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

Abstract

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.

Development and characterization of genic-SSR markers from different Asia lotus (Nelumbo nucifera) types by RNA-seq

“…In the contrast, the geographic sources of the samples were not consistent with the genetic distances among lotus individuals. These results were consistent with previous studies (Pan et al, 2010(Pan et al, , 2011Yang et al, 2012b). Table 1.…”

“…Genic-SSRs are generally thought to be less polymorphic than genomic-SSRs. This study supported this, and the polymorphism percentage (80.4%) was consistent with the 87% reported by Pan et al (2010) for lotus. The in-silico polymorphism analysis between WFL and CRL found 1627 common genic-SSRs, of which 48 (3%) possessed polymorphic features.…”

“…The PIC values for each SSR locus ranged from 0.19 to 0.87 with a mean value of 0.55. The informativeness value for these EST-SSR markers (0.55) was a little higher than the genomic SSR marker value (0.51) for N. nucifera reported by Kubo et al (2009);Pan et al (2010) also reported 23 genic-SSRs from EST sequences found in public databases. The PIC values ranged from 0.02 to 0.61 with a mean of 0.33 ± 0.17, and the number of alleles per locus ranged from two to five, with an average of 2.65 alleles.…”

“…After Pan et al (2010) reported the development of 23 EST-SSR markers using Nelumbo EST sequences in 2010, there have been no more reports of EST sequences being used for EST-SSRs development in the NCBI database. This study identified a total of 11,178 genic-SSR loci based on 105,834 nonredundant contigs.…”

“…Genetic diversity between China and Thailand lotus plants were evaluated using ISSR by Chen et al (2008) and Li et al (2010), which improved understanding of lotus germplasm classification. Although the development of a number of SSRs was reported by Tian et al (2008a), Kubo et al (2009), Pan et al (2010), and Xue et al (2012), few SSR markers have been exploited, particularly in Nelumbo molecular quantity genetic studies and molecular assistance breeding.…”