Cloning and Sequence Analysis of rbcS Gene of Wild Barley (Hordeum brevisubulatum) under Salt Stress

[Objective] The aim was to study the cloning and sequence analysis of rbcS gene of wild barley under salt stress. [Method] The tender leaf blade of wild barley under salt stress was taken as the experimental material. The primers were designed according to the homology of rbcS gene sequences of wheat and barely in Genbank; then PCR amplification,recovery,ligation,transformation and sequencing of rbcS gene were carried out. [Result] Two rbcS genes including rbcS1 and rbcS2 with the length of 1 252 and 908 bp respectively were cloned from the barely genome. rbcS1 and rbcS2 were both composed by two exons and one intron. The exons length of the two genes was the same of 525 bp,encoding 174 amino acids,and the homology between them was 96%; however,the intron length of rbcS1 and rbcS2 was 448 and 107 bp respectively.

Genetic diversity of wild barley (Hordeum vulgare ssp. spontaneum) and its utilization for barley improvement

Wild barley （Hordeum vulgare spp. spontaneum） is the undisputed progenitor of cultivated barley and offers considerable poten- tial as a genetic resource for barley improvement. Naturally occurring populations are distributed primarily across the Fertile Crescent, but secondary areas of diversity are also found in central Asia and Tibet. The pattern of its genetic diversity is non-random, and is structured on a global, regional and local scale. On a global scale, diversity follows ecogeography, while on a regional and local scale, it is more strongly determined by ecology. Populations growing in areas where the climate is unreliable and/or the soil is heterogeneous tend to retain the most diversity. A large number of accessions are currently conserved ex situ, but a high proportion of these have originated fi：om the Fertile Crescent. Given the likelihood of future climate change, conserving germplasm both in situ and ex situ in the various regions where the species＇ currently existing is a research priority. The value of H. vulgare spp. spontaneum for the improvement of cultivated barley lies largely in its potential contribution to the levels of attaina- ble pathogen resistance and abiotic stress tolerance.

Sequence Polymorphisms and Phylogenetic Relationships of hina Gene in Wild Barley from Tibet, China

The hina gene encodes a HINA protein in seeds of barley （Hordeum vulgare）, which was known to affect the grain hardness. 171 hina gene sequences from Tibetan wild barley accessions and worldwide were characterized. Across 1 452 nucleotides of 171 hina genes, 152 SNPs were detected, giving an average frequency of one SNP per 9.5 bases. There were 93 singleton variable sites （the nucleotide polymorphism only observed in a single accession）, 59 polymorphic sites （the polymorphisms found in two or more accessions） and 8 indels. A total of 18 haplotypes were defined, and most of the barley accessions shared one gene haplotype. H. spontaneum had a wider haplotype distribution. Through the analysis of median-joining network of the 18 haplotypes, 4 haplotype groups were found, which were testified by neighbor-joining tree based on the complete sequence alignment. Extremely low level of hina gene diversity was observed in Tibetan wild barley accessions, indicating that Tibet is unlikely a center of origin for cultivated barley.

Further molecular evidence for the Hordeum vulgare ssp. spontaneum in Tibet as ultimate progenitor of Chinese cultivated barley

106 aeeessions of Tibetan wild barley, including 50 accessions of the two-rowed wiht barley Hordeum vulgare ssp. sopntaneum （HS）, 27 accessions of the six-rowed bottle-shaped wild barley H. lagunculiforme （HL） and 29 accessions of the six-rowed wiht barley H. agriocrithon （HA） that separately represent different agrigeographical regions of Tibet, were used to study the genetic diversity and genetie differentiation using SSR markers selected from seven barley linkage groups. 229 allelic variants were identified with an average of 7.6 alleles/locus. The average of total number of alleles per locus in HA （6.4） is much higher than that in HS （3.9） and HL （3.4）. The genetie diversity and its standard deviation among the three subspecies were in the order of HS〉HL〉HA. Very significant genetic differentiation was observed among the three subspecies of wild barley. Comparisons of the results fiom this and previous studies showed a strong Oriental-Occidental differentiation of barley, and that Shannan region of Tibet might be the center of origin of the Tibetan two-rowed wild barley, thus supporting not only the hypothesis of a mono-phyletie origin of cultivated barley but also the proposition that the Tibetan two-rowed wild barley as ultimate progenitor of Chinese cultivated barley.