Table_2_Target SSR-Seq: A Novel SSR Genotyping Technology Associate With Perfect SSRs in Genetic Analysis of Cucumber Varieties.XLSX

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.

简单序列重复（Simple sequence repeats, SSR），又称微卫星（microsatellites），已被广泛应用于遗传分析、精细定位、数量性状位点（quantitative trait locus, QTL）定位以及标记辅助选择（marker-assisted selection, MAS）育种等技术领域。尽管已有大量研究表明，具有稳定基序与侧翼序列的完美型SSR在遗传研究中效率更高，但由于缺乏适用于SSR基因分型的高通量技术，其在众多作物中作为遗传靶标的应用受到了限制。本研究开发了一种名为靶向SSR测序（Target SSR-seq）的技术，该技术将完美型SSR的多重扩增与高通量测序相结合。依托高通量测序提供的高覆盖度，该方法可对数百份样本中的大量SSR位点进行基因分型，且结果准确率极高。本研究基于黄瓜的182份重测序数据集，共鉴定出844个完美型SSR，其中91个SSR被选中用于靶向SSR测序分析。最后，研究团队利用靶向SSR测序技术，对中国市场上易获取的382份核心黄瓜种质进行了基因分型，所用的122个SSR位点中包含31个用于品种鉴定的SSR位点。研究人员构建了PCR产物文库，并在Illumina HiSeq X Ten测序平台上完成测序。生物信息学分析显示，经过过滤的111个SSR位点均实现了准确的基因分型，平均覆盖度达1289×，且测序成本极低；此外，在382份黄瓜栽培品种中共检测到398个等位基因。遗传分析将供试材料划分为4个类群：华北型、华南型、欧洲型以及西双版纳型。此外，本研究筛选出一套包含16个核心SSR的位点组合，可用于区分382份黄瓜品种，其中42份被鉴定为骨干黄瓜品种。本研究证实，靶向SSR测序是一种用于遗传研究的新型高效技术手段。