| Lotus (Nelumbo Adands) is one of the earliest origin taxa in angiosperms. It has a long history of cultivation and diverse cultivars with edible, medicinal, ornamental values. However, classification and genetic relationship on many cultivars remain unclear. The approach only based on the traditional morphology is difficult to accurately identify a cultivar and its relationship with others. In addition, some wild lotus resources are becoming endangered Therefore, scientific evaluation and protective measures should be immediately taken on lotus germplasm. Although some molecular markers have been recently used to investigate genetic diversity and population relationship in Nelumbo, the application of EST-SSR markers and tag number are yet limited.SSR markers were developed from flower-bud transcriptome sequences of Nelumbo nucifera in this study, and 80 polymorphic EST-SSR markers to evaluate the genetic diversity of the 139 lotus accessions. The main results are as follows:1. In this study,68,593 unigenes were assembled from a mixed flower-bud cDNA pool, which was derived from three accessions of N. nucifera to develop SSR markers. A total of 6086 SSR loci were identified and distributed in 5482 unigenes, accounting for 8.0% of the transcriptome sequences, with an average of one SSR locus per 5.7 kb DNA. The distribution of SSR loci frequency was 8.9%. Di- and tri-nucleotide repeat motifs were the major abundant types among 5226 SSR loci (removing mononucleotide repeats) identified with a frequency of 65.2% and 31.7%, respectively. Moreover, the AG/CT (57.9%) and GAA/AGA/AAG (13.3%) motifs were predominant for the di-and tri-nucleotide repeats, respectively. A total of 575 primer pairs (fragment size ranging from 200 bp to 220 bp) were randomly synthesized from 3,059 SSR loci, of which 514 (89.4%) yielded PCR amplification products and 217 (42.2%) polymorphic SSR markers.2. The 80 polymorphic EST-SSR markers were screened from our study, and then applied to amplify the 139 lotus accessions. A total of 287 alleles were identified, and the number of alleles per locus and Polymorphic Information Content (PIC) value varied from 2 to 9 with a mean of 3.6 alleles and from 0.58 to 0.92 with an average value of 0.79, respectively. It indicated that these markers had a high level of informativeness and were effective in analysis of the genetic diversity in Nelumbo.3. Jaccard similarity coefficients of the amplification results were analyzed by NTSYS 2.11 software and the genetic similarity coefficient was ranged from 0.16 to 0.95. The clustering dendrogram constructed by UPGMA method indicated that 139 accessions of Nelumbo could be divided into four major groups at the similarity coefficient of 0.39. Group I included N. lutea; Group II included the majority of Asian-American lotus hybrids; Group III and group IV included the majority of N. nucifera. The Asian-American hybrids were closer to N. nucifera based on genetic relationship, which is consistent with the traditional classification result and the previous reports.A number of EST-SSR markers were developed in this study. Based on these markers, the genetic diversity of Nelumbo were evaluated to better understand genetic background and genetic relationship of lotus germplasm. The results would provide both theoretical and practical references for accurate classification and identification, effective protection and suitable utilization of lotus resources. A comprehensive set of EST-SSR markers developed by us will facilitate construction of linkage maps, gene mapping and cloning, marker-assisted selection breeding and genetic diversity analysis. |
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