Development and Characterization of Polymorphic EST-SSR and Genomic SSR Markers for Tibetan Annual Wild Barley

<div><p>Tibetan annual wild barley is rich in genetic variation. This study was aimed at the exploitation of new SSRs for the genetic diversity and phylogenetic analysis of wild barley by data mining. We developed 49 novel EST-SSRs and confirmed 20 genomic SSRs for 80 Tibetan annual wild barley and 16 cultivated barley accessions. A total of 213 alleles were generated from 69 loci with an average of 3.14 alleles per locus. The trimeric repeats were the most abundant motifs (40.82%) among the EST-SSRs, while the majority of the genomic SSRs were di-nuleotide repeats. The polymorphic information content (PIC) ranged from 0.08 to 0.75 with a mean of 0.46. Besides this, the expected heterozygosity (He) ranged from 0.0854 to 0.7842 with an average of 0.5279. Overall, the polymorphism of genomic SSRs was higher than that of EST-SSRs. Furthermore, the number of alleles and the PIC of wild barley were both higher than that of cultivated barley, being 3.12 <i>vs</i> 2.59 and 0.44 <i>vs</i> 0.37. Indicating more polymorphism existed in the Tibetan wild barley than in cultivated barley. The 96 accessions were divided into eight subpopulations based on 69 SSR markers, and the cultivated genotypes can be clearly separated from wild barleys. A total of 47 SSR-containing EST unigenes showed significant similarities to the known genes. These EST-SSR markers have potential for application in germplasm appraisal, genetic diversity and population structure analysis, facilitating marker-assisted breeding and crop improvement in barley.</p></div

Characterization of 20 genomic-SSR makers in barley.

<p>Characterization of 20 genomic-SSR makers in barley.</p

The dendrogram of the eight subpopulations according to the genetic distance using UPGMA clustering analysis.

<p>The dendrogram of the eight subpopulations according to the genetic distance using UPGMA clustering analysis.</p

The putative proteins identified by BLASTX of 49 unigene sequences containing polymorphic EST-SSRs.

<p>The putative proteins identified by BLASTX of 49 unigene sequences containing polymorphic EST-SSRs.</p

Characterization of 49 polymorphic EST-SSR makers in barley (<i>Hordeum vulgare</i>L.).

<p>Note: Na, number of alleles; Ne, number of effective alleles; Ho, observed heterozygosity; He, expected heterozygosity; PIC, polymorphic information content.</p

Δk and population structure.

<p>Estimation of the likelihood of clusters (k) for the most appropriate subpopulations (Δk) (A), and the population structure of 96 barley accessions in k = 8 clusters (B).</p

GO analyses show that miRNAs potentially target tissue forming-related biological processes.

<p>GO analyses show that miRNAs potentially target tissue forming-related biological processes.</p

Predicted secondary structures of novel cucumber miRNAs.

<p>The mature miRNA and miRNA^* sequences are written with red and blue capital letters, respectively.</p

Novel cucumber miRNAs identified by high-throughput sequencing.

<p>LM: length of the mature miRNA; LP: length of the miRNA precursor sequence; MFE: Minimal folding free energy; MFEI: Minimal folding free energy index</p><p>Frequency in leaves and roots: normalized sequencing frequencies in leaves and roots libraries, respectively.</p

Target plots (t-plots) of miRNA targets in different categories confirmed by degradome sequencing.

<p>(A) T-plot (top) and miRNA: mRNA alignments (bottom) for two category I targets, Csa020279 and Csa009014 transcripts. The arrow indicates signatures consistent with miRNA-directed cleavage. The solid lines and dot in miRNA: mRNA alignments indicate matched RNA base pairs and GU mismatch, respectively, and the red letter indicates the cleavage site. (B) As in (A) for Csa18310 and Csa008131, a category II target for csa-miR172 and csa-miR858. (C) As in (A) for Csa014411, a category III target for csa-miR169.</p