Genetic diversity and geographical differentiation of cultivated six-rowed naked barley landraces from the Qinghai-Tibet plateau of China detected by SSR analysis

Cultivated six-rowed naked barley (Hordeum vulgare ssp. hexastichon var. nudum Hsü) is the oldest cultivated barley in China. We used 35 simple sequence repeat (SSR) markers selected from seven barley linkage groups to study the genetic diversity, geographical differentiation and evolutionary relationships among 65 H. vulgare ssp. hexastichon landrace accessions collected from the Qinghai-Tibet plateau of China, 25 accessions from Tibet (TB), 20 from Qinghai (QH) and 20 from Ganzi (GZ) prefecture in Sichuan province. At the 35 SSR loci we identified 248 alleles among the 65 accessions, 119 (47.98%) of the alleles being common alleles. We also found that the TB accessions possessed 47 private alleles, about 1.5 times more than the 31 private alleles found in the QH accessions and about 5 times more than 9 private alleles found in the GZ accessions. Generally, the TB accessions showed significantly higher genetic diversity than either the QH or GZ accessions whereas no significant difference in genetic diversity was found between the QH and GZ accessions. Partitioning analysis of genetic diversity showed that about 81% of the total variation was due to within-subgroup diversity and about 19% was clearly accounted for by geographical differentiation among the three subgroups. The distributions of alleles for most loci (71.4%) were significantly different among the three subgroups and geographical differentiation could be found according to the distribution of SSR alleles. Cluster analysis indicated that most of the accessions could be clustered into groups which basically coincided with their geographical distribution. These results suggest that Tibet might be a center of genetic diversity for cultivated barley, the cultivated six-rowed naked barley on the Qinghai-Tibet plateau of China may have evolved in Tibet and spread to Qinghai and then to Ganzi prefecture of Sichuan province.


Introduction
Barley (Hordeum vulgare L.) is one of the oldest cultivated crops in the world and studying genetic diversity and evolutionary relationships in barley is important for the effective conservation and utilization of barley genetic resources.Early studies suggested that there were two original centers of cultivated barley, one in the Fertile Crescent (Ancient Egypt, the Levant and Mesopotamia) as indicated by the widespread dispersion of Hordeum spontaneum Koch, the nearest wild ancestor of two-and six-rowed cultivated barley, in this region (Harlan, 1976) and another in the Tibetan region of China on the Qinghai-Tibet plateau ('the roof of the world') supported by the discovery of Hordeum agriocrithon Åberg, a six-rowed wild barley considered an ancestor of two-and six-rowed cultivated barley (Vavilov, 1926;Åberg, 1938;Brücher and Åberg, 1950).
Many studies have demonstrated that Tibetan wild barley populations were clearly different from the Fertile Crescent wild barley in respect to their distribution, ecology, morphology, archaeology, cytogenetics and isozyme complement (Xu, 1975(Xu, , 1982;;Zhou, 1981;Shao, 1982;Yao, 1982).This supports the hypothesis of separate evolutionary systems leading to Tibetan two-rowed wild barley becoming the ultimate progenitor of Chinese cultivated barley, Tibetan six-rowed wild barley being an intermediate form in the processes of transforming two-rowed wild barley to six-rowed cultivated barley (Xu, 1982).Naked barley (Hordeum vulgare var.nudum), also called Qingke, is a major food used to produce 'Zanba' by ethnic Zangs inhabiting the Qinghai-Tibet plateau of China.Cultivated six-rowed naked barley (H.vulgare ssp.hexastichon var.nudum Hsü) is the earliest cultivated barley in China (Fu et al., 2000;Xu and Feng, 2001).Evidently, study on genetic diversity and geographical differentiation of cultivated six-rowed naked barley landraces from the Qinghai-Tibet plateau will be useful in understanding the evolutionary relationship of barley.
At present, most studies on the genetic diversity and evolution of cultivated barley from the Qinghai-Tibet plateau of China have dealt with morphology (Xu, 1986), botanical classification (Xu, 1982), cytogenetics (Yao, 1982;Shao, 1986) and isozymes (Shao, 1986;Dai and Zhang, 1989;Zhang et al., 1992aZhang et al., , 1994;;Sun et al., 1995).However, some researchers have used DNA molecular markers, including restriction fragment length polymorphisms (RFLP) (Zhang et al., 1992b(Zhang et al., , 1994)), random amplified polymorphic DNA (RAPD) (Hong et al., 2001) and ribosomal DNA spacer-length techniques (Li et al., 2003), although, except for Tibetan landraces, cultivated six-rowed naked barley landraces from the Qinghai-Tibet plateau have rarely been included in such studies.Simple sequence repeat (SSR), or microsatellite, analysis possess a number of advantages over other forms of genetic analysis, including a high level of polymorphisms, locus specificity, codominance, reproducibility, random distribution throughout the genome and is also methodologically simple (Saghai-Maroof et al., 1994;Feng et al., 2002).Techniques based on SSR technology are useful in evaluating and characterizing genetic diversity, phylogenetic development and evolution as well elucidating the relationships within and between species and populations of members of the genus Hordeum (Saghai-Maroof et al., 1994;Russell et al., 1997;Davila et al., 1998;De Bustos et al., 1999;Fernández et al., 2002;Zhang et al., 2002;Feng et al., 2003).
In the research described in this paper we used SSR markers covering the seven barley SSR linkage groups (Liu et al., 1996) to investigate the genetic diversity and geographical differentiation of 65 cultivated six-rowed naked barley landraces collected from the Qinghai-Tibet plateau of China.

Plant materials
In this study we used 65 landrace accessions (

Genomic DNA extraction
The cetyltrimethylammonium bromide (CTAB) method (Stein et al., 2001) was used to extract total DNA from about 300 mg of young leaf-tissue of each accession.The quality of the DNA was checked using agarose-gel electrophoresis and the DNA concentration estimated spectrophotometrically and the solution diluted with distilled water to a final working DNA concentration of 20 ng µL -1 .

PCR amplification, electrophoresis and silver staining
We selected 35 simple sequence repeats (SSRs) (Table 2), five from each chromosome, from the genetic maps described by Liu et al. (1996).The primers were synthesized by a commercial company (AuGCT Biotechnology, Beijing, China).The polymerase chain reaction (PCR) was carried out in a final volume of 15 µL containing 2 µL of the 20 ng µL -1 genomic DNA solution described above (template DNA), 1.5 µL of 10xPCR buffer containing 15 mM Mg 2+ , 1.5 µL of a 2.5 mM dNTP mixture, 0.5 units of rTaq DNA polymerase (TaKaRa Biotechnology, Dalian, China) and 1 µL of a 2 µM solution of the forward and reverse primers.Depending on the primer pair used, DNA amplifications were performed in a thermocycler using one of the following five PCR protocols: (1) A touchdown PCR reaction consisting of 18 cycles of a 94 °C denaturing step for 1 min and a 72 °C for 1 min extension, followed by annealing for 30 s with the temperatures decreasing by 1 °C every two cycles from 64 °C to 55 °C.The PCR reaction continued for 30 additional cycles at 94 °C for 1 min, 55 °C for 1 min and 72 °C for 1 min.The reaction ended with a 5-min extension at 72 °C.(2) A similar touchdown procedure to the above protocol except that the annealing temperatures were decreased from 69 °C to 60 °C for 18 cycles, at which temperature the reaction continued for 20 additional cycles.
(3) A normal PCR protocol consisting of one cycle of 94 °C for 3 min, 55 °C for 2 min and 72 °C for 1.5 min, followed by 30 cycles at 94 °C for 1 min, 55 °C for 2 min and 1 min and 72 °C for 2 min, followed by a final extension for 10 min at 72 °C.
After PCR amplification 5 µL of 98% (v/v) formamide electrophoresis loading buffer containing 0.25% (w/v) bromophenol blue and 0.25% (w/v) xylene cyanole FF were added to each reaction mixture.The PCR products were denatured and separated on 6% denaturing polyacrylamide gel with 8 M urea and 1xTBE buffer running at a constant power of 60 W for one hour.A 50 to 1031 bp DNA ladder (Gene Ruler TM 50bp DNA ladder, MBI Ferments) was used as a size standard and the DNA fragments were silver stained as described in Bassam et al. (1991).

Data analysis
The amplified DNA fragments of each SSR locus were assessed based on electrophoretic mobility using the Qbasic procedure of Rickwood et al. (1982) and the SSR profiles were scored for the presence (1) or absence (0) of clear bands.Genetic similarities were estimated using the DICE coefficient, 2a/(2a+b+c), where 'a' refers to alleles shared between two accessions and 'b' and 'c' to alleles present in either one of the two accessions compared (Rohlf, 1993).Similarity matrix cluster analysis was used to reveal associations among accessions based on the unweighted pair group method with arithmetic averages (UPGMA) implemented using the NTSYS-pc program (Rohlf, 1993).
Genetic diversity (H) was calculated with H = 1 -Σp i 2 in which p i is the frequency of the i th allele of the locus (Nei, 1973).For each locus, the frequencies of each allele in the entire sample were calculated as the expected allele frequencies, and the distribution of allelic frequencies among the three subgroups was tested using the Chi-square test (Rong et al., 1993).The genetic diversity of the entire sample (H T ) was partitioned into components reflecting genetic distance between subgroups (D ST ) and genetic polymorphism within subgroups (H S ), with genetic differentiation between subgroups (G ST ) being calculated as G ST = 1 -332 Genetic diversity and geographical differentiation of Qinghai-Tibetan naked barley Hs/H T (Nei, 1973).The comparisons of genetic diversity were carried out using the Z-test (Zhang and Allard, 1986;Zhang et al., 1992).

Allelic variation of SSRs
Total alleles, common alleles and the number of private alleles are shown in Table 3.A total of 248 alleles were detected at the 35 SSR loci, with an average of 7.09 alleles per locus in the entire sample.The number of alleles varied from 16 at the HVM68 locus to 2 each at the HVM3, HVM44 and HVM49 loci.Seven of the 35 loci showed more than 10 alleles per locus.The alleles for the 35 loci were distributed among the three geographical location accession subgroups (TB, QH and GZ) as follows: 193 alleles for TB, 180 for QH and 152 for GZ.No allele was detected at the HVM44 locus in the QH accessions and HVM14 locus in the GZ accessions and only one allele each was detected for the HVM34, HVM49 and HVM60 loci in the TB accessions, the HVM64 locus in the QH accessions and the HVM23, HVM49 and HVM64 loci in the GZ accessions.The average number of alleles per locus and standard deviation (SD) of the three subgroups were as follows: TB (5.51 ± 3.17) > QH (5.14 ± 2.88) > GZ (4.34 ± 2.72).Of the 248 alleles, 119 (47.98%) were common to the three subgroups (common alleles) and the average number of common alleles was 3.40 ± 2.66, with the highest number of a The numbers represent one of the five PCR conditions described in the materials and methods section.
common alleles ( 12) being detected at the HVM27 locus while no common alleles were detected at the HVM14, HVM44, HVM60 and HVM64 loci.The number of alleles to specific private alleles per locus varied significantly among the three geographical subgroups with an average of 1.34 in TB, 0.89 in QH and 0.26 in GZ.

Comparison of genetic diversity
Table 4 presents the statistics relating to the genetic variation found at each locus.The average genetic diversity for the entire sample (H T ) was 0.6594 whereas the mean value for the three geographical subgroups was as follows: TB = 0.6172; QH = 0.4993; and GZ = 0.4766.There was a large variation in genetic diversity among the loci, the lowest diversity (0.1719) occurring at the HVM70 locus and the highest (0.9862) at the HVM14 locus.Genetic diversity in excess of 0.90 was found in eight loci in the TB subgroup, six in the QH subgroup and five in the GZ subgroup.Table 4 also shows that significant genetic diversity between the any two arbitrary-selected subgroups strongly depended on the loci involved.Genetic diversity in 10 of the 35 loci was in the order TB < QH, with only the HVM74 showing no difference between TB and QH, while most loci (66.7%) showed a significant difference in the order TB > QH.We also found that 16 loci showed significantly larger genetic diversity in the TB and QH subgroups than in the GZ subgroup while only seven loci displayed obviously lower genetic diversity in the TB and QH subgroups than in the GZ subgroup.Similarly, 13 loci in the QH subgroup showed significantly larger genetic diversity than in the GZ subgroup whereas 11 loci in the QH subgroup presented significantly less genetic diversity than in the GZ subgroup.No difference was observed at the HVM70 locus in respect of the QH and GZ subgroups.Generally, the genetic diversity among the three subgroups was in the order TB > QH > GZ and, on average, genetic diversity in the TB subgroup accessions was significantly larger than in either the QH or GZ subgroups whereas there was no significant difference between the amount of genetic diversity in the QH and GZ subgroup accessions.

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Genetic diversity and geographical differentiation of Qinghai-Tibetan naked barley

Geographical differentiation and distribution of allelic frequencies
The total genetic diversity can be divided into withinand between-populations components, and in our case the proportion of each component varied from locus to locus.The genetic variation between subpopulations (G ST ) reflects the geographical differentiation of samples, the amount of differentiation among the three subgroups varying from 0.27% at the HVM26 locus to 55.52% at the HVM43 locus with an average of 18.58% and only five loci showing a G ST value of less than 5%.The Chi-square test for the distribution of the allelic frequencies of the 35 loci among the three subgroups (Table 4) showed that two loci (HVM4 and HVDHN7) displayed significant differences (p < 0.05) among the three subgroups and 23 loci highly Feng et al. 335 Table 4 -Genetic diversity, geographical differentiation (G ST ) and allele frequency distribution (χ 2 ) for the 35 simple sequence repeat (SSR) loci among cultivated six-rowed naked barley (nudum barley) collected from Tibet (TB), Qinghai (QH) and Ganzi (GZ) in the Qinghai-Tibet plateau of China.significant differences (p < 0.01), whereas ten loci showed no clear differences in their distribution among the three subgroups.These results clearly show that there exists significantly geographical differentiation among the three subgroups.

Cluster analysis
In order to reveal genetic relationships of 65 accessions of cultivated six-rowed naked barley (nudum barley) landraces from the Qinghai-Tibet plateau of China, the genetic similarity coefficients between accessions were calculated and a dendrogram was constructed depicting the relationships between the accessions (Figure 1).At a genetic similarity level of about 0.76 the accessions were clearly clustered into two large groups (Cluster I and cluster II) with all the TB accessions being located in cluster I while, except for accession QH132, the QH and GZ accessions were located in cluster II.At a genetic similarity of about 0.79 the accessions were clustered in five large groups (A, B, C, D and E, Figure 1), all the TB accessions being clustered in groups A, B and C while 13 of the 20 QH accessions and all 20 GZ accessions were clustered together in group D, with the four accessions from Chengduo county and the two accessions from Maqin county (both counties in Qinghai province, i.e.QH accessions) being separately clustered in group E (Figure 1).In group D the QH and GZ accessions were clustered into their own separate groups at a genetic similarity level of 0.8 (Figure 1).These results clearly reveal the geographical differentiation of nudum barley landraces in the Qinghai-Tibet plateau and their genetic relationship.

Discussion
In this study, we investigated the genetic variation among 65 nudum barley landraces at 35 SSR loci in the barley genome.There exists large genetic variation within and among the three geographical subgroups TB, QH and GZ.The genetic diversity was significantly higher in nudum barley from the TB region than it from either the QH or GZ regions while there was no significant difference in terms of genetic diversity between the QH and GZ barleys.The total number of alleles and their standard deviations (Table 3) were in the order TB > QH > GZ, while the average genetic distance followed the order TB (0.2071) > QH (0.2063) > GZ (0.1640).In addition, 18.58% of the total variation accounted for by differentiation among the three subgroups.These results appear to be in basic agreement with the Chi-square test on the distribution of allele frequencies (Table 4).The number of common and private alleles (Table 3) also reflected a certain geographical differentiation among the three subgroups, possibly due to the different geographical and ecological factors in the different regions of the Qinghai-Tibet plateau.As mentioned above, all the Tibetan accessions were clustered into one group (Cluster I) at a genetic similarity level of about 0.76 whereas the QH and GZ accessions (except the QH 132 ) were grouped in cluster II.
Overall, our nudum barley data suggests that Tibet might be an original center of evolution of cultivated sixrowed naked barley which then spread to Qinghai and Ganzi prefecture in Sichuan province.Vavilov (1955) 336 Genetic diversity and geographical differentiation of Qinghai-Tibetan naked barley pointed out that the Middle-Western mountains of China were an original center of cultivated six-rowed naked barley.Previous morphological, ecological, distributional, archaeological and isozyme studies as well as genetics have shown that H. spontaneum in Tibet was the ultimate ancestor of Chinese cultivated barley whereas H. agriocrithon was an intermediate form in the transformation from H. spontaneum to cultivated barley (Xu, 1982).Recently, our results on Tibetan wild barley using SSR markers also indicated that the Shannan region in Tibet might be the original center of Tibetan two-rowed wild barley (Feng et al., 2003) and Hordeum lagunculiforme in Tibet an intermediate form in the transformation from H. spontaneum to H. agriocrithon (unpublished data).Dai and Zhang (1989) studied the genetic diversity of six isozyme loci in cultivated barley from different agro-geographical regions in Tibet and found that the degree of genetic diversity was significantly higher in Tibetan cultivated barley than Ethiopia barley using the same 6 isozyme loci (Zhang et al., 1992a).Li et al. (2003) also supported the notion of Tibet being a center of genetic diversity for cultivated barley.
In conclusion, our SSR analysis of the 65 accessions of H. v. hexastichon var.nudum Hsü six-rowed naked barley from the Qinghai-Tibet plateau showed that the nudum barley landraces in Tibet possessed generally higher genetic diversity than those in Qinghai and Ganzi, with about 19% of the genetic diversity being accounted for by geographical differentiation among the three subgroups.This suggests that Tibet might be a center of genetic diversity of cultivated barley and that the cultivated six-rowed naked barley on the Qinghai-Tibet plateau of China might have evolved in Tibet and spread to Qinghai and then to Ganzi prefecture of Sichuan province.

Figure 1 -
Figure 1 -Unweighted pair group method with averages (UPGMA) genetic similarity dendrogram of 65 cultivated six-rowed naked barley landraces from the Qinghai-Tibet plateau of China.Key: X = Tibet; Q = Qinghai; and G = Ganzi.See materials and methods for more details.

Table 1
) of the cultivated six-rowed naked barley H. vulgare L. ssp.hexastichon var.nudum Hsü (hereafter denominated as nudum barley) from different geographical locations on the Qinghai-Tibet plateau of China, of which 25 accessions were from Tibet (TB), 20 from Qinghai (QH) and 20 from Ganzi (GZ) prefecture in Sichuan province.Seeds of the different accessions were kindly provided by the following people: TB accessions by Mr. QIANG Xiao-Lin (Institute of Agricultural Sciences, Tibetan Academy of Agricultural & livestock Sciences); QH accessions by Prof. SUN Li-Jun (Institute of Crop Germplasm Resources, Chinese Academy of Agricultural Sciences); and GZ accessions by Mr. YANG Kai-Jun (Ganzi Institute of Agricultural Sciences in Sichuan province).

Table 1 -
List and origin of cultivated six-rowed naked barley (nudum barley) landraces collected from the Qinghai-Tibet plateau of China used in this study.

Table 2 -
Primer sequences, PCR conditions, repeats and chromosomal locations of simple sequence repeat (SSR) markers of barley used in this study.

Table 3 -
Allele sizes in base-pairs (bp), total number of alleles and number of common and private simple sequence repeat (SSR) for three subgroups of cultivated six-rowed naked barley (nudum barley) collected from Tibet (TB), Qinghai (QH) and Ganzi (GZ) in the Qinghai-Tibet plateau of China.