (crambe) is an oilseed crop that has been recognized for its potential value in the chemical industry, particularly in terms of producing high-erucic acid content vegetable oil. However, as an understudied crop, improvement of crambe has been hampered by the lack of genetic and genomic information to enhance its yield, oil quality and resistance against biotic and abiotic stress. Development of molecular markers is therefore of great significance to facilitate genetic improvement of crambe. In this study, high-throughput sequencing was performed to generate sequences for the transcriptome and genome of a widely planted crambe cultivar, Galactica. A total of 186,778 expressed sequence tag (EST) contigs as 8,130,350 genomic contigs were assembled as well. Altogether, 82,523 pairs of primers were designed in the flanking sequences of the simple sequence repeat (SSR) within these contigs. Virtual PCR analysis showed that a fraction of these primers could be mapped onto the genomes of related species of Brassica, including Brassica rapa, B. oleraceae and B. napus. Genetic diversity analysis using a subset of 166 markers on 30 independent C. abyssinica accessions exhibited that 1) 95 % of the designed SSRs were polymorphic among these accessions; 2) the polymorphism information content (PIC) value of the markers ranged from 0.13 to 0.89; 3) the genetic distances (coefficient NEI72) between accessions varied from 0.06 to 0.36. Cluster analysis subsequent on the accessions demonstrated consistency with crambe breeding history. F-statistics analysis revealed a moderate level of genetic differentiation in C. abyssinica (Gst = 0.3934) and a accordingly low estimated gene flow (Nm = 0.7709). Application of high-throughput sequencing technology has facilitated SSR marker development, which was successfully employed in evaluating genetic diversity of C. abyssinica as demonstrated in our study. Results showed these molecular markers were robust and provided powerful tools for assessing genetic diversity and estimating crambe breeding history. Moreover, the SSR primers and sequence information developed in the study are freely available to the research community.