EST-SSR marker development based on RNA-sequencing of E. sibiricus and its application for phylogenetic relationships analysis of seventeen Elymus species

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. Electronic supplementary material The online version of this article (10.1186/s12870-019-1825-8) contains supplementary material, which is available to authorized users.

Development of EST-SSR Markers and Association Mapping with Floral Traits in Syringa Oblata

“…A total of 10,988 EST-SSR loci was identi ed from 9,864 transcriptome sequences, representing approximately 9.4% of the transcriptome in S. oblata, which was consistent with previous reports that the loci frequency ranged from 2.65-16.82% in dicotyledons [44]. Moreover, the distribution density was one SSR per 8.13 kb, which was lower than previous reports for Elymus sibiricus (1/6.2 kb) [45] and Mucuna pruriens (1/5.3 kb) [46], but higher than Medicago sativa (1/12.06 kb) [47] and Nelumbo nucifera (1/13.04 kb) [48]. The frequency of SSR loci varied greatly among different species, probably related to the size of the data set, the SSR search criteria, and the utilized mining tools for SSR search.…”

Genetic Divergence and Relationship Among Opisthopappus Species Identified by Development of EST-SSR Markers

“…These cluster analysis results indicated that the two species would seemingly be merged into one species, or the limited gene data offered from the above molecular markers might not be sufficient for phylogeny analysis. Zhang et al (2019) also pointed that the phylogenetic relationships and genetic components between species were unclear due to the limited detection capacity of specific or unique nuclear gene sequences. Therefore, the development of transferable and orthologous molecular markers between phylogenetically related species of Opisthopappus is essential and critical.…”

“…Recently, RNA sequencing (RNA-Seq) has been employed in the development of a high-throughput sequencing technology that has the ability to profile the entire transcriptome of any organism, particularly for non-model plants that lack sequenced genomic data (Gross et al, 2013;Wu et al, 2014;Du et al, 2015;Zhang et al, 2019). Meanwhile, transcriptome sequencing holds great potential as a platform for the generation of molecular markers (Martin et al, 2013).…”

“…Meanwhile, transcriptome sequencing holds great potential as a platform for the generation of molecular markers (Martin et al, 2013). Further, de novo RNA-Seq assembly can provide a cost-effective approach to identify molecular markers, such as microsatellite markers (SSRs) and singlenucleotide polymorphisms (SNPs) (Davey et al, 2011;Karan et al, 2012;Chen et al, 2013;Zhang et al, 2019). What is more, both of SSRs and SNPs markers are codominant and the reflection of specific segment or sequence information, which are recommended by UPOV-BMT molecular testing guidelines as optimal molecular markers for authenticity identification of varieties and database construction (UPOV-BMT, International Union for the Protection of New Varieties of Plants-Molecular Biology Technology Working Group 2007 3 ).…”

Transcriptome-wide analysis of expressed resistance gene analogs (RGAs) in mango

“…Similar in silico approaches in EST-SSR marker development were performed by other studies focusing on other plants, such as bamboo (Cai et al, 2019), mint (Kumar et al, 2015), castor (Thatikunta et al, 2016), Ethiopian potato (Gadissa et al, 2018) and Elymus species (Zhang et al, 2019). The EST-derived SSR markers generated in this study can be used for functional analysis of R genes, once verified by wet lab experiments.…”