Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 19 (1), 235

EST-SSR Marker Development Based on RNA-sequencing of E. Sibiricus and Its Application for Phylogenetic Relationships Analysis of Seventeen Elymus Species

Affiliations

EST-SSR Marker Development Based on RNA-sequencing of E. Sibiricus and Its Application for Phylogenetic Relationships Analysis of Seventeen Elymus Species

Zongyu Zhang et al. BMC Plant Biol.

Abstract

Background: Elymus L. is the largest genus in the tribe Triticeae Dumort., encompassing approximately 150 polyploid perennial species widely distributed in the temperate regions of the world. It is considered to be an important gene pool for improving cereal crops. However, a shortage of molecular marker limits the efficiency and accuracy of genetic breeding for Elymus species. High-throughput transcriptome sequencing data is essential for gene discovery and molecular marker development.

Results: We obtained the transcriptome dataset of E. sibiricus, the type species of the genus Elymus, and identified a total of 8871 putative EST-SSRs from 6685 unigenes. Trinucleotides were the dominant repeat motif (4760, 53.66%), followed by dinucleotides (1993, 22.47%) and mononucleotides (1876, 21.15%). The most dominant trinucleotide repeat motif was CCG/CGG (1119, 23.5%). Sequencing of PCR products showed that the sequenced alleles from different Elymus species were homologous to the original SSR locus from which the primer was designed. Different types of tri-repeats as abundant SSR motifs were observed in repeat regions. Two hundred EST-SSR primer pairs were designed and selected to amplify ten DNA samples of Elymus species. Eighty-seven pairs of primer (43.5%) generated clear and reproducible bands with expected size, and showed good transferability across different Elymus species. Finally, thirty primer pairs successfully amplified ninety-five accessions of seventeen Elymus species, and detected significant amounts of polymorphism. In general, hexaploid Elymus species with genomes StStHHYY had a relatively higher level of genetic diversity (H = 0.219, I = 0.330, %P = 63.7), while tetraploid Elymus species with genomes StStYY had low level of genetic diversity (H = 0.182, I = 0.272, %P = 50.4) in the study. The cluster analysis showed that all ninety-five accessions were clustered into three major clusters. The accessions were grouped mainly according to their genomic components and origins.

Conclusions: This study demonstrated that transcriptome sequencing is a fast and cost-effective approach to molecular marker development. These EST-SSR markers developed in this study are valuable tools for genetic diversity, evolutionary, and molecular breeding in E. sibiricus, and other Elymus species.

Keywords: E. sibiricus; EST-SSRs development; Elymus genus; Genetic relationship; Transcriptome sequencing; Transferability.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Characteristics of identified SSR. Six types of motif and their percentage (a), different types of tandem repeats and their percentage (b)
Fig. 2
Fig. 2
Comparative electropherogram analysis of two EST-SSR loci (c11036 and c69822) among different species of Elymus
Fig. 3
Fig. 3
Alignment of sequences obtained from selected PCR bands amplified by two primers (a, c11036; b, c69822) in seventeen Elymus species. The expected repeat motif types were marked in bold letters
Fig. 4
Fig. 4
A neighbor-joining (NJ) dendrogram tree showing the genetic relationship among Elymus accessions based on EST-SSRs. Only bootstrap values higher than 50% are presented. Three types of Elymus genome were represented by different colors, green (StH), red (StHY) and blue (StY). Besides, different geographic groups of E. nutans were annotated. The corresponding detailed information for the 95 Elymus accessions is shown in Table 4
Fig. 5
Fig. 5
Principal coordinate analysis (PCoA) for the first three axes generated from 95 Elymus accessions based on EST-SSR markers
Fig. 6
Fig. 6
Regression analysis between pairwise geographic distance and adjusted pairwise genetic distance of 95 Elymus accessions
Fig. 7
Fig. 7
Regression analysis between the effective number of alleles, Nei’s genetic diversity (H) and environmental factors (latitude and altitude) for StHY genome accessions
Fig. 8
Fig. 8
The structure analysis of 95 Elymus accessions based on Bayesian inferred from STRUCTURE program with 30 developed EST-SSRs. a STRUCTURE output with K = 2 and K = 8 showing the population structure among 480 Elymus individuals. Different vertical lines represent an individual genotype and different colors represent genetic stock. Besides, the structure analysis among 30 E. sibiricus accessions was performed based on K = 4; (b) The geographic distribution of the 95 Elymus accessions inferred with Structure across K = 8. The pie charts in the map represent the proportion of each accession and the size of each pie is proportional to sample size from 1 to 9 (Table 4); (c) The genetic distance among the StH, StHY and StY genomes. At K = 8, the proportion of each genome was described by using the pies, of which the protruding sectors belonged to the genome itself; (d) The mean ancestry in each of the eight clusters among 14 geographic groups of E. sibiricus and E. nutans. The percentage of the largest proportion was showed in the graph

Similar articles

See all similar articles

Cited by 1 PubMed Central articles

References

    1. Lu B. Diversity and conservation of the Triticeae genetic resources. Chinese Biodiversity. 1995;3:63–68.
    1. Dewey DR. The genomic system of classification as a guide to intergeneric hybridization with the perennial Triticeae. In: Gustafson JP, editor. Gene manipulation in plant improvement. New York: Columbia University Press; 1984.
    1. Löve À. Conspectus of the Triticeae. Feddes Repert. 1984;95:425–521.
    1. Zeng J, Cao W, Hucl P, Yang Y, Xue A, Chi D, Fedak G. Molecular cytogenetic analysis of wheat - Elymus repens introgression lines with resistance to Fusarium head blight. Genome. 2013;56:75–82. doi: 10.1139/gen-2012-0130. - DOI - PubMed
    1. Cainong JC, Bockus WW, Feng Y, Chen P, Qi L, Sehgal SK, Danilova TV, Koo D-H, Friebe B, Gill BS. Chromosome engineering, mapping, and transferring of resistance to Fusarium head blight disease from Elymus tsukushiensis into wheat. Theor Appl Genet. 2015;128:1019–1027. doi: 10.1007/s00122-015-2485-1. - DOI - PubMed
Feedback