Genome-wide development of simple sequence repeat (SSR) markers at 2-Mb intervals in lotus (Nelumbo Adans.)

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.