Locating introgressions of Hordeum bulbosum chromatin within the H. vulgare genome

Several disease-resistant recombinants between barley (Hordeum vulgare) and bulbous barley grass (H. bulbosum) have been obtained in recent years, but the process of characterization is often laborious and time-consuming. In order to improve the identification and chromosomal location of introgressed chromatin from H. bulbosum into the barley genome, we employed sequential genomic in situ hybridization (GISH) and fluorescence in situ hybridization (FISH). GISH enabled us to establish that an introgression was present in the disease-resistant recombinant line, and the subsequent use of FISH, with a short oligonucleotide sequence as probe, allowed us to locate the introgression on the long arm of barley chromosome 2H. These data were confirmed using RFLP probes that hybridize to barley chromosome 2HL. Received: 16 December 1998 / Accepted: 12 April 1999     

Characterisation of progeny from backcrosses of triploid hybrids between Hordeum vulgare L. (2x) and H. bulbosum L (4x) to H. vulgare

Interspecific hybridisations between Hordeum vulgare L. (cultivated barley) and H. bulbosum L. (bulbous barley grass) have been carried out to transfer desirable traits, such as disease resistance, from the wild species into barley. In this paper we report the results of an extensive backcrossing programme of triploid hybrids (H. vulgare 2x x H. bulbosum 4x) to two cultivars of H. vulgare. Progenies were characterised cytologically and by restriction fragment length polymorphism analysis and comprised (1) haploid and diploid H. vulgare plants, (2) hybrids and aneuploids, (3) single and double monosomic substitutions of H. bulbosum chromosomes into H. vulgare and (4) chromosomal rearrangements and recombinants. Five out of the seven possible single monosomic chromosome substitutions have now been identified amongst backcross progeny and will be valuable for directed gene introgression and genome homoeology studies. The presence amongst progeny of 1 plant with an H. vulgare-H. bulbosum translocated chromosome and one recombinant indicates the value of fertile triploid hybrids for interspecific gene introgression.     

Identification and mapping of a new leaf stripe resistance gene in barley (Hordeum vulgare L.)

Pyrenophora graminea is the seed-borne pathogen causal agent of barley leaf stripe disease. Near-isogenic lines (NILs) carrying resistance of the cv ”Thibaut” against the highly virulent isolate Dg2 were obtained by introgressing the resistance into the genetic background of the susceptible cv ”Mirco”. The segregation of the resistance gene was followed in a F₂ population of 128 plants as well as on the F₃ lines derived from the F₂ plants; the segregation fitted the 1:2:1 ratio for a single gene. By using NILs, a RAPD marker associated with the resistance gene was identified; sequence-specific (STS) primers were designed on the basis of the amplicon sequence and a RILs mapping population with an AFLP-based map were used to position this molecular marker to barley chromosome 1 S (7HS). STS and CAPS markers were developed from RFLPs mapped to the telomeric region of barley chromosome 7HS and three polymorphic PCR-based markers were developed. The segregation of these markers was followed in the F₂ population and their map position with respect to the resistance gene was determined. Our results indicate that the Thibaut resistance gene, which we designated as Rdg2a, maps to the telomeric region of barley chromosome 7HS and is flanked by the markers OPQ-9₇₀₀ and MWG 2018 at distances of 3.1 and 2.5 cM respectively. The suitability of the PCR-based marker MWG2018 in selection- assisted barley breeding programs is discussed. Received: 22 June 2000 / Accepted: 16 October 2000     

Localization of quantitative trait loci (QTL) for agronomic important characters by the use of a RFLP map in barley (Hordeum vulgare L.)

Two hundred and fifty doubled haploid lines were studied from a cross between two 2-row winter barley varieties. The lines were evaluated for several characters in a field experiment for 3 years on two locations with two replications. From a total of 431 RFLP probes 50 were found to be polymorphic and subsequently used to construct a linkage map. Quantitative trait loci (QTLs) were determined and localized for resistance against Rhynchosporium secalis and Erysiphe graminis, for lodging, stalk breaking and ear breaking tendency, for the physical state before harvest, plant height, heading date, several kernel parameters and kernel yield. The heritability of the traits ranged from 0.56 to 0.89. For each trait except for kernel thickness, QTLs have been localized that explain 5–52% of the genetic variance. Transgressive segregation occurred for all of the traits studied.     

Rph22: mapping of a novel leaf rust resistance gene introgressed from the non-host Hordeum bulbosum L. into cultivated barley (Hordeum vulgare L.)

A resistance gene (Rph22) to barley leaf rust caused by Puccinia hordei was introgressed from the non-host species Hordeum bulbosum into cultivated barley. The H. bulbosum introgression in line ‘182Q20’ was located to chromosome 2HL using genomic in situ hybridisation (GISH). Using molecular markers it was shown to cover approximately 20 % of the genetic length of the chromosome. The introgression confers a very high level of resistance to P. hordei at the seedling stage that is not based on a hypersensitive reaction. The presence of the resistance gene increased the latency period of the leaf rust fungus and strongly reduced the infection frequency relative to the genetic background cultivar ‘Golden Promise’. An F₂ population of 550 individuals was developed and used to create a genetic map of the introgressed region and to determine the map position of the underlying resistance gene(s). The resistance locus, designated Rph22, was located to the distal portion of the introgression, co-segregating with markers H35_26334 and H35_45139. Flanking markers will be used to reduce the linkage drag, including gene(s) responsible for a yield penalty, around the resistance locus and to transfer the gene into elite barley germplasm. This genetic location is also known to harbour a QTL (Rphq2) for non-hypersensitive leaf rust resistance in the barley cultivar ‘Vada’. Comparison of the ‘Vada’ and H. bulbosum resistances at this locus may lead to a better understanding of the possible association between host and non-host resistance mechanisms.     

The transfer of a powdery mildew resistance gene from Hordeum bulbosum L to barley (H. vulgare L.) chromosome 2 (2I)

Hordeum bulbosum L. is a source of disease resistance genes that would be worthwhile transferring to barley (H. vulgare L.). To achieve this objective, selfed seed from a tetraploid H. vulgare x H. bulbosum hybrid was irradiated. Subsequently, a powdery mildew-resistant selection of barley phenotype (81882/83) was identified among field-grown progeny. Using molecular analyses, we have established that the H. bulbosum DNA containing the powdery mildew resistance gene had been introgressed into 81882/83 and is located on chromosome 2 (2I). Resistant plants have been backcrossed to barley to remove the adverse effects of a linked factor conditioning triploid seed formation, but there remains an association between powdery mildew resistance and non-pathogenic necrotic leaf blotching. The dominant resistance gene is allelic to a gene transferred from H. bulbosum by co-workers in Germany, but non-allelic to all other known powdery mildew resistance genes in barley. We propose Mlhb as a gene symbol for this resistance.     

Molecular mapping of the Oregon Wolfe Barleys: a phenotypically polymorphic doubled-haploid population

A phenotypically polymorphic barley (Hordeum vulgare L.) mapping population was developed using morphological marker stocks as parents. Ninety-four doubled-haploid lines were derived for genetic mapping from an F₁ using the Hordeum bulbosum system. A linkage map was constructed using 12 morphological markers, 87 restriction fragment length polymorphism (RFLP), five random amplified polymorphic DNA (RAPD), one sequence-tagged site (STS), one intron fragment length polymorphism (IFLP), 33 simple sequence repeat (SSR), and 586 amplified fragment length polymorphism (AFLP) markers. The genetic map spanned 1,387 cM with an average density of one marker every 1.9 cM. AFLP markers tended to cluster on centromeric regions and were more abundant on chromosome 1 (7H). RAPD markers showed a high level of segregation distortion, 54% compared with the 26% observed for AFLP markers, 27% for SSR markers, and 18% for RFLP markers. Three major regions of segregation distortion, based on RFLP and morphological markers, were located on chromosomes 2 (2H), 3 (3H), and 7 (5H). Segregation distortion may indicate that preferential gametic selection occurred during the development of the doubled-haploid lines. This may be due to the extreme phenotypes determined by alleles at morphological trait loci of the dominant and recessive parental stocks. Several molecular markers were found to be closely linked to morphological loci. The linkage map reported herein will be useful in integrating data on quantitative traits with morphological variants and should aid in map-based cloning of genes controlling morphological traits. Received: 23 August 2000 / Accepted: 15 December 2000     

Marker development and characterisation of Hordeum bulbosum introgression lines: a resource for barley improvement

A set of 110 diploid putative introgression lines (ILs) containing chromatin introgressed from the undomesticated species Hordeum bulbosum L. (bulbous barley grass) into cultivated barley (Hordeum vulgare L.) has been identified using a high-copy number retrotransposon-like PCR marker, pSc119.1, derived from rye (Secale cereale L.). To evaluate these lines, 92 EST-derived markers were developed by marker sequencing across four barley cultivars and four H. bulbosum genotypes. Single nucleotide polymorphisms and insertions/deletions conserved between the two species were then used to develop a set of fully informative cleaved amplified polymorphic sequence markers or size polymorphic insertion/deletion markers. Introgressed chromatin from H. bulbosum was confirmed and genetically located in 88 of these lines using 46 of the EST-derived PCR markers. A total of 96 individual introgressions were detected with most of them (94.8%) extending to the most distal marker for each respective chromosome arm. Introgressions were detected on all chromosome arms except chromosome 3HL. Interstitial or sub-distal introgressions also occurred, with two located on chromosome 2HL and one each on 3HS, 5HL and 6HS. Twenty-two putative ILs that were positive for H. bulbosum chromatin using pSc119.1 have not had introgressions detected with these single-locus markers. When all introgressions are combined, more than 36% of the barley genetic map has now been covered with introgressed chromatin from H. bulbosum. These ILs represent a significant germplasm resource for barley improvement that can be mined for diverse traits of interest to barley breeders and researchers. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Localising QTLs for leaf rust resistance and agronomic traits in barley (Hordeum vulgare L.)

The Hordeum vulgare accession ’HOR 1063’ was crossed with the barley cultivar Krona, and 220 doubled haploid lines were produced based on this cross. A molecular map was constructed based on RFLP markers. Field trials were performed over 2 years and at two locations. In field trials, resistance to leaf rust by means of artificial infection, heading date, plant height and Kernel weight were assessed. For leaf rust resistance, 4 QTLs were localised, that explained 96.1% of the genetic variation. One QTL on chromosome 4H confirmed a position found in another genetic background and one mapped to the same position as Rph16 on chromosome 2H. All digenic effects decreased the effects of the respective QTLs. In addition to the denso-locus and the hex-v locus, other QTLs influencing heading date, plant length and kernel weight were found in this cross. Received: 16 July 1999 / Accepted: 7 September 1999     

Utility of barley and wheat simple sequence repeat (SSR) markers for genetic analysis of Hordeum chilense and tritordeum

A selection of 36 wheat and 35 barley simple sequence repeat markers (SSRs) were studied for their utility in Hordeum chilense. Nineteen wheat and nineteen barley primer pairs amplified consistent H. chilense products. Nine wheat and two barley SSRs were polymorphic in a H. chilense mapping population, producing codominant markers that mapped to the expected homoeologous linkage groups in all but one case. Thirteen wheat and 10 barley primer pairs were suitable for studying the introgression of H. chilense into wheat because they amplified H. chilense products of distinct size. Analysis of wheat/H. chilense addition lines showed that the H. chilense products derived from the expected homoeologous linkage groups. The results showed that wheat and barley SSRs provide a valuable resource for the genetic characterization of H. chilense, tritordeums and derived introgression lines. Received: 20 November 2000 / Accepted: 12 April 2001     

Mapping of Rym14 Hb,a gene introgressed from Hordeum bulbosum and conferring resistance to BaMMV and BaYMV in barley

Hordeum bulbosum represents the secondary gene pool of barley and constitutes a potential source of various disease resistances in barley breeding. Interspecific crosses of H. vulgare × H. bulbosum resulted in recombinant diploid-barley progeny with immunity to BaMMV after mechanical inoculation. Tests on fields contaminated with different viruses demonstrated that resistance was effective against all European viruses of the soil-borne virus complex (BaMMV, BaYMV-1, -2). Genetic analysis revealed that resistance was dominantly inherited. Marker analysis in a F5 mapping family was performed to map the introgression in the barley genome and to estimate its size after several rounds of recombination. RFLP anchor-marker alleles indicative of an H. bulbosum introgression were found to cover an interval 2.9 cM in length on chromosome 6HS. The soil-borne virus resistance locus harboured by this introgressed segment was designated Rym14^Hb. For marker-assisted selection of Rym14^Hb carriers, a diagnostic codominant STS marker was derived from an AFLP fragment amplified from leaf cDNA of homozygous-resistant genotypes inoculated with BaMMV.Communicated by F. Salamini     

Development and genetic mapping of 127 new microsatellite markers in barley

To enhance the marker density of existing genetic maps of barley (Hordeum vulgare L.), a new set of microsatellite markers containing dinucleotide motifs was developed from genomic clones. Out of 254 primer pairs tested, a total of 167 primer pairs were classifed as functional in a panel of six barley cultivars and three H. spontaneum accessions, and of those, 127 primer pairs resulting in 133 loci were either mapped or located onto chromosomes. The polymorphism information content (PIC) ranged from 0.05 to 0.94 with an average of 0.60. The number of alleles per locus varied from 1 to 9. On average, 3.9 alleles per primer pair were observed. The RFLP frameworks of two previously published linkage maps were used to locate a total of 115 new microsatellite loci on at least one mapping population. The chromosomal assignment of 48 mapped loci was corroborated on a set of wheat-barley chromosome addition lines; 18 additional loci which were not polymorphic in the mapping populations were assigned to chromosomes by this method. The microsatellites were located on all seven linkage groups with four significant clusters in the centromeric regions of 2H, 3H, 6H and 7H. These newly developed microsatellites improve the density of existing barley microsatellite maps and can be used in genetic studies and breeding research.Communicated by G. Wenzel     

Molecular linkage mapping in rye (Secale cereale L.)

A rye linkage map containing clones from rye, wheat, barley, oat and rice genomic and cDNA libraries, known-function genes and microsatellite markers, was created using an F₂ population consisting of 110 F₂-derived F₃ families. Both co-dominant and dominant markers were added to the map. Of all probes screened, 30.8% were polymorphic, and of those polymorphic 79.3% were mapped. The current map contains 184 markers present in all seven linkage groups covering only 727.3 cM. This places a marker about every 3.96 cM on average throughout the map; however, large gaps are still present. The map contains 60 markers that have been integrated from previous rye maps. Surprisingly, no markers were placed between the centromere and C1–1RS in the short arm of 1R. The short arm of chromosome 4 also lacked an adequate number of polymorphic markers. The population showed a remarkable degree of segregation distortion (72.8%). In addition, the genetic distance observed in rye was found to be very different among the maps created by different mapping populations. Received: 10 January 2000 / Accepted: 26 May 2000     

Genetic and geographic variation of bulbous barley (<Emphasis Type="Italic">Hordeum bulbosum</Emphasis> L.) assessed by RAPD markers

Genetic relationships among 21 barley accessions (17 of bulbous barley H. bulbosum L. and 4 of cultivated barley (H. vulgare L.) collected from different part of Turkey were investigated using Random Amplified Polymorphic DNA (RAPD). Eleven informative primers amplified 111 markers of which 98 (89.8%) were polymorphic. A dendogram was constructed using the UPGMA method based on the RAPD markers. The range of genetic similarity was from 0.111 to 0.815. The accessions were grouped into two main clusters based on the molecular data. The H. vulgare and H. bulbosum separated into two groups in the principle component analysis. The text was submitted by the authors in English.     

A barley RFLP map: alignment of three barley maps and comparisons to Gramineae species

Several gene linkage maps have been produced for cultivated barley. We have produced a new linkage map for barley, based on a cross between Hordeum vulgare subsp. spontaneum and Hordeum vulgare subsp. vulgare (Hvs x Hvv), having a higher level of polymorphism than most of the previous barley crosses used for RFLP mapping. Of 133 markers mapped in the Hvs x Hvv F₂ population, 69 were previously mapped on other barley maps, and 26 were mapped in rice, maize, or wheat. Two known gene clones were mapped as well as two morphological markers. The distributions of previously mapped markers were compared with their respective barley maps to align the different maps into one consensus map. The distributions of common markers among barley, wheat, rice and maize were also compared, indicating colinear linkage groups among these species.To be considered dual first authorsPublished with the approval of the Director of the Colorado State University/Agricultural Experiment Station.     

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.     

Construction of an RFLP map of barley

Summary In order to construct an RFLP map of barley, two populations were analyzed using 251 genomic and cDNA markers: one population comprised 71 F₁ antherderived double haploid (DH) individuals of an intraspecific cross (IGRI x FRANKA), and the other 135 individuals of an interspecific F₂/F₃ progeny (VADA x H. spontaneum). The distribution of nonrepetitive clones over the seven barley chromosomes revealed a maximum for chromosome 2H and a minimum for 6H. The polymorphism of the interspecific progeny (76%) clearly exceeded that of the intraspecific progeny (26%) although, based on their pedigrees, IGRI and FRANKA are only distantly related. The contribution of individual chromosomes of the DH parents to the overall polymorphism varied between 8% and 50%. A significant portion (44% versus 10% of the interspecific progeny) of the markers mapped on the DH offspring showed distorted segregation, caused mainly by the prevalence of variants originating from the parent that better responded to in vitro culture (IGRI). In contrast to the interspecific map, probes displaying skewed segregation were clustered on the DH map on discrete segments. The colinear arrangement of both maps covers a distance of 1,453 cM and identifies regions of varying map distances.     

Genetic linkage mapping in aspen (Populus tremula L. and Populus tremuloides Michx.)

A large number of simple sequence repeat (SSR) marker-containing genetic maps are available for several Populus species. For aspen however, no SSR-containing map has been published so far. In this study, genetic linkage mapping was carried out with an interspecific mapping pedigree of 61 full-sib hybrids of European × quaking aspen (Populus tremula L. × Populus tremuloides Michx.), using the two-way pseudo-testcross strategy. Amplified fragment-length polymorphism (AFLP) and SSR markers were used for mapping, resulting in the first SSR-containing genetic linkage maps for aspen. The maps allow comparisons with a Populus consensus map and other published genetic maps of the genus Populus. The maps showed good collinearity to each other and to the Populus consensus map and provide a direct link to the Populus trichocarpa genomic sequence. Sex as a morphological trait was assessed in the mapping population and mapped on a non-terminal position of linkage group XIX on the male P. tremuloides map.     

The distribution of RFLP markers on chromosome 2(2H) of barley in relation to the physical and genetic location of 5S rDNA

The 5S rDNA locus on the long arm of barley chromosome 2(2H) was genetically mapped in two crosses in relation to 30 other RFLP loci. Comparison of the genetic maps with the previously published physical position of the 5S rDNA, determined by in-situ hybridization, showed that there was a marked discrepancy between physical and genetic distance in both crosses, with recombination being less frequent in the proximal part of the arm. Pooled information from the present study and other published genetic maps showed that at least 26 of the 44 (59%) RFLPs that have been mapped on 2(2H)L lie distal to the 5S rDNA locus even though this region is only 27% of the physical length of the arm. The distribution of RFLP markers is significantly different from expected (P < 0.01), implying that the low-copy sequences used for RFLP analysis occur more frequently in distal regions of the arm and, or, that sequences in distal regions are more polymorphic.     

Comparison of wheat physical maps with barley linkage maps for group 7 chromosomes

Comparative genetic maps among the Triticeae or Gramineae provide the possibility for combining the genetics, mapping information and molecular-marker resources between different species. Dense genetic linkage maps of wheat and barley, which have a common array of molecular markers, along with deletion-based chromosome maps of Triticum aestivum L. will facilitate the construction of an integrated molecular marker-based map for the Triticeae. A set of 21 cDNA and genomic DNA clones, which had previously been used to map barley chromosome 1 (7H), were used to physically map wheat chromosomes 7A, 7B and 7D. A comparative map was constructed to estimate the degree of linkage conservation and synteny of chromosome segments between the group 7 chromosomes of the two species. The results reveal extensive homoeologies between these chromosomes, and the first evidence for an interstitial inversion on the short arm of a barley chromosome compared to the wheat homoeologue has been obtained. In a cytogenetically-based physical map of group 7 chromosomes that contain restriction-fragment-length polymorphic DNA (RFLP) and random amplified polymorphic DNA (RAPD) markers, the marker density in the most distal third of the chromosome arms was two-times higher than in the proximal region. The recombination rate in the distal third of each arm appears to be 8–15 times greater than in the proximal third of each arm where recombination of wheat chromosomes is suppressed.