The importance of wild barley from Qinghai-Tibet Plateau in the origin and domestication of cultivated barley has long been underestimated. Population-based phylogenetic analyses were performed to study the origin and genetic diversity of Chinese domesticated barley, and address the possibility that the Tibetan region in China was an independent center of barley domestication. Wild barley (Hordeum vulgare ssp. spontaneum) populations from Southwest Asia, Central Asia, and Tibet along with domesticated barley from China were analyzed using two nuclear genes. Our results showed that Tibetan wild barley distinctly diverged from Southwest Asian (Near East) wild barley, that Central Asian wild barley is related to Southwest Asian wild barley, and that Chinese domesticated barley shares the same haplotypes with Tibetan wild barley. Phylogenetic analysis showed a close relationship between Chinese domesticated barley and the Tibetan wild barley, suggesting that Tibetan wild barley was the ancestor of Chinese domesticated barley. Our results favor the polyphyletic origin for cultivated barley.
Citation: Ren X, Nevo E, Sun D, Sun G (2013) Tibet as a Potential Domestication Center of Cultivated Barley of China. PLoS ONE 8(5): e62700. https://doi.org/10.1371/journal.pone.0062700
Editor: David Caramelli, University of Florence, Italy
Received: February 4, 2013; Accepted: March 25, 2013; Published: May 3, 2013
Copyright: © 2013 Ren et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This project was supported in part by the National Natural Science Foundation of China (31228017), the Postdoctoral Science Foundation (2012M511627), an earmarked fund for China Agriculture Research System (CARS-5), a discovery grant (238425) from the Natural Sciences and Engineering Research Council of Canada, and a Senate Research Grant at Saint Mary’s University, Canada. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Barley, a founder crop of old World Neolithic food production, and one of the earliest domesticated crops , , is one of the main cereals of the Mediterranean belt of agriculture. The spread of crops from their domestication areas involved the dispersal of crop plants well beyond their progenitors’ native range and adaptation to new environments . It has been well demonstrated that cultivated barley (Hordeum vulgare ssp. vulgare) originated from wild barley (Hordeum vulgare ssp. spontaneum). The geographic distribution of wild barley in the near East Fertile Crescent was well defined, and the near East Fertile Crescent is considered as the only location where barley was domesticated by some scientists , –. However, the discovery of H. vulgare ssp. spontaneum in sites other than the Fertile Crescent such as Tibet, central Asia, Morocco, Libya, Egypt, Crete, and Ethiopia has challenged the prevalent single origin theory on the origin of barley –. It has been reported that Hordeum vulgare ssp. spontaneum is distributed in the east-Mediterranean basin and the west Asiatic countries, penetrating into the Aegean region and North Africa to Morocco, and extends eastwards to central Asian areas and Tibet of China , , . Recent molecular evidence suggested Central Asia, 1,500–3,000 km farther east from the Fertile Crescent , and Tibet of China  as additional centers of wild barley domestications, and supported multiple origins of cultivated barley. Morrell and Clegg , and Saisho and Purugganan  suggested that a second domestication occurred east of the Fertile Crescent that contributed to Central and East Asian barleys.
Since the discovery of Hordeum agriocrithon Åberg, the close wild relative of barley, and Hordeum vulgare ssp. spontaneum on the Qinghai-Tibet Plateau in China, the origin of Chinese cultivated barley have received more attention and debate –. Morphological, archaeological cytogenetic and isozyme data have demonstrated that wild barley on the Qinghai-Tibet Plateau was different from the Fertile Crescent wild barley , , –. Ecologically, the Near East wild barley is adapted to warm and dry climates, while the barley on Qinghai-Tibet Plateau is adapted to cold and dry environments . Recent DArT data supported that Tibet is one of the centers of domestication of cultivated barley . The position of Qinghai-Tibet Plateau wild barley in origin and domestication of cultivated barley has long been underestimated.
The resequencing of gene loci within diverse populations has implications for understanding the origins of barley domestication , . Protein content is the most important quality trait in barley. The protein content of barley affects the malting and brewing processes, and the operating efficiency of the brewery and the quality of malt used for making beer , . In barley, two protein coding genes, HvNAM-1 and HvNAM-2, have been identified and mapped on chromosomes 6H and 2H, respectively , . Allelic variation of the NAM-1 gene for three species of Hordeum representing wild and cultivated barley was analyzed . The expression of the NAM-1 gene should play a role in grain protein content regulation, and loss of functionality of the NAM-1 gene is related to lower grain protein content in Hordeum .
HTL (Hordeum thioredoxin-like) gene encodes a thioredoxin-like protein. Thioredoxins are universally distributed small-molecular-weight thermostable proteins that have the capacity to catalyze dithiol/disulphide exchange reactions , . Thioredoxins play an important role during various aspects of plant life, including enzymatic activation, photosynthesis, photorespiration, reactions associated with the citric acid cycle, lipid metabolism, electron transport ATP synthesis/transformation, membrane transport, translation, protein assembly/folding, nitrogen metabolism, sulfur metabolism, hormone synthesis, and stress responses –.
Genomic diversity provides the basis of evolutionary change by natural selection and domestication . Domestication and modern plant breeding practices have narrowed the genetic diversity in cultivated plants , . Wild barley provides a rich source of potential genetic variation for barley improvement. Gene sequence variations reflect the genetic and evolutionary history of organisms . This paper characterizes nuclear variation of Nam-1 and HTL genes in wild barley from Southwest Asia, Central Asia, and Tibet of China and cultivated barley from China. Population based analyses and phylogenetic analysis were performed to address if Tibet in China was an independent center of barley domestication, the origin and genetic diversity of Chinese domesticated barley.
Materials and Methods
A total of 103 barley accessions were used in this study including 45 wild barley from Southwest Asia, 18 wild barley from Central Asia, 20 wild barley from Tibet of China, and 20 cultivated barley from China (Table S1). No specific permissions were required for these locations/activities. The materials used in this study were provided by USDA (United States Department of Agriculture) and Huazhong Agricultural University barley germplasm collection. The seeds were planted in pots with sand-peat mixture and maintained in a greenhouse. DNA was extracted from young freeze-dried leaf tissue collected from 5 to 10 plants of each accession using the method of Stein et al. .
DNA Amplification and Sequencing
The single copy nuclear gene HTL sequences were amplified by polymerase chain reaction (PCR) using the primers TrxF and TrxR, following the protocols given in Kakeda et al. . Primers for amplification of Nam-1 gene were designed with the computer program “Primer 3” based on the sequence from Hordeum vulgare L. (Genebank accession number DQ869678). The forward and reverse primer sequences are Nam-1HF: 5′-TATCAAGCGCCGTAATTTCC-3′ and Nam-1HR: 5′-ATACTGCCGACGTTTCTGCT -3′, respectively. Amplification of DNA was carried out in 40 µl reaction mixture containing 60 ng template DNA, 0.2 µM of each primer, 1.5 mM MgCl2, 0.2 mM of each deoxynucleotide (dATP, dCTP, dGTP, dTTP), 1.5 unit of Taq DNA polymerase (Biolabs, New England) and distilled de-ionized water to the final volume. The mixture was amplified using the BioRad iCycler Thermal cycler. PCR condition was as follows: one cycle of 4 min at 95°C, 40 cycles of 1 min at 95°C, 1 min at 52°C, 2 min at 72°C, followed by 8 min at 72°C.
PCR products were purified using the QIAquickTM PCR purification kit (QIAGEN Inc), according to the manufacturer’s instruction, and then sequenced commercially at the Taihe Biotechnology Co Ltd (Beijing, China). To increase quality of the data, both forward and reverse strands were sequenced independently. In order to avoid the error induced by Taq DNA polymerase during PCR amplification, each sample was independently amplified three times and sequenced.
Multiple sequence alignments were performed using ClustalX . Nucleotide diversity and tests of neutral evolution were performed using the software program DnaSP 4.0  by Tajima’s  π, Watterson’s  θ and Fu and Li . Phylogenetic analysis was performed with the computer program PAUP* ver. 4 beta 10 . The neighbour-joining (NJ) method  using Tajima-Nei distance was used for phylogenetic construction.
Genetic Analysis of the Nam-1 Sequences from Four Populations
Sequences of the Nam-1 were compared from four populations: wild barley of Southwest Asia, Central Asian and Tibet of China, and cultivated barley from China (Fig. 1). The total length of the Nam-1 sequences amplified was 950 bp. A total of 10 haplotypes were identified in the four natural populations, of which six haplotypes were population-specific, while four haplotypes were shared between populations and only one haplotype was shared by four populations. Comparison of haplotypes among wild barley populations revealed three haplotypes specific to the Tibetan wild barley population and four haplotypes unique to the Southwest Asian wild barley population. However, no haplotype specific to the Central Asian wild barley population was found. Only two haplotypes were identified in the cultivated barley population from China and presented in the Tibetan wild barley population (Table 1).
The haplotype Hap 2 was the common one, shared among all four populations (Fig. 2A). A total of 5 haplotypes were identified in the Tibetan wild barley population with the majority of accessions (16 of 20) having either haplotype Hap 2 or Hap 7, which are also the two haplotypes present in the cultivated barley population from China. A total of 7 haplotypes were found in the Southwest Asia wild barley population, the majority of accessions were haplotype Hap 2 (46.7%), followed by Hap 5 (26.7%). In the Central Asian wild barley population, we only identified 2 haplotypes, and the majority of accessions (94.4%)) was haplotype Hap 2. The haplotype Hap 7 was unique to barley in China (wild and domesticated barleys); Hap 8 and Hap 10 were unique to the Tibetan wild barley populations, while Hap 1, Hap 4, Hap 5, and Hap 6 were unique to the Southwest Asian wild barley (Table 1).
The genetic diversity and the neutrality test for Nam-1 gene in different populations are summarized in Table 2. The highest haplotype diversity (Hd = 0.710) and per-site nucleotide diversity (θ = 0.00144±0.00070) were detected in the Southwest Asian wild barley population, followed by the Tibetan wild barley population. The highest nucleotide diversity (π = 0.00101) was detected in the Tibetan wild barley population. Both Tajima and Fu & Li’s neutrality tests were not significant (P>0.05) in all four natural populations. Positive values were obtained for the Tibetan wild barley and Chinese cultivated barley using both the Tajima and Fu & Li tests. In contrast, negative values for both tests were obtained for the Southwestern and Central Asian wild barley except Fu & Li tests for the Southwestern wild barley (Table 2).
Genetic Analysis of the HTL Sequences from Four Populations
The length of the HTL sequences amplified was 935 bp. A total of 12 haplotypes were identified in the four populations. Eight haplotypes were population-specific, while four haplotypes were shared between/among populations. No haplotype was shared by the four populations. A comparison of three populations of wild barley revealed only one each haplotype was unique to the Tibetan wild barley (Hap 4) and the Central Asian (Hap 2) population, while seven haplotypes were unique to the Southwest Asian wild barley. Two haplotypes were identified in the Chinese cultivated barley and were shared with the Tibetan wild barley population (Table 3).
Three wild barley populations harbored a different high-frequency haplotype. Only two haplotypes were detected in the Tibetan wild barley population with the majority of accessions (18 of 20) having one haplotype (Hap 5). The Chinese cultivated barley population also had only two haplotypes with the majority of accessions (15 of 20) having haplotype Hap 5 that was also detected in two accessions from Central Asia. Nine haplotypes were detected in the Southwest Asian wild barley population, 18 out of 45 accessions had haplotype Hap 9 that was also found in one Central Asian accession. A total of four haplotypes were observed in the Central Asian wild barley population with the majority of accessions (14 of 18) having haplotype Hap 1 that was also presented in 9 out 45 the Southwest Asian accessions (Table 3).
The genetic diversity analysis and the neutrality test results for the HTL gene in different populations are summarized in Table 4. The highest number of haplotypes (h = 9), highest nucleotide diversity (π = 0.00157), and greatest haplotype diversity (Hd = 0.785) and per-site nucleotide diversity (θ = 0.00196±0.00086) were observed in the Southwest Asian wild barley population, followed by the Central Asian wild barley population with four haplotypes, nucleotide diversity (π = 0.00075), and per-site nucleotide diversity (θ = 0.00093±0.00060). Nucleotide diversity (π), haplotype diversity (Hd) and per-site nucleotide diversity (θ) in wild barley of Tibet and cultivated barley from China were lower than those in other populations. Tajima and Fu & Li neutrality tests did not significantly depart from neutrality in all four natural populations. Positive values were obtained for Tajima and Fu & Li tests for both cultivated barley of China and wild barley of Tibet. In contrast, negative values were obtained for Tajima and Fu & Li tests for Central Asian wild barley population and Southwest Asian wild barley population (Table 4).
To investigate the relationships among haplotypes, phylogenetic analysis was performed using the neighbor-joining (NJ) method based on Tajima-Nei distance. The phylogenetic tree based on Nam-1 gene showed that the haplotypes detected in the Southwest Asian and Central Asian wild barley formed a cluster (Fig. 2A), and the haplotypes from Tibetan wild barley and Chinese cultivated barley formed a group, except Hap 9 shared between Tibetan and Southwest Asian wild barley (Fig 2A). Phylogenetic analysis based on HTL divided the haplotypes into three groups: group I and II with haplotypes from the Southwest Asian and Central Asian wild barley, group III was a mixture of haplotypes from Tibetan wild barley, Chinese cultivated barley, Central Asian wild barley, and Southwest Asian wild barley (Fig. 2B).
Phylogenetic analyses were also performed to reveal the relationships among all accessions from the four populations. The Nam-1 gene tree showed that a majority of accessions of Chinese cultivated barley were grouped together with Tibetan wild barley (Fig. 3A). The phylogenetic tree based on the HTL gene clearly grouped all Chinese cultivated barley with all Tibetan wild barley (Fig. 3B).
Origin of Chinese Domesticated Barley
Two hypotheses were suggested regarding the origin of Chinese cultivated barley. First, China was the original center of the six-rowed barley –. The results from isozyme and ribosomal DNA data supported that Tibetan six-row wild barley was a direct ancestor of six-row and two-row cultivated barley of China . Feng et al.  suggested that Chinese cultivated barley might originate from the two-rowed wild barley from Tibet and that the Tibetan six-rowed wild barley was an intermediate form in the process of transformation from two-rowed wild barley to cultivated barley. Analysis of genetic diversity of hordein in wild close relatives of barley from Tibet supported the hypothesis that the Qinghai-Tibet Plateau and its vicinity are the center for the cultivated barley in the Oriental region . The HvGlb1 sequence data favored this hypothesis . Recent DArT data indicated that some Chinese hulless six-rowed barley had been domesticated in the Tibetan Plateau and its vicinity, and suggested that Tibet is one of the centers of domestication of cultivated barley , while a second hypothesis suggested the Chinese cultivated barley was introduced from the Near East , . AFLP data favored this hypothesis . Sequence polymorphisms and phylogenetic relationships of the hina gene in wild barely from Tibet, China suggested that Tibet is unlikely a center of origin for cultivated barley . Our results showed that Chinese domesticated barley shared the same haplotypes with the Tibetan wild barley, and phylogenetic analysis showed a close relationship between Chinese domesticated barley and the Tibetan wild barley (Fig. 3), suggesting that Tibetan wild barley was the ancestor of Chinese domesticated barley. This suggestion was only based on the result from two genes. To further confirm this, we need to analyze more genes sequences.
Genetic Differentiation between Tibetan wild Barley and the Fertile Crescent Wild Barley
Genetic differentiation between the Oriental and Occidental barley has been reported using allozymes – and rDNA . The total number of alleles and the mean values of genetic diversity of the two-rowed wild barley from Tibet were obviously different from the values of two-rowed wild barley from Israel, Iran, and Turkey , . ISSRs and SSRs results indicated that the Tibetan wild barley and the wild barley from the Middle East were distinctly separate from each other . Recent DArT results showed that Tibetan wild barley distinctly diverged from the Near East wild barley . The current results of two nuclear gene sequences showed significant genetic differentiation among wild barley populations. For the Nam-1 gene, although wild barley populations shared a high-frequency haplotype, large amounts of unique haplotypes were detected in Tibetan and Southwest Asian barley. For the HTL gene, the distinct haplotypes were detected in Tibetan wild barley and Southwest Asian wild barley. Phylogenetic analysis also showed a certain degree of separation of haplotypes from Tibetan and Southwest Asian wild barley (Fig. 2). Our results provided evidence to further support that Tibetan wild barley distinctly diverged from the Southwest Asian (Near East) wild barley , and revealed that Central Asian wild barley was related to Southwest Asian wild barley. The above-mentioned facts indicated that the Tibetan wild barley is different from the wild barley of the Near East, which indirectly shows that Tibet is an original center and diversity center of cultivated barley. The present data favored the diphyletic origin for cultivated barley , , , , .
Loss of Alleles in Domesticated Barley
The domesticated lines harbored fewer haplotypes. Only two haplotypes were found for each gene tested here. Among the 10 haplotypes for Nam-1 and 12 haplotypes for HTL found in wild barley, only two of them for each gene were present in the domesticated lines, indicating that domesticated lines have lost most alleles of wild types. This is consistent with the analysis of haplotypes of wild and domesticated barley at seven loci –Adh2, Adh3, Amy1, Dhn9, GAPDH, PEPC and Waxy, which in total, 70 different haplotypes in 25 wild barley accessions were observed, while only 17 occurred in the 20 domesticated lines . The per-site nucleotide diversity (θ = 0.00089±0.00057) and nucleotide diversity (π = 0.00101) of Nam-1 gene in Tibetan wild barley were two-fold of those detected in the Chinese domesticated barley (Table 2). These values indicate a substantial loss of nucleotide diversity in domesticated barley in this gene. Reduction of genetic diversity in the cultivated gene pool of barley has been widely reported , , . The increase in Tajima’s D from wild (D = 0.37128) to domesticated barley (D = 1.43024, not significant) is also a signature of recent bottleneck .
It was notable that the per-site nucleotide diversity of the HTL gene in Tibetan wild barley is similar to that detected in the Chinese domesticated barley, while the nucleotide diversity (π) in the Tibetan wild barley was lower than that in the Chinese domesticated barley (Table 4). This might be caused by the natural of this gene. The HTL gene encodes a thioredoxin-like protein that plays an important role during various aspects of plant life as mentioned in the Introduction. The Chinese domesticated barley was cultivated in diversified environments, which could cause this functional gene mutation. The positive Tajima’s D value for the Chinese domesticated barley is indicative of over-dominant selection (D = 0.72261), although it was not significant, but accumulated nucleotide diversity in the domesticated barley for this gene.
In summary, our study has provided new insights into barley domestication and the origin of cultivated barley in China. The current results showed that the wild barley of Tibet was different from Southwest Asia. Wild barley of Tibet was the direct ancestor of cultivated barley of China. Wild barley is an important reservoir of genetic diversity and a potential source of beneficial alleles for barley breeding and improvement.
The code, accession number, origin and characteristic of 104 barley used in this study.
Conceived and designed the experiments: GS DS. Performed the experiments: XR GS. Analyzed the data: XR GS. Contributed reagents/materials/analysis tools: GS DS. Wrote the paper: XR EN DS GS.
Zohary D, Hopf M (2000) Domestication of plants in the Old World. 3rd ed. Oxford: Oxford University Press.
Zohary D, Hopf M, Weiss E (2012) Domestication of Plants in the Old World: The origin and spread of domesticated plants in Southwest Asia, Europe, and the Mediterranean Basin. 4th ed. Oxford: Oxford University Press.
- 3. Jones H, Leigh FJ, Mackay I, Bower MA, Smith LMJ, et al. (2008) Population-based resequencing reveals that the flowering time adaptation of cultivated barley originated east of the Fertile Crescent. Mol Biol Evol 25: 2211–2219.
- 4. Harlan JR, Zohary D (1966) Distribution of wild wheats and barley. Science 153: 1074–1080.
- 5. Nevo E, Beiles A, Gutterman Y, Storch N, Kaplan D (1984) Genetic resources of wild cereals in Israel and vicinity: II. Phenotypic variation within and between populations of wild barley, Hordeum spontaneum. Euphytica 33: 737–756.
- 6. Nevo E, Beiles A, Zohary D (1986) Genetic resources of wild barley in the Near East: Structure, evolution and application in breeding. Biol J Linn Soc Lond 27: 355–380.
- 7. Badr A, Mueller K, Schafer-Pregl R, El Rabey H, Effgen S, et al. (2000) On the origin and domestication of barley (Hordeum vulgare). Mol Biol Evol 17: 499–510.
- 8. Nevo E (2006) Genome evolution of wild cereal diversity and prospects for crop improvement. Plant Genet Resour 4: 36–46.
Molina-Cano JL, Igartua E, Casas AM, Moralejo MA (2002) New views on the origin of cultivated barley. In: Slafer GA, Molina-Cano JL, Savin R, Araus JL, Romagosa I (eds) Barley science: recent advances from molecular biology to agronomy of yield and quality. Binghamton: The Haworth Press.
- 10. Molina-Cano JL, Russell JR, Moralejo MA, Escacena JL, Arias G, et al. (2005) Chloroplast DNA microsatellite analysis supports a polyphyletic origin for barley. Theor Appl Genet 110: 613–619.
- 11. Azhaguvel P, Komatsuda TA (2007) A phylogenetic analysis based on nucleotide sequence of a marker linked to the brittle rachis locus indicates a diphyletic origin of barley. Ann Bot 100: 1009–1015.
- 12. Morrell PL, Clegg MT (2007) Genetic evidence for a second domestication of barley (Hordeum vulgare) east of the Fertile Crescent. Proc Natl Acad Sci USA 104: 3289–3294.
- 13. Orabi J, Jahoor A, Backes G (2009) Genetic diversity and population structure of wild and cultivated barley from West Asia and North Africa. Plant Breed 128: 606–614.
- 14. Bjornstad A, Abay F (2010) Multivariate patterns of diversity in Ethiopian barleys. Crop Sci 50: 1579–1586.
von Bothmer R, Komatsuda T (2011) in Barley: Production, Improvement, and Uses, ed Ullrich SE (Wiley-Blackwell, Chichester, UK), 14–62.
Dai F, Nevo E, Wu D, Comadran J, Zhou M, et al.. (2012) Tibet is one of the centers of domestication of cultivated barley. Proc Natl Acad Sci USA www.pnas.org/cgi/doi/10.1073/pnas.1215265109.
von Bothmer R, Jacobsen N, Baden C, Jorgensen RB, Linde-Laursen I (1995) An ecogeographical study of the genus Hordeum. Systematic and ecogeographical studies on crop genepools, 7, 2nd edn. Rome: IPGRI.
Nevo E (2012) Evolution of wild barley and barley improvement. Advance in Barley Sciences, Proceedings of 11th International Barley Genetics Symposium. 1–16.
- 19. Saisho D, Purugganan MD (2007) Molecular phylogeography of domesticated barley traces expansion of agriculture in the old world. Genetics 177: 1765–1776.
- 20. Shao QQ, Li CS, Baschan CR (1975) Origin and evolution of cultivated barley-wild barleys from the south-western part of China. Acta Genet Sin 2: 123–127.
- 21. Xu TW (1975) On the origin and phylogeny of cultivated barley with reference to the discovery of Ganzi wild two-rowed barley Hordeum spontaneum C. Koch. Acta Genet Sin 2: 129–137.
Shao QQ (1982) Wild Barley of Tibet. 1st edition. Beijing: Scientific Press. 1–86.
- 23. Xu TW (1984) Consanguinity and taxonomy of the genus Horduum. J Sichuan Agricultural College 2: 11–18.
- 24. Giles BE, von Bothmer R (1985) The progenitor of barley (Hordeum vulgare ssp. spontaneum) – its importance as a gene resource. Sveriges Utsadesforenings Tidskrift 95: 71–79.
- 25. Xu TW (1985) The wild close relatives of cultivated barley in Qinghai-Tibet Plateau. Barley Newslet 29: 105–106.
- 26. Yang JL, Yen C (1985) Distribution of weedrace barley in China and the center of origin of cultivated barley. Utsadesforenings Tidskrift 95: 71–79.
- 27. Zhou ZQ (1981) Comparison between the two-rowed wild barley of Qing-Zang plateau and of Israel. Acta Genet Sin 8: 344–349.
- 28. Xu TW (1982) Origin and evolution of cultivated barley in China. Acta Genet Sin 9: 440–446.
- 29. Yao Z (1982) Study on N-bands of chromosomes in wild and cultivated barley. Acta Genet Sin 9: 160–164.
- 30. See D, Kanazin V, Kephart K, Blake T (2002) Mapping genes controlling variation in barley grain protein concentration. Crop Sci 42: 680–685.
- 31. Emebiria LC, Moodya DB, Horsleyb R, Panozzoa J, Readc BJ (2005) The genetic control of grain protein content variation in a doubled haploid population derived from a cross between Australian and North American two-rowed barley lines. J Cereal Sci 41: 107–114.
- 32. Uauy C, Distelfeld A, Fahima T, Blechl A, Dubcovsky J (2006) A NAC gene regulating senescence improves grain protein, zinc, and iron content in wheat. Science 314: 1298–1301.
- 33. Distelfeld A, Korol A, Dubcovsky J, Uauy C, Blake T, et al. (2008) Colinearity between the barley grain protein content (GPC) QTL on chromosome arm 6HS and the wheat Gpc-B1 region. Mol Breeding 22: 25–38.
- 34. Jamar C, Loffet F, Frettinger P, Ramsay L, Fauconnier ML, et al. (2010) NAM-1 gene polymorphism and grain protein content in Hordeum. J Plant Physiol 167: 497–501.
- 35. Buchanan BB, Schermann P, Decottignies P, Lozano RM (1994) Thioredoxin: a multifunctional regulatory protein with a bright future in technology and medicine. Arch Biochem Biophys 314: 257–260.
- 36. Juttner J, Olde D, Langridge P, Baumann U (2000) Cloning and expression of a distinct subclass of plant thioredoxins. Eur J Biochem 267: 7109–7117.
- 37. Gelhaye E, Rouhier N, Jacquot JP (2004) The thioredoxin h system of higher plants. Plant Physiol Biochem 42: 265–271.
- 38. Raines C (2005) Preface to redox regulation of leaf metabolism. J Exp Bot 56: 416.
- 39. Balmer Y, William HV, Manieri W, Schurmann P, William JH, et al. (2006) A complete ferredoxin thioredoxin system regulates fundamental processes in amyloplasts. Proc Natl Acad Sci USA 103: 2988–2993.
- 40. Nuruzzaman M, Gupta M, Zhang C, Wang L, Xie W, et al. (2008) Sequence and expression analysis of the thioredoxin protein gene family in rice. Mol Genet Genomics 280: 139–151.
- 41. Ross-Ibarra J, Morrell PL, Gaut BS (2007) Plant domestication, a unique opportunity to identify the genetic basis of adaptation. Proc Natl Acad Sci USA 104: 8641–8648.
- 42. Maeso I, Roy SW, Irimia M (2012) Widespread recurrent evolution of genomic features. Genome Biol Evol 4: 486–500.
- 43. Stein N, Herren G, Keller B (2001) A new DNA extraction method for high-throughput marker analysis in a large-genome species such as Triticum aestivum. Plant Breed 120: 354–356.
- 44. Kakeda K, Taketa S, Komatsuda T (2009) Molecular phylogeny of the genus Hordeum using thioredoxin-like gene sequences. Breeding Sci 59: 595–601.
- 45. Thompson JD, Gibson TJ, Plewniak F, Jeanmouguin F, Higgins DG (1997) The Clustal X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25: 4876–4882.
Rozas J, Sánchez-DelBarrio JC, Messeguer X, Rozas R (2005) DNA Sequence Polymorphism, Version 4.10.4. DNAsp4 Computer Software. Barcelona University, Barcelona, Spain.
- 47. Tajima F (1989) Statistical method for testing the neutral mutation of hypothesis by DNA polymorphism. Genetics 123: 585–595.
- 48. Watterson GA (1975) On the number of segregation sites in genetical models without recombination. Theor Popul Bio 7: 256–276.
- 49. Fu YX, Li WH (1993) Statistical tests of neutrality of mutations. Genetics 133: 693–709.
Swofford DL (2003) PAUP Version 4 Beta 10. Phylogenetic Analysis Using Parsimony - Macintosh Version. Sinauer Associates, Sunderland, MA.
- 51. Saitou N, Nei M (1987) The neighbour-joining method: a new method for reconstructing phylogenetic trees. J Mol Evol 4: 406–425.
- 52. Vavilov NI (1926) Studies on the origin of cultivated plants. Bull Appl Bot Genet Plant Breed USSR 16: 1–248.
- 53. Vavilov NI (1951) The origin, variation, immunity and breeding of cultivated plants. Chron Bot 13: 1–366.
- 54. Takahashi R (1955) The origin and evolution of cultivated barley. Advance Genet 7: 227–266.
Xu TW (1986) Origin and phylogeny of cultivated barley in China. In: Proceedings of Chinese Barley Science. Beijing: Chin Agri Sci Tech Press. 34–44.
- 56. Zhang Q, Yang GP, Dai XK, Su JZ (1994) A comparative analysis of genetic polymorphism in wild and cultivated barley from Tibet using isozyme and ribosomal DNA markers. Genome 37: 631–638.
- 57. Feng ZY, Liu XJ, Zhang YZ, Ling HQ (2006) Genetic diversity analysis of Tibet wild barley using SSR markers. Acta Genet Sin 33: 917–928.
- 58. Yin YQ, Ma DQ, Ding Y (2003) Analysis of genetic diversity of hordein in wild close relatives of barley from Tibet. Theor Appl Genet 107: 837–842.
- 59. Jin X, Cai S, Han Y, Wang J, Wei K, et al. (2011) Genetic variants of HvGlb1 in Tibetan annual wild barley and cultivated barley and their correlation with malt quality. J Cereal Sci 53: 59–64.
Harlan JR (1971) On the origin of barley: A second look. In BarleyGenetics II 45–49.
Harlan JR (1976) Barley, Hordeum vulgare. In: Simmonds N (ed) Evolution of Crop Plants. London and New York: Academic Press. 93–98.
- 62. Yang SD, Wei YM, Qi PF, Zheng YL (2008) Sequence polymorphisms and phylogenetic relationships of hina Gene in wild barley from Tibet, China. Agri Sci China 7: 796–803.
- 63. Kahler AL, Allard RW (1981) Worldwide patterns of genetic variation among four esterase loci in barley (Hordeum vulgare L.). Theor Appl Genet 59: 101–111.
- 64. Zhang QF, Saghai Maroof MA, Allard RW (1990) Effects on adaptedness of variations in ribosomal DNA copy number in populations of wild barley (Hordeum vulgare ssp. spontaneum). Proc Natl Acad Sci USA 87: 8741–8745.
- 65. Zhang QF, Dai XK, Saghai Maroof MA (1992) Comparative assessment of genetic variation at 6 isozyme loci in barley from two centers of diversity: Ethiopia and Tibet. Acta Genet Sin 19: 236–243.
- 66. Zhang QF, Saghai-Maroof MA, Yang GP (1992) Ribosomal DNA polymorphisms and the Oriental-Occidental genetic differentiation in cultivated barley. Theor Appl Genet 84: 682–687.
- 67. Saghai-Maroof MA, Biyashev RM, Yang GP, Zhang QF, Allard RW (1994) Extraordinarily polymorphic microsatellite DNA in barley: species diversity, chromosomal locations, and population dynamics. Proc Natl Acad Sci USA 91: 5466–5470.
- 68. Ivandic V, Hackett CA, Nevo E, Keith R, Thomas WTB (2002) Analysis of simple sequence repeats (SSRs) in wild barley from the Fertile Crescent: associations with ecology, geography and flowering time. Plant Mol Biol 48: 511–527.
- 69. Wang AH, Yu ZY, Ding Y (2009) Genetic diversity analysis of wild close relatives of barley from Tibet and the Middle East by ISSR and SSR markers. C R Biologies 332: 393–403.
- 70. Kilian B, Okan H, Kohl J, Von HA, Barale F, et al. (2006) Haplotype structure at seven barley genes: relevance to gene pool bottlenecks, phylogeny of ear type and site of barley domestication. Mol Genet Genomics 276: 230–241.
- 71. Ellis RP, Forster BP, Robinson D, Handley LL, Gordon DC, et al. (2000) Wild barley: a source of genes for crop improvement in the 21st century. J Exp Bot 51: 9–17.
Nevo E (2004) Population genetic structure of wild barley and wheat in the Near East Fertile Crescent: regional and local adaptive. in: Gupta PK, Varshney (Eds.), Cereal Genomics. Kluwer Academic Publishers, 135–163.