Resistance genes in barley (Hordeum vulgare L.) and their identification with molecular markers

Current information on barley resistance genes available from scientific papers and on-line databases is summarised. The recent literature contains information on 107 major resistance genes (R genes) against fungal pathogens (excluding powdery mildew), pathogenic viruses and aphids identified in Hordeum vulgare accessions. The highest number of resistance genes was identified against Puccinia hordei, Rhynchosporium secalis, and the viruses BaYMV and BaMMV, with 17, 14 and 13 genes respectively. There is still a lot of confusion regarding symbols for R genes against powdery mildew. Among the 23 loci described to date, two regions Mla and Mlo comprise approximately 31 and 25 alleles. Over 50 R genes have already been localised and over 30 mapped on 7 barley chromosomes. Four barley R genes have been cloned recently: Mlo, Rpg1, Mla1 and Mla6, and their structures (sequences) are available. The paper presents a catalogue of barley resistance gene symbols, their chromosomalocation and the list of available DNA markers useful in characterising cultivars and breeding accessions.     

Inheritance and molecular mapping of a gene conferring seedling resistance against Puccinia hordei in the barley cultivar Ricardo

Genetic studies were undertaken to determine the inheritance and genomic location of uncharacterised seedling resistance to leaf rust, caused by Puccinia hordei, in the barley cultivar Ricardo. The resistance was shown to be conferred by a single dominant gene, which was tentatively designated RphRic. Bulk segregant analysis (BSA) and genetic mapping of an F₃ mapping population using multiplex-ready SSR genotyping and Illumina GoldenGate SNP assay located RphRic in chromosome 4H. Given that this is the first gene for leaf rust resistance mapped on chromosome 4H, it was designated Rph21. The presence of an additional gene, Rph2, in Ricardo, was confirmed by the test of allelism. The seedling gene Rph21 has shown effectiveness against all Australian pathotypes of P. hordei tested since at least 1992 and hence represents a new and useful source of resistance to this pathogen.     

Sequence analysis and gene identification in a set of mapped RFLP markers in barley (Hordeum vulgare).

The "Igri/Franka" (I/F) map ranks among the most comprehensive genetic linkage maps of barley (Hordeum vulgare), containing a large number of markers derived from cDNA and genomic PstI clones. Fourty-three cDNA clones and 259 genomic clones were at least partially sequenced and compared with the major data bases of protein and nucleic acid sequences. Of the cDNA clones, 53% show significant similarity to known sequences in protein data bases. A comparison of sequences from genomic clones to nucleic acid sequence data bases revealed similarities for 9% of the clones. For cDNA sequences analyzed the same way, significant similarities were observed for 35% of the clones. These results show that genomic PstI clones, although containing genes at a significant frequency, represent an inappropriate source for an efficient, systematic gene identification in barley. Sequence information obtained in the context of the present study provides a resource for the conversion of these markers into sequence-tagged site (STS) markers and their use in PCR assays.     

A novel dwarfing mutation in a green revolution gene from Brassica rapa

Mutations in the biosynthesis or signaling pathways of gibberellin (GA) can cause dwarfing phenotypes in plants, and the use of such mutations in plant breeding was a major factor in the success of the Green Revolution. DELLA proteins are GA signaling repressors whose functions are conserved in different plant species. Recent studies show that GA promotes stem growth by causing degradation of DELLA proteins via the ubiquitin-proteasome pathway. The most widely utilized dwarfing alleles in wheat (Triticum aestivum; e.g. Rht-B1b and Rht-D1b) encode GA-resistant forms of a DELLA protein that function as dominant and constitutively active repressors of stem growth. All of the previously identified dominant DELLA repressors from several plant species contain N-terminal mutations. Here we report on a novel dwarf mutant from Brassica rapa (Brrga1-d) that is caused by substitution of a conserved amino acid in the C-terminal domain of a DELLA protein. Brrga1-d, like N-terminal DELLA mutants, retains its repressor function and accumulates to high levels, even in the presence of GA. However, unlike wild-type and N-terminal DELLA mutants, Brrga1-d does not interact with a protein component required for degradation, suggesting that the mutated amino acid causes dwarfism by preventing an interaction needed for its degradation. This novel mutation confers nondeleterious dwarf phenotypes when transferred to Arabidopsis (Arabidopsis thaliana) and oilseed rape (Brassica napus), indicating its potential usefulness in other crop species.     

Inheritance of organelle DNA markers in a pea cross associated with nuclear-cytoplasmic incompatibility

An unusual biparental mode of plastid inheritance was found in pea, in a cross associated with nuclear-cytoplasmic incompatibility manifested as deficiency of chlorophyll pigmentation. Plastid DNA marker trnK and mitochondrial DNA marker cox1 were analyzed in F1 progeny that received cytoplasm from an accession of a wild subspecies Pisum sativum ssp. elatius. Plants with sectors of green tissue on leaves and seed cotyledons with green patches on an otherwise chlorotic background were found to carry paternally inherited plastid DNA, suggesting that photosynthetic function was affected by nuclear-cytoplasmic conflict and required proliferation of paternally inherited plastids for normal performance. The paternally inherited plastid DNA marker was also observed in the roots. The presence of the paternal marker in cotyledons, roots and leaves was independent of each other. Inheritance of the mitochondrial DNA marker cox1 appeared to be of the maternal type.     

Inheritance of citrus nematode resistance and its linkage with molecular markers

Eleven RAPD markers linked to a gene region conferring resistance to citrus nematodes in an intergen-eric backcross family were identified. Two sequence- characterized amplified region markers linked to a citrus tristeza virus resistance gene and one selected resistance gene candidate marker were evaluated for their association with citrus nematode resistance. A nematode-susceptible citrus hybrid, LB6-2 [Clementine mandarin (Citrus reticulata)×Hamlin orange (C. sinensis)], was crossed with the citrus nematode-resistant hybrid Swingle citrumelo (C. paradisi×Poncirus trifoliata) to produce 62 hybrids that were reproduced by rooted cuttings. The plants were grown in a greenhouse and inoculated with nematodes isolated from infected field trees. The hybrids segregated widely for this trait in a continuous distribution, suggesting possible polygenic control of the resistance. Bulked segregant analysis was used to identify markers associated with resistance by bulking DNA samples from individuals at the phenotypic distribution extremes. Linkage relationships were established by the inheritance of the markers in the entire population. A single major gene region that contributes to nematode resistance was identified. The resistance was inherited in this backcross family from the grandparent Poncirus trifoliata as a single dominant gene. QTL analysis revealed that 53.6% of the phenotypic variance was explained by this major gene region. The existence of other resistance-associated loci was suggested by the continuous phenotypic distribution and the fact that some moderately susceptible hybrids possessed the resistance-linked markers. The markers may be useful in citrus rootstock breeding programs if it can be demonstrated that they are valid in other genetic backgrounds. Received: 4 May 1999 / Accepted: 21 September 1999     

A novel approach for the identification of bacterial taxa-specific molecular markers

AIMS: To develop and establish a methodology for an oriented and fast identification of species taxa-specific molecular markers useful for the identification of micro-organisms. METHODS AND RESULTS: From the complete microbial genomes available in Pfam database, taxa-specific protein domains were identified which lead to the selection of taxa-specific loci. This strategy was used to identify six genetic markers: four specific for Pseudomonas syringae pv. tomato, one specific for P. syringae pv. syringae and one specific for P. putida. The discriminatory potential of these loci was evaluated by Southern hybridization using several pseudomonad species and pathovars, by dot-blot hybridization and by multiplex PCR optimized for the simultaneous detection of P. putida, P. syringae pv. syringae and P. syringae pv. tomato. Sensitivity assays indicated a detection limit of approximately 10 pg of chromosomal DNA template needed for each bacterium. CONCLUSIONS: The proposed methodology was efficient on the selection of six Pseudomonas-specific markers able to discriminate Pseudomonas at the species and pathovar level. SIGNIFICANCE AND IMPACT OF THE STUDY: The oriented search of taxa-specific molecular probes described in this work, which can be easily extended to other groups of bacteria, will improve the accuracy and expedite the identification of micro-organisms by DNA-based molecular methods.     

Comparative mapping of a gibberellic acid-insensitive dwarfing gene (Dwf2) on chromosome 4HS in barley

 We report the genetic mapping of Dwf2, a dominant gibberellic acid (GA₃)-insensitive dwarfing gene which has been previously described to cause a very short growth habit in barley (Hordeum vulgare) mutant ‘93/B694’. Using RFLP and microsatellite markers we performed segregation analysis in an F₂ population comprising 86 individuals developed from a cross of ‘93/B694’ (Dwf2) with ‘Bonus M2’ (dwf2). Dwf2 was mapped on the short arm of barley chromosome 4H proximal to microsatellite marker XhvOle (5.7 cM) and distal to RFLP marker Xmwg2299 (18.3 cM). The genetic localization of the Dwf2 gene at a homoeologous position to the multiallelic Rht-B1 and Rht-D1 loci in wheat suggests synteny of GA-insensitive dwarfing genes within the Triticeae. Moreover, the extremely prostrate growth habit exhibited in barley ‘93/B694’ (Dwf2) resembles that of wheat plants carrying the genes Rht-B1c (Rht3) or Rht-D1c (Rht10). Received: 1 July 1998 / Accepted: 17 September 1998     

Morphological and molecular identification of Colletotrichum siamense,a novel leaf pathogen associated with Sterculia lanceolata recorded in China

Sterculia lanceolata, an important tropical woody plant, has high ornamental and medicinal value. To our knowledge, only brown root disease in this plant has been reported. In Nanning, Guangxi, China, an outbreak of leaf blight disease was observed on S. lanceolata in June 2019, with the leaf infection rate ranging from 80% to 100%. The disease seriously affected the leaves of trees and caused economic loss. Eight isolates were recovered from the infected leaves of different trees, and the pathogenicity was then determined by the methods of mycelial disc and conidial suspension, fulfilling Koch's postulates. According to the morphological and molecular biological characteristics of isolates, the pathogen causing leaf blight on S. lanceolata was identified as Colletotrichum siamense. Accurate identification of the pathogen provides a reliable basis for the control of the disease.     

Use of locus-specific AFLP markers to construct a high-density molecular map in barley

 By using 25 primer combinations, 563 AFLP markers segregating in a recombinant inbred population (103 lines, F₉) derived from L94/Vada were generated. The 38 AFLP markers in common to the existing AFLP/RFLP combined Proctor/Nudinka map, one STS marker, and four phenotypic markers with known map positions, were used to assign present AFLP linkage groups to barley chromosomes. The constructed high-density molecular map contains 561 AFLP markers, three morphological markers, one disease resistance gene and one STS marker, and covers a 1062-cM genetic distance, corresponding to an average of one marker per 1.9 cM. However, extremely uneven distributions of AFLP markers and strong clustering of markers around the centromere were identified in the present AFLP map. Around the centromeric region, 289 markers cover a genetic distance of 155 cM, corresponding to one marker per 0.5 cM; on the distal parts, 906 cM were covered by 277 markers, corresponding to one marker per 3.3 cM. Three gaps larger than 20 cM still exist on chromosomes 1, 3 and 5. A skeletal map with a uniform distribution of markers can be extracted from the high-density map, and can be applied to detect and map loci underlying quantitative traits. However, the application of this map is restricted to barley species since hardly any marker in common to a closely related Triticum species could be identified. Received: 16 June 1997 / Accepted: 9 October 1997     

Genetic analysis and fine mapping of a novel semidominant dwarfing gene LB4D in rice

A dwarf mutant, designated LB4D, was obtained among the progeny of backcrosses to a wild rice introgression line. Genetic analysis of LB4D indicated that the dwarf phenotype was controlled by a single semidominant dwarfing gene, which was named LB4D. The mutants were categorized as dn-type dwarf mutants according to the pattern of internode reduction. In addition, gibberellin (GA) response tests showed that LB4D plants were neither deficient nor insensitive to GA. This study found that tiller formation by LB4D plants was decreased by 40% compared with the wild type, in contrast to other dominant dwarf mutants that have been identified, indicating that a different dwarfing mechanism might be involved in the LB4D dominant mutant. The reduction of plant height in F(1) plants ranged from 27.9% to 38.1% in different genetic backgrounds, showing that LB4D exerted a stronger dominant dwarfing effect. Using large F(2) and F(3) populations derived from a cross between heterozygous LB4D and the japonica cultivar Nipponbare, the LB4D gene was localized to a 46 kb region between the markers Indel 4 and Indel G on the short arm of chromosome 11, and four predicted genes were identified as candidates in the target region.     

RAPD markers in diversity detection and variety identification of Tibetan hulless barley

RAPD markers were generated from 6 groups of Tibetan hulless barley (23 varieties): Lasagoumang, QB, Zangqing, Guoluo, Dongqing, and Hymalayia. Of the 48 fragments generated by 5 selected primers (among 68 primers), 44 appeared to be polymorphic (92%). Cluster analysis was performed (RAPDistance 1.04). The 23 varieties were divided into 2 groups, and the molecular foundation of genetic diversity was explored. In addition, to identify the varieties, one DNA fingerprint was constructed based on 17 bands amplified with S32 and 2 bands with S18. A specific RAPD fragment can be used to select for high and low β-glucan content varieties. Supported by the important program of Ministry of Science and Technology of P.R. China, “The Construction of Key Animal & Plant Resource System of Southwest China”.     

Mapping of quantitative trait loci affecting Drechslera teres resistance in barley with molecular markers

 Resistance loci for seedling-stage resistance to net blotch disease (Drechslera teres) in barley were mapped with molecular markers in an F₂ population derived from a cross between the susceptible barley cultivar ‘Arena’ and the resistant Ethiopian landrace ‘Hor 9088’. Disease reactions were scored with first and second leaves of 2-week-old plants 7 and 9 days after inoculation with a single spore-derived isolate. For linkage analysis, 22 RFLP markers and 284 AFLP markers were used. The seven linkage groups covered 1153.3 cM with an average marker interval of 3.76 cM. The resistance was determined to be inherited in a quantitative manner. Altogether, 12 QTLs were mapped with positions depending on the leaf used for testing and the time period after infection. Heritability in the broad sense ranged between 0.21 and 0.37. Received: 26 May 1998 / Accepted: 9 June 1998     

Development of DNA markers associated with beer foam stability for barley breeding

Traits conferring brewing quality are important objectives in malting barley breeding. Beer foam stability is one of the more difficult traits to evaluate due to the requirement for a relatively large amount of grain to be malted and then the experimental costs for subsequent brewing trials. Consequently, foam stability tends to be evaluated with only advanced lines in the final stages of the breeding process. To simplify the evaluation and selection for this trait, efficient DNA makers were developed in this study. Previous studies have suggested that the level of both of the foam-associated proteins Z4 and Z7 were possible factors that influenced beer foam stability. To confirm the relationship between levels of these proteins in beer and foam stability, 24 beer samples prepared from malt made from 10 barley cultivars, were examined. Regression analyses suggested that beer proteins Z4 and Z7 could be positive and negative markers for beer foam stability, respectively. To develop DNA markers associated with contents of proteins Z4 and Z7 in barley grain, nucleotide sequence polymorphisms in barley cultivars in the upstream region of the translation initiation codon, where the promoter region might be located were compared. As a result, 5 and 23 nucleotide sequence polymorphisms were detected in protein Z4 and protein Z7, respectively. By using these polymorphisms, cleaved amplified polymorphic sequence (CAPS) markers were developed. The CAPS markers for proteins Z4 and Z7 were applied to classify the barley grain content of 23 barley cultivars into two protein Z4 (pZ4-H and pZ4-L) and three protein Z7 (the pZ7-H, pZ7-L and pZ7-L2) haplotypes, respectively. Barley cultivars with pZ4-H showed significantly higher levels of protein Z4 in grain, and those with pZ7-L and pZ7-L2 showed significantly lower levels of protein Z7 in grain. Beer foam stability in the cultivars with pZ4-H and pZ7-L was significantly higher than that with pZ4-L and pZ7-H, respectively. Our results indicate that these CAPS markers provide an efficient selection tool for beer foam stability in barley breeding programs.     

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.     

Quantitative trait loci and molecular markers associated with wheat allelopathy

Wheat (Triticum aestivum L.) has been examined for allelopathic potential against annual ryegrass (Lolium rigidum). The bioassay technique, 'equal-compartment-agar-method', was employed to evaluate seedling allelopathy in a doubled-haploid (DH) population derived from cv Sunco (weakly allelopathic) and cv Tasman (strongly allelopathic). A significant difference in allelopathic activity was found among the DH lines, which inhibited the root length of ryegrass across a range from 23.7 to 88.3%. The phenotypic data showed that wheat allelopathic activity was distributed normally within this DH population and a substantial transgressive segregation for seedling allelopathic activity was also found. Analysis of restriction fragment length polymorphism (RFLP), amplified fragment length polymorphism (AFLP) and microsatellite (SSRs) markers identified two major QTLs on chromosome 2B associated with wheat allelopathy. The linkage analysis of genetic markers and the QTLs may improve genetic gains for the allelopathic activity through marker-assisted selection in wheat breeding. The development of wheat allelopathic cultivars could reduce the over-reliance of weed control on synthetic herbicides.Communicated by J. Dvorak