A. Kilian

A molecular, isozyme and morphological map of the barley (Hordeum vulgare) genome

A map of the barley genome consisting of 295 loci was constructed. These loci include 152 cDNA restriction fragment length polymorphism (RFLP), 114 genomic DNA RFLP, 14 random amplified polymorphic DNA (RAPD), five isozyme, two morphological, one disease resistance and seven specific amplicon polymorphism (SAP) markers. The RFLP-identified loci include 63 that were detected using cloned known function genes as probes. The map covers 1,250 centiMorgans (cM) with a 4.2 cM average distance between markers. The genetic lengths of the chromosomes range from 124 to 223 cM and are in approximate agreement with their physical lengths. The centromeres were localized to within a few markers on all of the barley chromosomes except chromosome 5. Telomeric regions were mapped for the short (plus) arms of chromosomes 1, 2 and 3 and the long (minus) arm of chromosomes 7.

The barley stem rust-resistance gene Rpg1 is a novel disease-resistance gene with homology to receptor kinases.

Stem rust caused by Puccinia graminis f. sp. tritici was among the most devastating diseases of barley in the northern Great Plains of the U.S. and Canada before the deployment of the stem rust-resistance gene Rpg1 in 1942. Since then, Rpg1 has provided durable protection against stem rust losses in widely grown barley cultivars (cvs.). Extensive efforts to clone Rpg1 by synteny with rice provided excellent flanking markers but failed to yield the gene because it does not seem to exist in rice. Here we report the map-based cloning and characterization of Rpg1. A high-resolution genetic map constructed with 8,518 gametes and a 330-kb bacterial artificial chromosome contig physical map positioned the gene between two crossovers ≈0.21 centimorgan and 110 kb apart. The region including Rpg1 was searched for potential candidate genes by sequencing low-copy probes. Two receptor kinase-like genes were identified. The candidate gene alleles were sequenced from resistant and susceptible cvs. Only one of the candidate genes showed a pattern of apparently functional gene structure in the resistant cvs. and defective gene structure in the susceptible cvs. identifying it as the Rpg1 gene. Rpg1 encodes a receptor kinase-like protein with two tandem protein kinase domains, a novel structure for a plant disease-resistance gene. Thus, it may represent a new class of plant resistance genes.

TOWARDS MAP-BASED CLONING OF THE BARLEY STEM RUST RESISTANCE GENES RPG1 AND RPG4 USING RICE AS AN INTERGENOMIC CLONING VEHICLE

The barley stem rust resistance genes Rpg1 and rpg4 were mapped in barley on chromosomes 1P and 7M, respectively and the syntenous rice chromosomes identified as 6P and 3P by mapping common probes in barley and rice. Rice yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC) and cosmid clones were used to isolate probes mapping to the barley Rpg1 region. The rice BAC isolated with the pM13 probe was a particularly excellent source of probes. A high-resolution map of the Rpg1 region was established with 1400 gametes yielding a map density of 3.6 markers per 0.1 cM. A detailed physical map was established for the rice BAC fragment containing the Rpg1-flanking markers pM13 and B24. This fragment covers a barley genetic distance of 0.6 cM and a rice DNA physical distance of ca. 70 kb. The distribution of barley cross-overs in relation to the rice DNA physical distances was extremely uneven. The barley genetic distance between the pM13 marker and Rpg1 was 0.1 cM per ca. 55 kb, while on the proximal side it was 0.5 cm per ca. 15 kb. Three probes from the distal end of the pM13 BAC mapped 3.0 cm proximal of Rpg1 and out of synteny with rice. These experiments confirm the validity of using large insert rice clones as probe sources to saturate small barley (and other large genome cereals) genome regions with markers. They also establish a note of caution that even in regions of high microsynteny, there may be small DNA fragments that have transposed and are no longer in syntenous positions.

Mapping of β-glucan content and β-glucanase activity loci in barley grain and malt

Genetic study of β-glucan content and β-glucanase activity has been facilitated by recent developments in quantitative trait loci (QTL) analysis. QTL for barley and malt β-glucan content and for green and finished malt β-glucanase activity were mapped using a 123-point molecular marker linkage map from the cross of Steptoe/Morex. Three QTL for barley β-glucan, 6 QTL for malt β-glucan, 3 QTL for β-glucanase in green malt and 5 QTL for β-glucanase in finished malt were detected by interval mapping procedures. The QTL with the largest effects on barley β-glucan, malt βglucan, green malt β-glucanase and finished malt βglucanase were identified on chromosomes 2,1,4 and 7, respectively. A genome map-based approach allows for dissection of relationships among barley and malt βglucan content, green and finished malt β-glucanase activity, and other malting quality parameters.

Verification of barley seed dormancy loci via linked molecular markers

Seed dormancy is a relatively complex trait in barley (Hordeum vulgare L.). Several dormancy loci were identified previously by quantitative trait locus analysis. Three reciprocal crosses were made in the present study between parents carrying specific dormancy alleles via linked molecular markers to verify individual dormancy locus effects and potential expression. Analyses of F2 progenies revealed that the dormancy allele at the locus flanked by the markers Ale and ABC302 on the long arm of chromosome 7 had a major effect on dormancy, and was at least partly epistatic to the dormancy locus in the ABC309-MWG851 interval near the telomere of the long arm of chromosome 7. In the absence of the dormancy allele in the Ale-ABC302 interval, the allele in the ABC309-MWG851 interval exerted moderate to large effects on dormancy. Cytoplasmic effects on dormancy were also observed. The germination percentages of progeny with relatively high levels of dormancy were more variable than those of non-dormant or less-dormant progeny, apparently due to environmental effects. Removal of the dormancy allele in the Ale-ABC302 interval, or introducing the dormancy allele in the ABC309-MWG851 interval, should suffice for adjusting dormancy levels in breeding programs to suit various production situations and end uses. The verification of dormancy loci via linked molecular markers allows manipulation of these loci in applied breeding programs.

Reconstruction of the Synthetic W7984 × Opata M85 wheat reference population

Reference populations are valuable resources in genetics studies for determining marker order, marker selection, trait mapping, construction of large-insert libraries, cross-referencing marker platforms, and genome sequencing. Reference populations can be propagated indefinitely, they are polymorphic and have normal segregation. Described are two new reference populations who share the same parents of the original wheat reference population Synthetic W7984 (Altar84/ Aegilops tauschii (219) CIGM86.940) x Opata M85, an F(1)-derived doubled haploid population (SynOpDH) of 215 inbred lines and a recombinant inbred population (SynOpRIL) of 2039 F(6) lines derived by single-plant self-pollinations. A linkage map was constructed for the SynOpDH population using 1446 markers. In addition, a core set of 42 SSR markers was genotyped on SynOpRIL. A new approach to identifying a core set of markers used a step-wise selection protocol based on polymorphism, uniform chromosome distribution, and reliability to create nested sets starting with one marker per chromosome, followed by two, four, and six. It is suggested that researchers use these markers as anchors for all future mapping projects to facilitate cross-referencing markers and chromosome locations. To enhance this public resource, researchers are strongly urged to validate line identities and deposit their data in GrainGenes so that others can benefit from the accumulated information.

Physical mapping of the barley stem rust resistance gene rpg4

Abstract. The barley stem rust resistance gene rpg4 was physically and genetically localized on two overlapping BAC clones covering an estimated 300-kb region of the long arm of barley chromosome 7(5H). Initially, our target was mapped within a 6.0-cM region between the previously described flanking markers MWG740 and ABG391. This region was then saturated by integrating new markers from several existing barley and rice maps and by using BAC libraries of barley cv. Morex and rice cv. Nipponbare. Physical/genetic distances in the vicinity of rpg4 were found to be 1.0xa0Mb/cM, which is lower than the average for barley (4xa0Mb/cM) and lower than that determined by translocation breakpoint mapping (1.8xa0Mb/cM). Synteny at high resolution levels has been established between the region of barley chromosome 7(5H) containing the rpg4 locus and the subtelomeric region of rice chromosome 3 between markers S16474 and E10757. This 1.7-cM segment of the rice genome was covered by two overlapping BAC clones, about 250xa0kb of total length. In barley the markers S16474 and E10757 genetically delimit rpg4, lying 0.6xa0cM distal and 0.4xa0cM proximal to the locus, respectively.

The distinctness and diversity of Ethiopian barleys

Abstractu2002The relative diversity and distinctness of Ethiopian barleys has been investigated using (1) morphology/isozyme/hordein polymorphisms and (2) RFLP markers. In the former a set of 51 landraces from over the whole of Ethiopia was compared with Iranian landraces based on data from previous studies and new hordein data. The two sets of landraces were found to have a comparable diversity. The Ethiopian ones are more diverse morphologically (5 traits), are similar in numbers of alleles per protein locus (17 loci) and in genetic differentiation, but are less diverse in average heterozygosity per locus and degree of polymorphism. However, on the basis of the hordein data the two sources of germplasm are very distinct. The correlation between morphological and protein diversity is very low. In the RFLP study 28 probes evenly distributed across the genome were used to analyse 43 Ethiopian landraces (represented by one single genotype) and to compare them with modern cultivars from North America, Europe and Japan, as well as 3 landraces from Iran, 1 from Nepal, and 1 accession of H. spontaneum from Afghanistan. The major finding was that the Ethiopian germplasm appears to be significantly less diverse than the modern germplasm but that it is genotypically very distinct. The apparent contradiction between a high diversity of useful genes coming from Ethiopia and an apparently low diversity at the DNA level is discussed.

Cloning and Mapping of a Putative Barley NADPH-Dependent HC-Toxin Reductase

The NADPH-dependent HC-toxin reductase (HCTR), encoded by Hm1 in maize, inactivates HC-toxin produced by the fungus Cochliobolus carbonum, and thus confers resistance to the pathogen. The fact that C. carbonum only infects maize (Zea mays) and is the only species known to produce HC-toxin raises the question: What are the biological functions of HCTR in other plant species? An HCTR-like enzyme may function to detoxify toxins produced by pathogens which infect other plant species (R. B. Meeley, G. S. Johal, S. E. Briggs, and J. D. Walton, Plant Cell, 4:71-77, 1992). Hm1 homolog in rice (Y. Hihara, M. Umeda, C. Hara, Q. Liu, S. Aotsuka, K. Toriyama, and H. Uchimiya, unpublished) and HCTR activity in barley, wheat, oats and sorghum have been reported (R. B. Meeley and J. D. Walton, Plant Physiol. 97:1080-1086, 1993). To investigate the sequence conservation of Hm1 and HCTR in barley and the possible relationship of barley Hm1 homolog to the known disease resistance genes, we cloned and mapped a barley (Hordeum vulgare) Hm1-like gene. A putative full-length cDNA clone, Bhm1-18, was isolated from a cDNA library consisting of mRNA from young leaves, inflorescences, and immature embryos. This 1,297-bp clone encodes 363 amino acids which show great similarity (81.6%) with the amino acid sequence of HM1 in maize. Two loci were mapped to barley molecular marker linkage maps with Bhm1-18 as the probe; locus A (Bhm1A) on the long arm of chromosome 1, and locus B (Bhm1B) on the short arm of chromosome 1 which is syntenic to maize chromosome 9 containing the Hm2 locus. The Bhm1-18 probe hybridized strongly to a Southern blot of a wide range of grass species, indicating high conservation of HCTR at the DNA sequence level among grasses. The HCTR mRNA was detected in barley roots, leaves, inflorescences, and immature embryos. The conservation of the HCTR sequence, together with its expression in other plant species (R. B. Meeley and J. D. Walton, Plant Physiol. 97:1080-1086, 1993), suggest HCTR plays an important functional role in other plant species.

RFLP markers linked to the durable stem rust resistance gene Rpg1 in barley

The gene, Rpg1, conferring stable resistance in barley to the wheat stem rust pathogen (Puccinia graminis f. sp. tritici) was mapped using two doubled haploid populations. Rpg1 mapped to the extreme subteleomeric region of barley chromosome 1P 0.3 and 1.1 cM proximal from the molecular markers ABG704 and plastocyanin (Plc), respectively, and 2.2 cM distal from MWG036B. The closest marker, ABG704, was sequenced and PCR-based markers were developed.