Development of Novel EST-SSRs from Sacred Lotus (Nelumbo nucifera Gaertn) and Their Utilization for the Genetic Diversity Analysis of N-nucifera

Expressed sequence tags (ESTs) provide a valuable resource for the development of simple sequence repeat (SSR) or microsatellite markers. This study identified SSRs within ESTs from Nelumbo nucifera (lotus or sacred lotus), developed markers from them, and assessed the potential of those markers for diversity analysis. Within 2207 ESTs from N. nucifera downloaded from GenBank, 1483 unigenes (303 contigs and 1180 singletons) were identified. After eliminating for redundancy, 125 SSR-containing ESTs were derived, and 71 unique SSRs were detected with an average density of one SSR per 13.04 kb. Dinucleotide repeats were the dominant motif in N. nucifera, whereas the sequences AG/TC/GA/CT, AAG/TTC/GAT/AGA, and AAAGCC were the most frequent of di-, tri-, and hexanucleotide motifs, respectively. The AG/TC (40.85%) and AAG (5.63%) motifs were predominant for the di- and trinucleotide repeats, respectively. Sixty-two SSR-containing ESTs were suitable for primer design. From these sequences, 23 EST-SSR markers were developed and were applied to 39 cultivated varieties of N. nucifera, 10 accessions of wild N. nucifera, and 1 accession of Nelumbo lutea (American lotus). Genetic diversity and genetic relationships were examined by constructing unweighted pair-group method with arithmetic average dendrograms and principal coordinates analysis plots based on SSR polymorphisms. Results indicated genetic differentiation between cultivated and wild lotus and between seed lotus cultivars and rhizome lotus cultivars. These EST-SSR markers will be useful for further studies of the evolution and diversity of Nelumbo.