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

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

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

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

Difference in Yield and Physiological Features in Response to Drought and Salinity Combined Stress during Anthesis in Tibetan Wild and Cultivated Barleys

<div><p>Soil salinity and drought are the two most common and frequently co-occurring abiotic stresses constraining crop growth and productivity. Greenhouse pot experiments were conducted to investigate the tolerance potential and mechanisms of Tibetan wild barley genotypes (XZ5, drought-tolerant; XZ16, salinity/aluminum tolerant) during anthesis compared with salinity-tolerant <i>cv</i> CM72 in response to separate and combined stresses (D+S) of drought (4% soil moisture, D) and salinity (S). Under salinity stress alone, plants had higher Na⁺ concentrations in leaves than in roots and stems. Importantly, XZ5 and XZ16 had substantially increased leaf K⁺ concentrations; XZ16 was more efficient in restricting Na⁺ loading in leaf and maintained a lower leaf Na⁺/K⁺ ratio. Moreover, a significant decrease in cell membrane stability index (CMSI) and an increase in malondialdehyde (MDA) were accompanied by a dramatic decrease in total biomass under D+S treatment. We demonstrated that glycine-betaine and soluble sugars increased significantly in XZ5 and XZ16 under all stress conditions, along with increases in protease activity and soluble protein contents. Significant increases were seen in reduced ascorbate (ASA) and reduced glutathione (GSH) contents, and in activities of H⁺K⁺-, Na⁺K⁺-, Ca⁺⁺Mg⁺⁺-, total- ATPase, and antioxidant enzymes under D+S treatment in XZ5 and XZ16 compared to CM72. Compared with control, all stress treatments significantly reduced grain yield and 1000-grain weight; however, XZ5 and XZ16 were less affected than CM72. Our results suggest that high tolerance to D+S stress in XZ5 and XZ16 is closely related to the lower Na⁺/K⁺ ratio, and enhanced glycine-betaine and soluble protein and sugar contents, improved protease, ATPase activities and antioxidative capacity for scavenging reactive oxygen species during anthesis. These results may provide novel insight into the potential responses associated with increasing D+S stress in wild barley genotypes.</p> </div

MDA content and CMSI in flag leaves of Tibetan wild and cultivated barley genotypes.

<p>Barley plants were exposed to drought, salinity alone and combined stresses during anthesis stage at 4% soil moisture level. Error bars represent SD values (n=4). Different letters indicate significant differences (P<0.05) among the treatments within each genotype. CMSI, cell membrane stability index; MDA, malondialdehyde;.</p

The antioxidant enzyme activities in cultivated and Tibetan wild barley genotypes.

<p>Barley plants were exposed to alone and combined stresses of drought and salinity on superoxide dismutase (SOD, A) , peroxidase (POD, B), catalase (CAT, C) and ascorbate peroxidase (APX, D) in flag leaves of three barley genotypes during anthesis stage at 4% soil moisture level. Error bars represent SD values (n=4). Different letters indicate significant differences (P<0.05) among the treatments within each genotype. Control, drought, salinity and D+S correspond to normal growth condition without drought and salinity stress, drought stress (D, seedlings were subjected to drought stress for 20 d during anthesis by withholding irrigation until the soil moisture content (SMC) reduced to 4%), salinity stress (S, 1 L 200 mM NaCl solution was added per pot at anthesis stage and then kept humid throughout the experimental period), combined stresses of drought and salinity (D+S, 1 L 200 mM NaCl solution was added per pot at anthesis stage, then subjected to drought stress until SMC reduced to 4%), respectively. </p

The correlation between relative dry weight and ionic concentration.

<p>The correlation between Na⁺, K⁺ concentration and Na⁺/K⁺ ratios and relative leaf (A, D and G), stem (B, E and H) and root (C, F and I) dry weight based on three genotypes: CM72, XZ16 and XZ5. ns’ not significant, * and ** indicate significant at 0.05 and 0.01, respectively.</p