Genome-Wide Identification of SSR and SNP Markers Based on Whole-Genome Re-Sequencing of a Thailand Wild Sacred Lotus (Nelumbo nucifera)

Genomic resources such as single nucleotide polymorphism (SNPs), insertions and deletions (InDels) and SSRs (simple sequence repeats) are essential for crop improvement and better utilization in genetic breeding. However, the resources for the sacred lotus (Nelumbo nucifera Gaertn.) are still limited. In the present study, to dissect large-scale genomic molecular marker resources for sacred lotus, we re-sequenced a Thailand sacred lotus cultivar ‘Chiang Mai wild lotus’ and compared with the reported lotus genome ‘Middle lake wild lotus’. A total of 3,180,059 SNPs, 328, 251 InDels and 14,191 SVs were found between the two genomes. The functional impact analyses of these SNPs indicated that they may be involved in metabolic processes, binding, catalytic activity, etc. Mining the genome sequences for SSRs showed that 191,657 SSRs were identified with a frequency of one SSR per 4.23 kb and 103,656 SSR primer pairs were designed. Furthermore, 14, 502 EST-SSRs were also indentified using the available RNA-seq data in the NCBI. A subset of 150 SSRs (genomic and EST-SSRs) was randomly selected for validation and genetic diversity analysis. The genotypes could be easily distinguished using these SSR markers and the ‘Chiang Mai wild lotus’ was obviously differentiated from the other Chinese accessions. This study provides considerable amounts of genomic resources and markers for the quantitative trait locus (QTL) identification and molecular selection of the species, which could have a potential role in various applications in sacred lotus breeding.单核苷酸多态性（single nucleotide polymorphism, SNPs）、插入缺失（insertions and deletions, InDels）与简单序列重复（simple sequence repeats, SSRs）等基因组资源，是作物遗传育种改良与高效利用的核心支撑。然而，莲（Nelumbo nucifera Gaertn.）的此类基因组资源仍较为匮乏。本研究为解析莲的大规模基因组分子标记资源，对泰国莲品种‘清迈野生莲’开展重测序，并与已发表的莲基因组‘中湖野生莲’进行比对分析。两组基因组间共鉴定出3180059个SNPs、328251个InDels以及14191个结构变异（structural variations, SVs）。对上述SNPs的功能影响分析显示，其可能参与代谢过程、分子结合、催化活性等生物学活动。通过挖掘基因组序列中的SSRs，共鉴定出191657个SSRs，平均每4.23 kb便存在1个SSRs，并设计得到103656对SSRs引物。此外，借助NCBI数据库中已有的RNA测序（RNA-seq）数据，本研究还鉴定出14502个表达序列标签-简单序列重复（expressed sequence tag simple sequence repeats, EST-SSRs）。本研究随机选取150个SSRs（涵盖基因组SSRs与EST-SSRs）用于验证实验与遗传多样性分析，结果表明，利用这些SSR标记可轻松区分不同莲种质，且‘清迈野生莲’与其他中国莲种质存在显著分化。本研究为莲的数量性状位点（quantitative trait locus, QTL）定位与分子选择育种提供了大量基因组资源与分子标记，有望在莲遗传育种的各类应用中发挥关键作用。