Liu Fengluan, Xi Lin, Fu Naifeng
Shanghai Key Laboratory of Plant Functional Genomics and Resources, Shanghai Chenshan Botanical Garden, No. 3888 Chenhua Road, Songjiang District, Shanghai, 201602, China.
Department of Plant Systems Biology, University of Hohenheim, 70599, Stuttgart, Germany.
BMC Genomics. 2025 Jan 3;26(1):4. doi: 10.1186/s12864-024-11191-4.
Despite the rapid advancement of high-throughput sequencing, simple sequence repeats (SSRs) remain indispensable molecular markers for various applied and research tasks owing to their cost-effectiveness and ease of use. However, existing SSR markers cannot meet the growing demand for research on lotus (Nelumbo Adans.) given their scarcity and weak connections to the lotus genome.
Using whole-genome resequencing, active SSR loci were identified throughout the genomes of eight typical Asian lotus. After that, high polymorphism SSR molecular markers were mined from each 2n + 0.5 Mb site on each chromosome (e.g., Chr.1-2.5, 4.5, 6.5 Mb) through four steps: online primer design, primer pair evaluation, agarose gel electrophoresis testing using six Asian lotus, one American lotus, and two their hybrids, and DNA sequence alignment. Finally, the polymerase chain reaction (PCR) efficiency of several SSR markers was validated in 20 Asian temperate lotus, eight Asian tropical lotus, and one American lotus.
A total of 463 SSR markers were developed based on each 2n + 0.5 Mb site of the eight lotus chromosomes (totaling 821.29 Mb). These markers were evenly distributed throughout the lotus genome at a density of 1 SSR per 1.76 Mb. The chromosomal locations of the SSR markers were determined precisely, and the specificity of the primer pairs for each site was verified by sequencing the PCR products. We further provided a set of genome-wide SSR loci, covering 129 per Mb, identified from eight representative Asian lotus, allowing other researchers to independently discover specific SSR markers for particular experiments.
These SSR markers, which have a density of 1 SSR marker per 1.76 Mb in this study, will act as a bridge connecting lotus phenotypes with the genome. This work reveals a novel and convenient strategy for developing highly polymorphic SSR markers at any location throughout the lotus genome, and it sheds light on the development of SSR molecular markers in other plant species.
尽管高通量测序技术迅速发展，但简单序列重复（SSR）因其成本效益高且使用方便，在各种应用和研究任务中仍然是不可或缺的分子标记。然而，现有的SSR标记由于其稀缺性以及与莲基因组的联系薄弱，无法满足对莲（Nelumbo Adans.）研究日益增长的需求。
利用全基因组重测序，在八个典型亚洲莲的基因组中鉴定出活跃的SSR位点。之后，通过四个步骤从每条染色体上的每个2n + 0.5 Mb位点（例如，Chr.1 - 2.5、4.5、6.5 Mb）挖掘高多态性SSR分子标记：在线引物设计、引物对评估、使用六个亚洲莲、一个美洲莲及其两个杂种进行琼脂糖凝胶电泳测试，以及DNA序列比对。最后，在20个亚洲温带莲、8个亚洲热带莲和1个美洲莲中验证了几个SSR标记的聚合酶链反应（PCR）效率。
基于八个莲染色体的每个2n + 0.5 Mb位点（总计821.29 Mb）共开发了463个SSR标记。这些标记以每1.76 Mb一个SSR的密度均匀分布在莲基因组中。精确确定了SSR标记的染色体位置，并通过对PCR产物测序验证了每个位点引物对的特异性。我们还提供了一组全基因组SSR位点，每兆碱基有129个，这些位点是从八个代表性亚洲莲中鉴定出来的，使其他研究人员能够为特定实验独立发现特定的SSR标记。
本研究中密度为每1.76 Mb一个SSR标记的这些SSR标记，将成为连接莲表型与基因组的桥梁。这项工作揭示了一种在莲基因组任何位置开发高多态性SSR标记的新颖且便捷的策略，并为其他植物物种SSR分子标记的开发提供了思路。
