Development and Characterization of Simple Sequence Repeat (SSR) Markers Based on RNA-Sequencing of Medicago sativa and In silico Mapping onto the M. truncatula Genome

Abstract

Sufficient codominant genetic markers are needed for various genetic investigations in alfalfa since the species is an outcrossing autotetraploid. With the newly developed next generation sequencing technology, a large amount of transcribed sequences of alfalfa have been generated and are available for identifying SSR markers by data mining. A total of 54,278 alfalfa non-redundant unigenes were assembled through the Illumina HiSeqTM 2000 sequencing technology. Based on 3,903 unigene sequences, 4,493 SSRs were identified. Tri-nucleotide repeats (56.71%) were the most abundant motif class while AG/CT (21.7%), AGG/CCT (19.8%), AAC/GTT (10.3%), ATC/ATG (8.8%), and ACC/GGT (6.3%) were the subsequent top five nucleotide repeat motifs. Eight hundred and thirty- seven EST-SSR primer pairs were successfully designed. Of these, 527 (63%) primer pairs yielded clear and scored PCR products and 372 (70.6%) exhibited polymorphisms. High transferability was observed for ssp falcata at 99.2% (523) and 71.7% (378) in M. truncatula. In addition, 313 of 527 SSR marker sequences were in silico mapped onto the eight M. truncatula chromosomes. Thirty-six polymorphic SSR primer pairs were used in the genetic relatedness analysis of 30 Chinese alfalfa cultivated accessions generating a total of 199 scored alleles. The mean observed heterozygosity and polymorphic information content were 0.767 and 0.635, respectively. The codominant markers not only enriched the current resources of molecular markers in alfalfa, but also would facilitate targeted investigations in marker-trait association, QTL mapping, and genetic diversity analysis in alfalfa.

The development of 204 novel EST-SSRs and their use for genetic diversity analyses in cultivated alfalfa

“…1). This result showed a high level of polymorphism in alfalfa, as suggested previously (Liu et al, 2007;Wang et al, 2013Wang et al, , 2014. The estimates of polymorphisms are comparable to those reported for 27 EST-SSRs with the same statistical method in alfalfa (mean alleles per locus, Ho, He and H'c of 6.11, 0.64, 0.64 and 1.23, respectively;Liu et al, 2013).…”

“…Only unigenes longer than 1 kb were included in the EST-SSR detection. To avoid possible duplicates of published EST-SSRs (Liu et al, 2013;Wang et al, 2013Wang et al, , 2014, a local BLASTX was performed using the published EST-SSR sequences against the 1494 SSR-containing unigene sequences (E-value <10E-10). The parameters were adjusted to identify perfect di-, tri-, tetra-, penta-and hexa-nucleotide motifs with a minimum of 6, 5, 5, 5 and 5 repeats, respectively.…”

RNA sequencing, de novo assembly, and functional annotation of an endangered Nymphalid butterfly, Fabriciana nerippe Felder, 1862

“…; Wang et al . ). Expressed sequence tag (EST) and transcriptome‐derived molecular markers correspond to genes with a predicted function and lack introns and intragenic regions; hence they are more robust for analysis.…”

“…Simple sequence repeats (SSRs) and single nucleotide polymor-phism (SNP) markers have been popular for comparative genome analysis and conservation genetics (Selkoe & Toonen 2006). The growing numbers of such de novo microsatellite markers showing a high degree of transferability means that researchers have undertaken evolutionary and comparative studies (Fernandez-Silva et al 2013;Wang et al 2014). Expressed sequence tag (EST) and transcriptome-derived molecular markers correspond to genes with a predicted function and lack introns and intragenic regions; hence they are more robust for analysis.…”

RNA-Sequencing Reveals Biological Networks during Table Grapevine (‘Fujiminori’) Fruit Development

“…Because of improvements in Next Generation Sequencing technologies, the development of SSR markers for application has become much easier [55, 57]. In this study, 4,934 SSR loci were identified from 4,337 genes (Fig 7, S13 Table) that can help in the development of phylogenetic analysis of grapevine and other fruit trees.…”