Distribution of <Emphasis Type="Italic">Hordoindoline</Emphasis> genes in the genus <Emphasis Type="Italic">Hordeum</Emphasis>

Hordoindoline (Hin) genes, which are known to comprise Hina, Hinb-1, and Hinb-2, are associated with grain hardness in barley. However, the interspecific variation in the Hin genes in the genus Hordeum has not been studied in detail. We examined the variation in Hin genes and used it to infer the phylogenetic relationships between the genes found in two H. vulgare subspecies (cultivated barley and H. vulgare subsp. spontaneum) and 10 wild relatives (H. bogdanii, H. brachyantherum, H. bulbosum, H. chilense, H. comosum, H. marinum, H. murinum, H. patagonicum, H. pusillum, and H. roshevitzii). The Hina and Hinb genes of these species were amplified by PCR. We found two Hinb genes in three wild species (H. bogdanii, H. brachyantherum, and H. roshevitzii) and preliminarily named them Hinb-A and Hinb-B. Cluster analysis showed that the 17 Hinb genes present in Hordeum formed two distinct clusters (named A and B). Seven Hinb genes were included in Cluster-A, and 10 Hinb genes were included in Cluster-B. All Hinb-A genes were included in Cluster-A, while all of the Hinb-B genes were included in Cluster-B. In contrast, the Hinb-1 and Hinb-2 genes in H. vulgare were included in Cluster-B. These results suggest that the Hinb genes duplicated during the early stages of diversification in the genus Hordeum. On the other hand, the Hinb-1 and Hinb-2 genes in H. vulgare seem to have been generated by a duplication of the Hinb gene after the split of the lineages leading to H. vulgare and H. bulbosum.     

Ribosomal DNA repeat unit polymorphism in 25 Hordeum species

Summary Tandemly repeated DNA sequences containing structural genes encoding ribosomal RNA (rDNA) were investigated in 25 species of Hordeum using the wheat rDNA probe pTA71. The rDNA repeat unit lengths were shown to vary between 8.5 and 10.7 kb. The number of length classes (1–3) per accession generally corresponded to the number of nucleolar organizing regions (NORs). Intraspecific variation was found in H. parodii, H. spontaneum and H. leporinum, but not in H. bulbosum. Restriction analysis showed that the positions of EcoRI, SacI and certain BamHI cleavage sites in the rRNA structural genes were highly conserved, and that repeat unit length variation was generally attributable to the intergenic spacer region. Five rDNA BamHI restriction site maps corresponded to the following groups of species: Map A — H. murinum, H. glaucum, H. leporinum, H. bulbosum, H. marinum, H. geniculatum; Map B — H. leporinum; Map C — H. vulgare, H. spontaneum, H. agriocrithon; Map D — H. chilense, H. bogdanii; and Map E — remaining 14 Hordeum species. The repeat unit of H. bulbosum differed from all other species by the presence of a HindIII site. The closer relationship of H. bulbosum to H. leporinum, H. murinum and H. glaucum than to H. vulgare was indicated by their BamHI restriction maps.Contribution No. 1169, Plant Research Centre     

Phylogenetic analysis of the genus Hordeum using repetitive DNA sequences

A set of six cloned barley (Hordeum vulgare) repetitive DNA sequences was used for the analysis of phylogenetic relationships among 31 species (46 taxa) of the genus Hordeum, using molecular hybridization techniques. in situ hybridization experiments showed dispersed organization of the sequences over all chromosomes of H. vulgare and the wild barley species H. bulbosum, H. marinum and H. murinum. Southern blot hybridization revealed different levels of polymorphism among barley species and the RFLP data were used to generate a phylogenetic tree for the genus Hordeum. Our data are in a good agreement with the classification system which suggests the division of the genus into four major groups, containing the genomes I, X, Y, and H. However, our investigation also supports previous molecular studies of barley species where the unique position of H. bulbosum has been pointed out. In our experiments, H. bulbosum generally had hybridization patterns different from those of H. vulgare, although both carry the I genome. Based on our results we present a hypothesis concerning the possible origin and phylogeny of the polyploid barley species H. secalinum, H. depressum and the H. brachyantherum complex.     

Analysis of the genetic diversity of wild Spanish populations of the genusHordeum through the study of their endosperm proteins

A study was made of the genetic variability of 101 barley populations belonging to the four, wild, Spanish species of the genusHordeum (the autogamousH. marinum subspp.marinum andgussoneanum, andH. murinum subspp.murinum andleporinum, plus the allogamous speciesH. bulbosum andH. secalinum). Electrophoresis of endosperm proteins was performed using a large number of individuals from each population sampled, in order to determine intra- and interpopulational variation. The distribution of variability observed by population and taxonomic unit, is closely related to the breeding system.Hordeum bulbosum showed the greatest intrapopulational variability andH. marinum subsp.gussoneanum the least. In contrast to the autogamous species, the allogamous species showed low levels of interpopulational variation.     

Transferability and polymorphism of barley EST-SSR markers used for phylogenetic analysis in Hordeum chilense

Background

Hordeum chilense, a native South American diploid wild barley, is a potential source of useful genes for cereal breeding. The use of this wild species to increase genetic variation in cereals will be greatly facilitated by marker-assisted selection. Different economically feasible approaches have been undertaken for this wild species with limited direct agricultural use in a search for suitable and cost-effective markers. The availability of Expressed Sequence Tags (EST) derived microsatellites or simple sequence repeat (SSR) markers, commonly called as EST-SSRs, for barley (Hordeum vulgare) represents a promising source to increase the number of genetic markers available for the H. chilense genome.

Results

All of the 82 barley EST-derived SSR primer pairs tested for transferability to H. chilense amplified products of correct size from this species. Of these 82 barley EST-SSRs, 21 (26%) showed polymorphism among H. chilense lines. Identified polymorphic markers were used to test the transferability and polymorphism in other Poaceae family species with the aim of establishing H. chilense phylogenetic relationships. Triticum aestivum-H. chilense addition lines allowed us to determine the chromosomal localizations of EST-SSR markers and confirm conservation of the linkage group.

Conclusion

From the present study a set of 21 polymorphic EST-SSR markers have been identified to be useful for diversity analysis of H. chilense, related wild barleys like H. murinum, and for wheat marker-assisted introgression breeding. Across-genera transferability of the barley EST-SSR markers has allowed phylogenetic inference within the Triticeae complex.     

Comparisons of the hybrids Hordeum chilensexH. vulgare,H. chilensexH. bulbosum,H. chilensexSecale cereale and the amphidiploid of H. chilensexH. vulgare

Summary Diploid hybrids between Hordeum chilense and three other species, namely H. vulgare, H. bulbosum and Secale cereale, are described together with the amphidiploid of H. chilensexH. vulgare. Both the diploid hybrid and the amphidiploid of H. chilensexH. vulgare were chromosomally unstable, H. chilensexH. bulbosum was less so, while H. chilensexS. cereale was stable. Differential amphiplasty was found in all combinations. No homoeologous pairing was found in the Hordeum hybrids but in H. chilensexS. cereale there was chromosome pairing both within the two genomes and between the genomes.     

Pollen size in Hordeum L.: correlation between size,ploidy level,and breeding system

The size of pollen in the genus Hordeum (Poaceae) is correlated with ploidy level and breeding system. Generally, the pollen size increases with the ploidy level, and outbreeding species possess significantly larger pollen than inbreeders. In H. roshevitzii (2x), H. pusilplum (2x), H. murinum subsp. murinum (4x), and H. parodii (6x) pollen, heteromorphisms occur between the central and the lateral florets of the triplet. In all four taxa, pollen from florets in which the anthers are exserted is larger than pollen from florets where the anthers remain inside the floret. The biological consequences of heteromorphic pollen are discussed, and a model for the evolution of breeding strategies in Hordeum is suggested.     

The distribution, organization and evolution of two abundant and widespread repetitive DNA sequences in the genus Hordeum

The genomic organization and chromosomal distributions of two abundant tandemly repeated DNA sequences, dpTa1 and pSc119.2, were examined in six wild Hordeum taxa, representing the four basic genomes of the genus, by Southern and fluorescence in situ hybridization. The dpTa1 probe hybridized to between 30 and 60 sites on the chromosomes of all five diploid species studied, but hybridization patterns differed among the species. Hybridization of the pSc119.2 sequence to the chromosomes and Southern blots of digested DNA detected signals in Hordeum bulbosum, Hordeum chilense, Hordeum marinum and Hordeum murinum 4x, but not in Hordeum murinum 2x and Hordeum vulgare ssp. spontaneum. A maximum of one pSc119.2 signal was observed in the terminal or subterminal region of each chromosome arm in the species carrying this sequence. The species carrying the same I-genome differed in the presence (Hordeum bulbosum) or absence (Hordeum spontaneum) of pSc119.2. The presence of pSc119.2 in the tetraploid cytotype of Hordeum murinum, but its absence in the diploid cytotype, suggests that the tetraploid is not likely to be a simple autotetraploid of the diploid. Data about the inter- and intra-specific variation of the two independent repetitive DNA sequences give information about both the interrelationships of the species and the evolution of the repetitive sequences. Received: 17 March 1999 / Accepted: 16 June 1999     

Molecular characterization and diversity of the Pina and Pinb genes in cultivated and wild diploid wheat

Grain hardness is one of the most important characteristics of wheat quality. Soft endosperm is associated with the presence of two proteins in the wild form, puroindoline a and b. The puroindoline genes and their derived proteins are present in the putative wheat diploid ancestors which are thought to be the donors of the A, B and D genomes in common and durum wheat. In this study, we investigated the variability of grain hardness in einkorn, along with the nucleotide diversity of Pina and Pinb genes in a collection of einkorn wheat and T. urartu, in addition to studying the neutrality and linkage disequilibrium of these genes. Various alleles were detected for Pina and Pinb genes including three novel alleles for the Pinb locus: Pinb-A ^m 1i, Pinb-A ^m 1j and Pinb-A ^m 1k. Some differences were found in grain hardness between the different genotypes. The neutrality test showed a different pattern of variation between the two Pin genes. The genetic analysis of a diploid wheat collection has demonstrated that these species are a potential source of novel puroindoline variants. Our data suggest that, although further studies must be carried out, these variants could be used to expand the range of grain texture in durum and common wheat, which would permit the development of new materials adapted to novel uses in the baking and pasta industry.     

RAPD variation in wild populations of four species of the genus Hordeum (Poaceae)

 The genetic variation of 102 natural populations of wild barley growing in Spain was assessed using RAPDs (random amplified polymorphic DNA). The plant material included the annual species H. marinum subsp. marinum (22 populations) and subsp. gussoneanum (14), H. murinum subsp. murinum (7) and subsp. leporinum (35), and the perennial species H. bulbosum (17) and H. secalinum (7). Ten of the tested 64 arbitrary 10-mer primers amplified polymorphic DNA in all taxonomic units. Analyses was performed within and between populations, species and subspecies. The primers gave a total of 250 RAPD products. The level of polymorphism varied between taxonomic units depending on the primers employed and the plant reproductive system. In general, the most variable were the allogamous species H. secalinum and H. bulbosum and the autogamous H. marinum subsp. marinum. Among the amplified bands, 69 (27%) were shared by at least two different taxonomic units. The remaining bands were specific. The results demonstrate differences in the degree of similarity between taxonomic units. Jaccard’s similarity coefficients for interval measure within and between populations were used to produce a cluster diagram using the unweighted pair-group method (UPGMA). The different populations of the species and subspecies of Hordeum fell into three groups. The first group contained the populations belonging to both subspecies of H. marinum, plus those of H. secalinum. The populations of H. marinum subsp. gussoneanum were very closely associated. Those of H. marinum subsp. marinum were grouped in a broad cluster. The second group, occupying the innermost position of the tree, was very closely associated with the populations of both subspecies of H. murinum. The third branch segregated H. bulbosum. A series of RAPD markers were investigated by cleaving the amplified products of the same size with restriction endonucleases that recognize targets of 4- or 6-bp. The production of equivalent fragments following cleavage by the same enzyme would seem to demonstrate their homology in samples from different individuals, populations or taxonomic units. Received: 18 May 1997 / Accepted: 22 August 1997     

Chromosome 5H of <Emphasis Type="Italic">Hordeum</Emphasis> species involved in reduction in grain hardness in wheat genetic background

Grain hardness is an important factor affecting end-use quality in wheat. Mutations of the puroindoline genes, which are located on chromosome 5DS, control a majority of grain texture variations. Hordoindoline genes, which are the puroindoline gene homologs in barley, are located on chromosome 5HS and are also responsible for grain texture variation. In this study, we used three types of wheat–barley species (Hordeum vulgare, H. vulgare ssp. spontaneum, and H. chilense) chromosome addition lines and studied the effect of chromosome 5H of these species on wheat grain characteristics. The 5H chromosome addition lines showed significantly lower grain hardness and higher grain weight than the corresponding wheat parents. The effect of enhancing grain softness was largest in the wheat–H. chilense line regardless of having an increase in grain weight similar to those in the wheat–H. vulgare and wheat–H. spontaneum lines. Our results indicated that chromosome 5H of the Hordeum species plays a role in enhancing grain softness and increasing grain weight in the wheat genetic background, and the extent of effect on grain hardness depends on the type of Hordeum species. Protein analysis of hordoindolines indicated that profiles of 2D-electrophoresis of hordoindolines were different among Hordeum species and hordoindolines in the addition lines appeared to be most abundant in wheat–H. chilense line. The differences in enhancing grain softness among the Hordeum species might be attributed to the quantity of hordoindolines expressed in the 5H chromosome addition lines. These results suggested that the barley hordoindolines located on chromosome 5HS play a role in reducing grain hardness in the wheat genetic background.     

Genetic diversity in three perennial grasses from the Semipalatinsk nuclear testing region of Kazakhstan after long-term radiation exposure

The extent and structure of the genetic diversity of plant populations from the Semipalatinsk region of Kazakhstan, adjacent to a former major nuclear test site, have been studied using RAPD (Random Amplified Polymorphic DNA) marker analyses. The DNAs from three perennial species, Stipa capillata, Hordeum bogdanii, and Agropyron pectinatum, each collected from heavily (HPZ), moderately (MPZ) and lightly polluted zones (LPZ) have been analyzed using RAPDs. The results show a significantly higher level of variability in plants collected from the highest radiation pollution area compared with the moderately and lightly radiation contaminated zones for A. pectinatum and H. bogdanii. Variation was five times as higher in heavily exposed H. bogdanii, and two times higher in A. pectinatum populations compared to their lightly contaminated populations. H. bogdanii appears to be very sensitive to radiation and as such is a good indicator species for mapping radiation pollution at nuclear test sites or nuclear accidents.     

Electrophoretic Variation in Esterases, Peroxidases and Acid Phosphatases in Some Native Greek Taxa of the Genera Hordeum and Taeniatherum

Horizontal starch gel electrophoresis has been applied to thestudy of esterase, peroxidase and acid phosphatase patternsin seven taxa, namely Hordeum diploids (2n=14) (H. marinum,H. marinum I and H. hystrix), tetraploids (2n=28) (H. bulbosumand H. murinum subsp. leporinum) and Taeniatherum (2n=14) (T.caput-medusae and T. caput-medusae I) in order to elucidatetheir phylogenetic relationships. On the basis of our experimentalresults the seven taxa may be placed in the following threegroups; (1) diploid Hordeum (H. marinum, H. marinum I, H. hystrix);(2) tetraploid Hordeum (H. bulbosum, H. murinum subsp. leporinum);(3) Taeniatherum (T. caput-medusae, T. caput-medusae I). Esterase, peroxidase and acid phosphatase patterns of the twoHordeum diploid taxa (H. marinum and H. marinum I) are verysimilar suggesting their close phylogenetic relationship; thesame is true for both the taxa of the genus Taeniatherum (T.caput-medusae and T. caput-medusae I). The taxa of the Taeniatherumgroup compared with the diploid Hordeum (H. marinum, H. marinumI, H. hystrix) and the tetraploid Hordeum (H. bulbosum, H. murinumsubsp. leporinum) show a lower degree of phylogenetic relationshipand seem to be equally distant from them. The tetraploid Hordeumgroup shows a higher phylogenetic relationship with diploidHordeum group than with the Taeniatherum group. These results confirm that the genus Taeniatherum, previouslyconsidered as part of the genus Hordeum, should be regardedas a separate genus. Gramineae (Poaceae), Hordeum L., Taeniatherum Nevski., esterase, peroxidase and acid phosphatase patterns, phylogenetic relationships     

A barley Hordoindoline mutation resulted in an increase in grain hardness

Barley seed proteins, Hordoindolines, are homologues of wheat Puroindolines, which are associated with grain hardness. Barley Hordoindoline genes are known to comprise Hina and Hinb, and Hinb consists of two Hinb genes, Hinb-1 and Hinb-2. Two types of allele were found for Hina, Hinb-1 and Hinb-2 genes, respectively, among Japanese two- and six-rowed barley lines. One of the alleles of Hinb-2 (Hinb-2b) had a frame-shift mutation resulting in an in-frame stop codon. For two-rowed barley lines, grain hardness was significantly higher among lines with the Hinb-2b than those with the wild type Hinb-2 gene (Hinb-2a). Protein spots corresponding to HINa, HINb-1, and HINb-2 were identified by 2D-gel electrophoresis among barley lines with Hinb-2a. Among the lines with Hinb-2b, HINa and HINb-1 were expressed at similar levels as those in the wild type, but HINb-2 was not detected. A DNA (cleaved amplified polymorphic sequence) marker was developed to distinguish between the Hinb-2a and Hinb-2b gene sequences. Analysis of grain hardness among F₂ lines derived from a cross between a line with Hinb-2a (Shikoku hadaka 115) and a line with the Hinb-2b (Shikoku hadaka 84) showed significantly higher grain hardness in the mutant lines. From these results, the Hinb-2b frame-shift (null) mutation might play a critical role in barley grain hardness. The DNA marker will be useful in barley breeding to select lines having harder grain texture.     

Phylogenetic analysis in some Hordeum species (Triticeae; Poaceae) based on two single-copy nuclear genes encoding acetyl-CoA carboxylase

We investigate the phylogenetic relationship, and evolutionary history of 18 diploid and polyploid Hordeum species including 22 taxa based on two single-copy nuclear ACC1 and ACC2 genes using maximum parsimony, maximum likelihood, and Bayesian inference. The results of molecular phylogenetic analysis demonstrated genetic relationships among taxa and origin of polyploids. Our phylogenetic analyses revealed a clear alloploid origin of Hordeum capense, with Eurasian Hordeum marinum subsp. gussoneanum as the Xa genome donor and diploid Asian Hordeum roshevitzii as the H genome donor. The formation of hexaploid Hordeum lechleri likely involves hybridization between tetraploid Hordeum brachyantherum subsp. brachyantherum and a diploid possessing the I genome. The Acc1 and Acc2 gene data analyses suggested that Siberian Hordeum bogdanii might have be the common ancestor of the diploid New World Hordeum species. Perennial diploid South American species, Hordeum comosum was the first-diverging group within the clade of diploid American species in the analyses.     

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.     

Molecular evolution and phylogeny of the RPB2 gene in the genus Hordeum

Background and Aims

It is known that the miniature inverted-repeat terminal element (MITE) preferentially inserts into low-copy-number sequences or genic regions. Characterization of the second largest subunit of low-copy nuclear RNA polymerase II (RPB2) has indicated that MITE and indels have shaped the homoeologous RPB2 loci in the St and H genome of Eymus species in Triticeae. The aims of this study was to determine if there is MITE in the RPB2 gene in Hordeum genomes, and to compare the gene evolution of RPB2 with other diploid Triticeae species. The sequences were used to reconstruct the phylogeny of the genus Hordeum.

Methods

RPB2 regions from all diploid species of Hordeum, one tetraploid species (H. brevisubulatum) and ten accessions of diploid Triticeae species were amplified and sequenced. Parsimony analysis of the DNA dataset was performed in order to reveal the phylogeny of Hordeum species.

Key Results

MITE was detected in the Xu genome. A 27–36 bp indel sequence was found in the I and Xu genome, but deleted in the Xa and some H genome species. Interestingly, the indel length in H genomes corresponds well to their geographical distribution. Phylogenetic analysis of the RPB2 sequences positioned the H and Xa genome in one monophyletic group. The I and Xu genomes are distinctly separated from the H and Xa ones. The RPB2 data also separated all New World H genome species except H. patagonicum ssp. patagonicum from the Old World H genome species.

Conclusions

MITE and large indels have shaped the RPB2 loci between the Xu and H, I and Xa genomes. The phylogenetic analysis of the RPB2 sequences confirmed the monophyly of Hordeum. The maximum-parsimony analysis demonstrated the four genomes to be subdivided into two groups.Key words: Molecular evolution, RPB2, Hordeum, transposable element, phylogeny     

Allelic variation and distribution independence of Puroindoline b-B2 variants and their association with grain texture in wheat

In this study, we identify the allelic variation of the Pinb-B2v3 variant, which could be divided into three different alleles, Pinb-B2v3a, Pinb-B2v3b and Pinb-B2v3c. The result of χ² tests showed that the distribution of Puroindoline b-2 variants has different frequencies in common and durum wheats. Analysis of the association of Pinb-B2v with grain hardness indicated that wheat cultivars with Pinb-B2v3b possessed relatively higher single kernel characterization system (SKCS) hardness indices in soft wheat in the 2006–2007 cropping season. Further analysis of SKCS hardness among different Puroindoline B-b2 variants by an F₈ recombinant inbred line (RIL) population containing 350 RILs indicated that lines with Pinb-2v3b were on average 5.4 SKCS hardness index units harder than those carrying the Pinb-2v2 haplotype. Derived cleaved amplified polymorphic sequence markers were developed for identification of Pinb-B2v3b and Pinb-B2v3c alleles and will be useful for screening early generation materials by marker-assisted selection during wheat breeding. The results of quantitative real-time PCR indicated that the relative expression level of Pinb-B2v3b was significantly higher than those of Pinb-B2v2, Pinb-B2v3a and Pinb-B2v3c, that four Pinb-B2 alleles showed the highest relative expression level on the 14th day after anthesis during grain development, and that relative expression levels of Pinb-B2v3b and Pinb-B2v2 in leaf were significantly higher than those in root, suggesting that PINB-2 are possibly not seed-specific proteins and that the expression level of Pinb-B2v3 was possibly positively correlated with grain hardness.     

A review of the occurrence of Grain softness protein-1 genes in wheat (Triticum aestivum L.)

Grain softness protein-1 (Gsp-1) is a small, 495-bp intronless gene found throughout the Triticeae tribe at the distal end of group 5 chromosomes. With the Puroindolines, it constitutes a key component of the Hardness locus. Gsp-1 likely plays little role in grain hardness, but has direct interest due to its utility in phylogeny and its role in arabinogalactan peptides. Further role(s) remain to be identified. In the polyploid wheats, Triticum aestivum and T. turgidum, the gene is present in a homoeologous series. Since its discovery, there have been conflicting reports and data as to the number of Gsp-1 genes and the level of sequence polymorphism. Little is known about allelic variation within a species. In the simplest model, a single Gsp-1 gene is present in each wheat and Aegilops tauschii genome. The present review critically re-examines the published and some unpublished data (sequence available in the NCBI nucleotide and MIPS Wheat Genome Databases). A number of testable hypotheses are identified, and include the level of polymorphism that may represent (and define) different Gsp-1 alleles, the existence of a fourth Gsp-1 gene, and the apparent, at times, high level of naturally-occurring or artifactual gene chimeras. In summary, the present data provide firm evidence for at most, three Gsp-1 genes in wheat, although there are numerous data that suggest a more complex model.     

Unlocking the secondary gene‐pool of barley with next‐generation sequencing

Crop wild relatives (CWR) provide an important source of allelic diversity for any given crop plant species for counteracting the erosion of genetic diversity caused by domestication and elite breeding bottlenecks. Hordeum bulbosum L. is representing the secondary gene pool of the genus Hordeum. It has been used as a source of genetic introgressions for improving elite barley germplasm (Hordeum vulgare L.). However, genetic introgressions from H. bulbosum have yet not been broadly applied, due to a lack of suitable molecular tools for locating, characterizing, and decreasing by recombination and marker‐assisted backcrossing the size of introgressed segments. We applied next‐generation sequencing (NGS) based strategies for unlocking genetic diversity of three diploid introgression lines of cultivated barley containing chromosomal segments of its close relative H. bulbosum. Firstly, exome capture‐based (re)‐sequencing revealed large numbers of single nucleotide polymorphisms (SNPs) enabling the precise allocation of H. bulbosum introgressions. This SNP resource was further exploited by designing a custom multiplex SNP genotyping assay. Secondly, two‐enzyme‐based genotyping‐by‐sequencing (GBS) was employed to allocate the introgressed H. bulbosum segments and to genotype a mapping population. Both methods provided fast and reliable detection and mapping of the introgressed segments and enabled the identification of recombinant plants. Thus, the utilization of H. bulbosum as a resource of natural genetic diversity in barley crop improvement will be greatly facilitated by these tools in the future.