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

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

Characterization of 20 genomic-SSR makers in barley.

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

Various primers used in this experiment.

<p>Various primers used in this experiment.</p

The AmidP drives the GUS expression in the vascular vein of leaves resembling a pattern of the sink-to-source transition.

<p>A. GUS expression is detected in cotyledons and the distal tip of young leaves of 10-d seedlings. B. The AmidP drives expression in the germinating seed joint of above- and under-ground part. C–D. GUS activity is detected in sepals of flowers (C), as shown in an amplified flower indicated with a red arrow (D). E–J. X-Gluc staining is detected throughout the vascular veins of a cotyledon (E), expanded source leaves (F, G, and H) and progresses basipetally down transition leaves (I and J).</p

The construction and results of the 5′- and 3′- deletions of <i>VVE</i> motif between −1500 and −1324 of the AmidP.

<p>A. Sequence and element site analysis of the <i>VVE</i> motif in the AmidP. 4 nucleotides (grey box) are arbitrarily added to form an <i>Eco</i>RI acting site. Element sites for known transcription factors indicated as arrows are detected by AthaMap web tools (see “Materials and methods”). Vertical dotted line indicated deletion sites. B. Schematic diagram of the chimeric constructs. The numbers above the bars indicate the residual region of the <i>VVE</i> motif after 5′- or 3′- deletions. All the fused constructs are obtained by ligation of the pFGC-MiniGUS and the PCR products precut by <i>Eco</i>RI and <i>Bam</i>HI respectively. C–K. Representative histochemical stained cotyledon demonstrates the strength and specificity of GUS activities in the transgenic Arabidopsis of 5M1 (C), 5M2 (D), 5M3 (E), 5M4 (F), 3M1 (G), 3M2 (H), 3M3 (I), 3M4 (J), and 3M5 (K).</p

Truncation analysis of the AmidP in cotyledons of transgenic Arabidopsis seedlings.

<p>A. Promoter truncation, the primers binding sites and endonucleases sites are illustrated. The 1504 bp cloned sequence for the AmidP includes 4 nucleotides which are added to form an <i>Eco</i>RI acting site. All the fused structures are obtained by ligation of the pFGC-DR and the PCR products precut by <i>Eco</i>RI and <i>Nco</i>I respectively, except that of the P3-DR which is self-ligated with the P1-DR digested by <i>Bam</i>HI and <i>Bgl</i>II. B-G. GUS activities in the transgenic Arabidopsis cotyledons of P1-DR (B), P2-DR (C), P3-DR (D), P4-DR (E), P5-DR (F), and P6-DR (G).</p