A DNA marker closely linked to the <Emphasis Type="Italic">vrs1</Emphasis> locus (row-type gene) indicates multiple origins of six-rowed cultivated barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.)

The origin of six-rowed cultivated barley was studied using a DNA marker cMWG699 closely linked to the vrs1 locus. Restriction patterns of the PCR-amplified product of the cMWG699 locus were examined in 280 cultivated (Hordeum vulgare ssp. vulgare) and 183 wild (H. vulgare ssp. spontaneum) barleys. Nucleotide sequences of the PCR products were also examined in selected accessions. Six-rowed cultivated barleys were divided into two distinct groups, types I and II. Type I six-rowed cultivated barley was distributed widely while type II six-rowed cultivated barley was found only in the Mediterranean region. The type I sequence was also found in a wild barley accession from Turkmenistan whereas the type II sequence was also found in a two-rowed cultivated barley from North Africa and a wild barley from Morocco. These results suggested that the six-rowed type I and II barleys were derived from two-rowed type I and II barleys, respectively, by independent mutations at the vrs1 locus. Received: 3 November 2000 / Accepted: 17 April 2001     

Genetic Diversity Analysis of Tibetan Wild Barley Using SSR Markers

One hundred and six accessions of wild barley collected from Tibet, China, including 50 entries of the two-rowed wild barley Hordeum vulgare ssp. spontaneum (HS), 29 entries of the six-rowed wild barley Hordeum vulgare ssp. agriocrithon (HA), and 27 entries of the six-rowed wild barley Hordeum vulgare ssp. agriocrithon var. lagunculiforme (HL), were analyzed using 30 SSR markers selected from the seven barley linkage groups for studying genetic diversity and evolutionary relationship of the three subspecies of Tibetan wild barley to cultivated barley in China. Over the 30 genetic loci that were studied, 229 alleles were identified among the 106 accessions, of which 70 were common alleles. H. vulgare ssp. spontaneum possesses about thrice more private alleles (2.83 alleles/locus) than HS (0.93 alleles/locus), whereas almost no private alleles were detected in HL. The genetic diversity among-subspecies is much higher than that within-subspecies. Generally, the genetic diversity among the three subspecies is of the order HS > HL > HA. Phylogenetic analysis of the 106 accessions showed that all the accessions of HS and HA was clustered in their own groups, whereas the 27 accessions of HL were separated into two groups (14 entries with group HS and the rest with group HA). This indicated that HL was an intermediate form between HS and HA. Based on this study and previous works, we suggested that Chinese cultivated barley might evolve from HS via HL to HA.     

Elucidation of the origin of ‘agriocrithon’ based on domestication genes questions the hypothesis that Tibet is one of the centers of barley domestication

Wild barley forms a two‐rowed spike with a brittle rachis whereas domesticated barley has two‐ or six‐rowed spikes with a tough rachis. Like domesticated barley, ‘agriocrithon’ forms a six‐rowed spike; however, the spike is brittle as in wild barley, which makes the origin of agriocrithon obscure. Haplotype analysis of the Six‐rowed spike 1 (vrs1) and Non‐brittle rachis 1 (btr1) and 2 (btr2) genes was conducted to infer the origin of agriocrithon barley. Some agriocrithon barley accessions (eu‐agriocrithon) carried Btr1 and Btr2 haplotypes that are not found in any cultivars, implying that they are directly derived from wild barley through a mutation at the vrs1 locus. Other agriocrithon barley accessions (pseudo‐agriocrithon) carried Btr1 or Btr2 from cultivated barley, thus implying that they originated from hybridization between six‐rowed landraces carrying btr1Btr2 and Btr1btr2 genotypes followed by recombination to produce Btr1Btr2. All materials we collected from Tibet belong to pseudo‐agriocrithon and thus do not support the Tibetan Plateau as being a center of barley domestication. Tracing the evolutionary history of these allelic variants revealed that eu‐agriocrithon represents six‐rowed barley lineages that were selected by early farmers, once in south‐eastern Turkmenistan (vrs1.a1) and again in the eastern part of Uzbekistan (vrs1.a4).     

Comparison of the internal transcribed spacer region (ITS) of the ribosomal RNA genes in wild and cultivated two and six-rowed barleys (Hordeum vulgare L.)

The ITS region of the ribosomal RNA genes from two and six-rowed cultivated barley (Hordeum vulgare subsp. distichon and H. v. subsp. hexastichon, respectively), and its two and six-rowed wild relatives (H. v. subsp. spontaneum and H. v. subsp. agriocrithon, respectively) was isolated and sequenced. The entire ITS region is 598 bp in the two-rowed taxa (H. v. subsp. distichon and H. v. subsp. spontaneum) and 599 bp in the six-rowed ones (agriochriton and hexastichon). The ITS1 is 217 bp in the six-rowed barleys (H. v. subsp. agriochriton and H. v. subsp. hexastichon) and 218 bp in the two-rowed barleys (H. v. subsp. distichon and H. v. subsp. spontaneum). The 5.8S region is 163 bp in all studied H. vulgare taxa. The ITS2 region is 217 bp in the two-rowed barleys (H. v. subsp. distichon and H. v. subsp. spontaneum) and 219 bp in the six rowed ones (H. v. subsp. hexastichon and H. v. subsp. agriochriton). The ITS sequence data of the studied taxa and that of three other wild Hordeum species (H. murinum, H. marinum and H. chilense) were aligned and a phylogeny tree was reconstructed using the Lasergene Program. H. v. subsp. spontaneum was appeared as the ancestor of all other H. vulgare taxa.     

Discovery and assay of single-nucleotide polymorphisms in barley (Hordeum vulgare)

The least ambiguous genetic markers are those based on completely characterized DNA sequence polymorphisms. Unfortunately, assaying allele states by allele sequencing is slow and cumbersome. The most desirable type of genetic marker would be unambiguous, inexpensive to assay and would be assayable singly or in parallel with hundreds of other markers (multiplexable). In this report we sequenced alleles at 54 barley (Hordeum vulgare ssp. vulgare) loci, 38 of which contained single-nucleotide polymorphisms (SNPs). Many of these 38 loci contained multiple polymorphisms, and a total of 112 polymorphisms were scored in five barley genotypes. The polymorphism data set was analyzed both by using the individual mutations as cladistic characters and by reducing data for each locus to haplotypes. We compared the informativeness of these two approaches by consensus tree construction and bootstrap analysis. Both approaches provided similar results. Since some of the loci sequenced contained insertion/deletion events and multiple point mutations, we thought that these multiple-mutated loci might represent old alleles that predated the divergence of barley from H. spontaneum. We evaluated sequences from a sample of H. spontaneum accessions from the Eastern Mediterranean, and observed similar alleles present in both cultivated barley and H. spontaneum, suggesting either multiple domestication events or multiple transfers of genes between barley and its wild ancestor.     

Distribution of MWG699 polymorphism in Spanish European barleys.

The STS marker MWG699/TaqI is closely linked to the vrs1 locus and has been proposed as a marker of domestication in barley. This study included 257 cultivated barleys of both two- and six-rowed varieties, mainly from the western Mediterranean region. These included many landraces from the Spanish barley core collection, Moroccan landraces, and a set of accessions from other European countries. Restriction analysis of amplified DNA revealed three alleles, as previously described. Most of the two-rowed entries had the same allele, type K. Six-rowed entries showed both types A and D. Indeed, type D was widespread among Spanish landraces and commercial varieties from central Europe. It was also found in some two-rowed landraces originating from Spain and Morocco. Barleys with the D haplotype were predominantly winter types, whereas the A haplotype was evenly distributed among spring and winter types. These results support the existence of two different genetic sources among six-rowed Spanish landraces.     

Mapping of powdery mildew resistance genes in a newly determined accession of Hordeum vulgare ssp. spontaneum

The accession PI466197 of wild barley (Hordeum vulgare ssp. spontaneum) with a newly identified resistance to powdery mildew caused by Blumeria graminis f.sp. hordei was studied with the aim to localise the genes determining resistance on a barley genetic map using DNA markers. Molecular analysis was performed in the F₂ population of the cross between the winter variety ‘Tiffany’ and the resistant accession PI466197, consisting of 113 plants. DNA markers, 17 simple sequence repeats (SSRs), four sequence-tagged sites (STSs) and one cleaved amplified polymorphic sequence (CAPS) marker developed from the Mla locus sequence were used for genetic mapping and a two-locus model of resistance was shown. One of the resistance genes originating from H. vulgare ssp. spontaneum PI466197 was localised between the markers RGH1aE1 and Bmac0213 on the short arm of chromosome 1H, which is the position consistent with the Mla locus. The other gene was proven to be highly significantly linked with GBMS247, Bmac0134 and MWG878 on the short arm of chromosome 2H. The flanking markers were Bmac0134 and MWG878, assigned 4 and 8 cM from the resistance gene, respectively. Until now, no gene conferring powdery mildew resistance originating from H. vulgare has been located on the short arm of barley chromosome 2H.     

A phylogenetic analysis based on nucleotide sequence of a marker linked to the brittle rachis locus indicates a diphyletic origin of barley

BACKGROUND AND AIMS: Barley (Hordeum vulgare ssp. vulgare) cultivation started between 9500 and 8400 years ago, and was a major part of ancient agriculture in the Near East. The brittle rachis is a critical trait in the domestication process. METHODS: A DNA sequence closely linked to the brittle rachis complex was amplified and resequenced in a collection of cultivated barleys, wild barleys (H. vulgare ssp. spontaneum) and weedy brittle rachis varieties (H. vulgare ssp. vulgare var. agriocrithon). The sequence was used to construct a phylogenetic tree. KEY RESULTS: The phylogeny separated the W- (btr1-carrying) from the E- (btr2-carrying) cultivars. The wild barleys had a high sequence diversity and were distributed throughout the W- and E-clades. Some of the Tibetan var. agriocrithon lines were closely related to the E-type and others to the W-type cultivated barleys, but an Israeli var. agriocrithon line has a complex origin. CONCLUSIONS: The results are consistent with a diphyletic origin of barley. The W- and E-type cultivars are assumed to have evolved from previously diverged wild barley via independent mutations at Btr1 and Btr2.     

Dialect-I,species-specific repeated DNA sequence from barley,Hordeum vulgare

Summary Dialect-1, species-specific repetitive DNA sequence of barley Hordeum vulgare, was cloned and analysed by Southern blot and in situ hybridization. Dialect-1 is dispersed through all barley chromosomes with copy number 5,000 per genome. Two DNA fragments related to Dialect-1 were revealed in phage library, subcloned and mapped. All three clones are structurally heterogenous and it is suggested that the full-length genomic repeat encompassing Dialect-1 is large in size. The Dialect-1 DNA repeat is represented in the genomes of H. vulgare and ssp. agriocrithon and spontaneum in similar form and copy number; it is present in rearranged form with reduced copy number in the genomes of H. bulbosum and H. murinum, and it is absent from genomes of several wild barley species as well as from genomes of wheat, rye, oats and maize. Dialect-1 repeat may be used as a molecular marker in taxonomic studies and for identification of barley chromosomes in interspecies hybrids.     

Analysis of the barley chromosome 2 region containing the six-rowed spike gene <Emphasis Type="Italic">vrs1</Emphasis> reveals a breakdown of rice–barley micro collinearity by a transposition

In cultivated barley (Hordeum vulgare ssp. vulgare), six-rowed spikes produce three times as many seeds per spike as do two-rowed spikes. The determinant of this trait is the Mendelian gene vrs1, located on chromosome 2H, which is syntenous with rice (Oryza sativa) chromosomes 4 and 7. We exploited barley–rice micro-synteny to increase marker density in the vrs1 region as a prelude to its map-based cloning. The rice genomic sequence, covering a 980 kb contig, identified barley ESTs linked to vrs1. A high level of conservation of gene sequence was obtained between barley chromosome 2H and rice chromosome 4. A total of 22 EST-based STS markers were placed within the target region, and the linear order of these markers in barley and rice was identical. The genetic window containing vrs1 was narrowed from 0.5 to 0.06 cM, which facilitated covering the vrs1 region by a 518 kb barley BAC contig. An analysis of the contig sequence revealed that a rice Vrs1 orthologue is present on chromosome 7, suggesting a transposition of the chromosomal segment containing Vrs1 within barley chromosome 2H. The breakdown of micro-collinearity illustrates the limitations of synteny cloning, and stresses the importance of implementing genomic studies directly in the target species. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

On the origin of Spanish two-rowed barleys

To investigate the phylogenetic origin of Spanish two-rowed barleys, we studied 44 accessions of old land-races both morphologically and biochemically to ascertain their similarity with 51 entries of old cultivars and land-races of widespread origin across Europe. They were also compared with 20 accessions of Hordeum spontaneum from the Mediterranean basin and other regions of its distribution range, 14 accessions of Moroccan cultivated six-rowed barley land-races, and different six-rowed Spanish and two-and six-rowed European cultivars. CM-(trypsin inhibitors and subunits of the barley tetrameric -amylase inhibitor) proteins and hordeins, all of which are endosperm proteins, were used as biochemical markers. The appearance of separate clusters of the Spanish barleys in the numerical classifications for both protein systems as a result of the existence of characteristic gene combinations that do not exist in entries from other origins permitted us to postulate the existence of local ancestors for most of the Spanish two-rowed barleys studied, and, therefore, a possible in situ domestication.     

应用微卫星标记研究西藏野生大麦的遗传多样性

以西藏不同地区的106份野生大麦为材料,其中包括50份野生二棱大麦（HS）,27份野生瓶形大麦（HL）和29份野生六棱大麦（HA）,用Liu等（1996）发表的SSR连锁图的每个连锁群的两个臂的不同位置上选取3～5个共30个SSR标记,研究了西藏3类野生大麦的遗传多样性。结果表明,这3类野生大麦在遗传组成及等位变异频率分布上存在着明显的遗传分化。在总样本中,共检测到229个等位变异,平均每个SSR位点检测到7．6个等位变异,其中70个为这3类野生大麦间共同的等位变异,等位变异数在这3类野生大麦间有明显的差异,亚种问的遗传多样性明显高于亚种内的遗传多样性。其遗传多样性大小顺序为HS〉HL〉HA。聚类分析表明,野生二棱大麦、野生六棱大麦分别聚在不同的两类,而野生瓶形大麦中各有约50％的材料分别聚在这两类。根据本研究及前人研究结果,我们认为中国栽培大麦是从野生二棱大麦经野生瓶形大麦向野生六棱大麦进化的。该结果支持了栽培大麦起源的“野生二棱大麦单系起源论”的观点。     

Genome-Wide Association Analysis of Aluminum Tolerance in Cultivated and Tibetan Wild Barley

Tibetan wild barley (Hordeum vulgare L. ssp. spontaneum), originated and grown in harsh enviroment in Tibet, is well-known for its rich germpalsm with high tolerance to abiotic stresses. However, the genetic variation and genes involved in Al tolerance are not totally known for the wild barley. In this study, a genome-wide association analysis (GWAS) was performed by using four root parameters related with Al tolerance and 469 DArT markers on 7 chromosomes within or across 110 Tibetan wild accessions and 56 cultivated cultivars. Population structure and cluster analysis revealed that a wide genetic diversity was present in Tibetan wild barley. Linkage disequilibrium (LD) decayed more rapidly in Tibetan wild barley (9.30 cM) than cultivated barley (11.52 cM), indicating that GWAS may provide higher resolution in the Tibetan group. Two novel Tibetan group-specific loci, bpb-9458 and bpb-8524 were identified, which were associated with relative longest root growth (RLRG), located at 2H and 7H on barely genome, and could explain 12.9% and 9.7% of the phenotypic variation, respectively. Moreover, a common locus bpb-6949, localized 0.8 cM away from a candidate gene HvMATE, was detected in both wild and cultivated barleys, and showed significant association with total root growth (TRG). The present study highlights that Tibetan wild barley could provide elite germplasm novel genes for barley Al-tolerant improvement.     

Analysis of genetic diversity of hordein in wild close relatives of barley from Tibet

We analyzed genetic diversity in the storage protein hordein encoded at Hor-1, Hor-2 and Hor-3 loci in seeds from 211 accessions of wild close relatives of barley, Hordeum vulgare ssp. agriocrithon and H. vulgare ssp. spontaneum. Altogether 32, 27 and 13 different phenotypes were found for Hor-1, Hor-2 and Hor-3, respectively. A comparison of our results with those of previous studies indicates that Tibetan samples reflect the highest diverse level of hordein phenotypes when compared to samples from Israel and Jordan. This high degree of polymorphism supports the hypothesis that Tibet is one of the original centers of H. vulgare L.Communicated by H.F. Linskens     

Cytogenetic diversity of SSR motifs within and between Hordeum species carrying the H genome: H. vulgare L. and H. bulbosum L.

Non-denaturing FISH (ND-FISH) was used to compare the distribution of four simple sequence repeats (SSRs)—(AG) _n , (AAG) _n , (ACT) _n and (ATC) _n —in somatic root tip metaphase spreads of 12 barley (H. vulgare ssp. vulgare) cultivars, seven lines of their wild progenitor H. vulgare ssp. spontaneum, and four lines of their close relative H. bulbosum, to determine whether the range of molecular diversity shown by these highly polymorphic sequences is reflected at the chromosome level. In both, the cultivated and wild barleys, clusters of AG and ATC repeats were invariant. In contrast, clusters of AAG and ACT showed polymorphism. Karyotypes were prepared after the identification of their seven pairs of homologous chromosomes. Variation between these homologues was only observed in one wild accession that showed the segregation of a reciprocal translocation involving chromosomes 5H and 7H. The two subspecies of H. vulgare analysed were no different in terms of their SSRs. Only AAG repeats were found clustered strongly on the chromosomes of all lines of H. bulbosum examined. Wide variation was seen between homologous chromosomes within and across these lines. These results are the first to provide insight into the cytogenetic diversity of SSRs in barley and its closest relatives. Differences in the abundance and distribution of each SSR analysed, between H. vulgare and H. bulbosum, suggest that these species do not share the same H genome, and support the idea that these species are not very closely related. Southern blotting experiments revealed the complex organization of these SSRs, supporting the findings made with ND-FISH.     

The Horn of Africa as a centre of barley diversification and a potential domestication site

According to a widely accepted theory on barley domestication, wild barley (Hordeum vulgare ssp. spontaneum) from the Fertile Crescent is the progenitor of all cultivated barley (H. vulgare ssp. vulgare). To determine whether barley has undergone one or more domestication events, barley accessions from three continents have been studied (a) using 38 nuclear SSR (nuSSRs) markers, (b) using five chloroplast SSR (cpSSR) markers yielding 5 polymorphic loci and (c) by detecting the differences in a 468 bp fragment from the non-coding region of chloroplast DNA. A clear separation was found between Eritrean/Ethiopian barley and barley from West Asia and North Africa (WANA) as well as from Europe. The data from chloroplast DNA clearly indicate that the wild barley (H. vulgare ssp. spontaneum) as it is found today in the “Fertile Crescent” might not be the progenitor of the barley cultivated in Eritrea (and Ethiopia). Consequently, an independent domestication might have taken place at the Horn of Africa. Jihad Orabi and Gunter Backes have contributed equally to this work.     

Characterization and mapping of a Prbs gene controlling spike development in Hordeum vulgare L.

The barley mutant, poly-row-and-branched spike (prbs) showed altered inflorescence morphology: complete conversion of the rudimentary lateral spikelets in two-rowed barley into fully developed fertile spikelets similar to the six-rowed phenotype, and additional spikelets in the middle of spike. Moreover, branched spikes emerged in progeny from a cross between the mutant and a six-rowed barley cultivar. Morphological observation of the development of immature spikes of the mutant and descendants with branched spikes showed that the Prbs gene is involved in spikelet development in the triple-mound stage. In mutant prbs, new meristems initiated at the flanks of lateral spikelets and middle spikelet meristems were converted to branch meristems, developing branched spikes. These observations suggested that the Prbs gene plays a crucial role in spikelet initiation and identity maintenance. The Prbs gene may be an important modifier in inflorescence differentiation from a panicle into a spike. The branched spikes emerging in hybrids from a cross between the mutant and six-rowed barley cultivar were not conferred by the gene vrs1 or Int-c, which decide spike morphology in six-rowed barley. These results imply that although six-row genes vrs1 and Int-c and prbs have similar effects on lateral spikelet development, they have different functions in branched spikes. The Prbs gene was mapped to chromosome 3H between SSR marker Bmag0023 and marker Cbic60 at a genetic distance of 3.3 and 5.4 centimorgans (cM), respectively.     

Comparative high resolution map of the six-rowed spike locus 1 (vrs1) in several populations of barley, Hordeum vulgare L

Multiple alleles at the vrs1 locus control the development and fertility of the lateral spikelets of barley (Hordeum vulgare L.), which is a key character in the study of yield, utilization and domestication. In this study, six linkage maps of the vrs1 locus were constructed, using different mapping populations developed from nine different barley cultivars (H. vulgare subsp. vulgare) or mutant and wild barley (H. vulgare subsp. spontaneum). A total of 8387 chromosomes (gametes) were sampled for analysis based on a hypothesis that orders of marker loci were the same over the different parental lines. The results showed that four markers and the vrs1 locus in all cases were arranged in the same order, which was in a good agreement with the hypothesis. This makes the linkage maps suitable for the positional cloning of the alleles at the vrs1 locus.     

Haplotype diversity and population structure in cultivated and wild barley evaluated for Fusarium head blight responses

Fusarium head blight (FHB) is a threat to barley (Hordeum vulgare L.) production in many parts of the world. A number of barley accessions with partial resistance have been reported and used in mapping experiments to identify quantitative trait loci (QTL) associated with FHB resistance. Here, we present a set of barley germplasm that exhibits FHB resistance identified through screening a global collection of 23,255 wild (Hordeum vulgare ssp. spontaneum) and cultivated (Hordeum vulgare ssp. vulgare) accessions. Seventy-eight accessions were classified as resistant or moderately resistant. The collection of FHB resistant accessions consists of 5, 27, 46 of winter, wild and spring barley, respectively. The population structure and genetic relationships of the germplasm were investigated with 1,727 Diversity Array Technology (DArT) markers. Multiple clustering analyses suggest the presence of four subpopulations. Within cultivated barley, substructure is largely centered on spike morphology and growth habit. Analysis of molecular variance indicated highly significant genetic variance among clusters and within clusters, suggesting that the FHB resistant sources have broad genetic diversity. The haplotype diversity was characterized with DArT markers associated with the four FHB QTLs on chromosome 2H bin8, 10 and 13 and 6H bin7. In general, the wild barley accessions had distinct haplotypes from those of cultivated barley. The haplotype of the resistant source Chevron was the most prevalent in all four QTL regions, followed by those of the resistant sources Fredrickson and CIho4196. These resistant QTL haplotypes were rare in the susceptible cultivars and accessions grown in the upper Midwest USA. Some two- and six-rowed accessions were identified with high FHB resistance, but contained distinct haplotypes at FHB QTLs from known resistance sources. These germplasm warrant further genetic studies and possible incorporation into barley breeding programs.