Characterization of Flower-Bud Transcriptome and Development of Genic SSR Markers in Asian Lotus (Nelumbo nucifera Gaertn.)

BACKGROUND Asian lotus (Nelumbo nucifera Gaertn.) is the national flower of India, Vietnam, and one of the top ten traditional Chinese flowers. Although lotus is highly valued for its ornamental, economic and cultural uses, genomic information, particularly the expressed sequence based (genic) markers is limited. High-throughput transcriptome sequencing provides large amounts of transcriptome data for promoting gene discovery and development of molecular markers. RESULTS In this study, 68,593 unigenes were assembled from 1.34 million 454 GS-FLX sequence reads of a mixed flower-bud cDNA pool derived from three accessions of N. nucifera. A total of 5,226 SSR loci were identified, and 3,059 primer pairs were designed for marker development. Di-nucleotide repeat motifs were the most abundant type identified with a frequency of 65.2%, followed by tri- (31.7%), tetra- (2.1%), penta- (0.5%) and hexa-nucleotide repeats (0.5%). A total of 575 primer pairs were synthesized, of which 514 (89.4%) yielded PCR amplification products. In eight Nelumbo accessions, 109 markers were polymorphic. They were used to genotype a sample of 44 accessions representing diverse wild and cultivated genotypes of Nelumbo. The number of alleles per locus varied from 2 to 9 alleles and the polymorphism information content values ranged from 0.6 to 0.9. We performed genetic diversity analysis using 109 polymorphic markers. A UPGMA dendrogram was constructed based on Jaccard's similarity coefficients revealing distinct clusters among the 44 accessions. CONCLUSIONS Deep transcriptome sequencing of lotus flower buds developed 3,059 genic SSRs, making a significant addition to the existing SSR markers in lotus. Among them, 109 polymorphic markers were successfully validated in 44 accessions of Nelumbo. This comprehensive set of genic SSR markers developed in our study will facilitate analyses of genetic diversity, construction of linkage maps, gene mapping, and marker-assisted selection breeding for lotus.