Abstract
Simple sequence repeats (SSR) – also known as microsatellites – have been used extensively in genetic analysis, fine mapping, quantitative trait locus (QTL) mapping, as well as marker-assisted selection (MAS) breeding and other techniques. Despite a plethora of studies reporting that perfect SSRs with stable motifs and flanking sequences are more efficient for genetic research, the lack of a high throughput technology for SSR genotyping has limited their use as genetic targets in many crops. In this study, we developed a technology called Target SSR-seq that combined the multiplexed amplification of perfect SSRs with high throughput sequencing. This method can genotype plenty of SSR loci in hundreds of samples with highly accurate results, due to the substantial coverage afforded by high throughput sequencing. We also detected 844 perfect SSRs based on 182 resequencing datasets in cucumber, of which 91 SSRs were selected for Target SSR-seq. Finally, 122 SSRs, including 31 SSRs for varieties identification, were used to genotype 382 key cucumber varieties readily available in Chinese markets using our Target SSR-seq method. Libraries of PCR products were constructed and then sequenced on the Illumina HiSeq X Ten platform. Bioinformatics analysis revealed that 111 filtered SSRs were accurately genotyped with an average coverage of 1289× at an extremely low cost; furthermore, 398 alleles were observed in 382 cucumber cultivars. Genetic analysis identified four populations: northern China type, southern China type, European type, and Xishuangbanna type. Moreover, we acquired a set of 16 core SSRs for the identification of 382 cucumber varieties, of which 42 were isolated as backbone cucumber varieties. This study demonstrated that Target SSR-seq is a novel and efficient method for genetic research.
Highlights
Simple sequence repeats (SSR) – otherwise known as microsatellites – exist ubiquitously throughout prokaryotic and eukaryotic genomes (Tóth et al, 2000)
We developed a novel method called Target SSR-seq, which combined the high throughput sequencing system X Ten platform with genome-wide perfect SSRs that harbored stable motifs and flanking sequences derived from 182 resequencing datasets of a core collection of cucumber lines
To select a core SSR set for variety identification, we developed a new Perl method to choose the best discernibility group based on the principle of minimum numbers of SSRs representing the maximum genetic diversity
Summary
Simple sequence repeats (SSR) – otherwise known as microsatellites – exist ubiquitously throughout prokaryotic and eukaryotic genomes (Tóth et al, 2000). Based on their universal distribution and high density in a multitude of genomes, SSRs have been analyzed as second-generation molecular markers. Many SSRs used in previous studies were often less polymorphic and failed to yield the expected PCR products. This limited the use and accuracy of SSR markers for genotyping in genetic research (Gao et al, 2012; Hu et al, 2014; Li et al, 2017)
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