Molecular marker analyses of powdery mildew resistance in barley

Powdery mildew, caused byEryisphe graminis f. sp.hordei, is one of the most important diseases of barley (Hordeum vulgare). A number of loci conditioning resistance to this disease have been reported previously. The objective of this study was to use molecular markers to identify chromosomal regions containing genes for powdery mildew resistance and to estimate the resistance effect of each locus. A set of 28 F₁ hybrids and eight parental lines from a barley diallel study was inoculated with each of five isolates ofE. graminis. The parents were surveyed for restriction fragment length polymorphisms (RFLPs) at 84 marker loci that cover about 1100 cM of the barley genome. The RFLP genotypes of the F₁s were deduced from those of the parents. A total of 27 loci, distributed on six of the seven barley chromosomes, detected significant resistance effects to at least one of the five isolates. Almost all the chromosomal regions previously reported to carry genes for powdery mildew resistance were detected, plus the possible existence of 1 additional locus on chromosome 7. The analysis indicated that additive genetic effects are the most important component in conditioning powdery mildew resistance. However, there is also a considerable amount of dominance effects at most loci, and even overdominance is likely to be present at a number of loci. These results suggest that quantitative differences are likely to exist among alleles even at loci which are considered to carry major genes for resistance, and minor effects may be prevalent in cultivars that are not known to carry major genes for resistance.     

Conversion of an RFLP marker for the barley stem rust resistance geneRpg1 to a specific PCR-amplifiable polymorphism

TheRpg1 gene in barley has provided satisfactory levels of stem rust resistance for the last 50 years. The appearance of a new race of stem rust that is virulent toRpg1 has resulted in efforts to incorporate new stem rust resistance genes into barley. Marker-assisted selection may provide the only means of combining this useful gene with resistance genes for which no virulent races have been identified. Several RFLP markers have been identified as linked to theRpg1 locus. One of these, ABG704 was converted into a post-amplification restriction polymorphism. To generate a specific PCR-amplifiable polymorphism the sequence of the ABG704 locus from four barley cultivars was determined. Primers were developed that can detect a single-base difference between resistant and susceptible cultivars. The successful conversion of an RFLP marker to an allele-specific PCR-based marker not only demonstrates that this type of conversion is possible for cereals, but also results in an immediately useful marker for application to plant breeding programmes.     

Use of RFLP markers for the identification of alleles of the Pm3 locus conferring powdery mildew resistance in wheat (Triticum aestivum L.)

The objective of this study was to identify molecular markers linked to genes for resistance to powdery mildew (Pm) in wheat using a series of Chancellor near-isogenic-lines (NILs), each having one powdery mildew resistance gene. A total of 210 probes were screened for their ability to detect polymorphism between the NILs and the recurrent parent. One of these restriction fragment length polymorphism (RFLP) markers (Xwhs179) revealed polymorphism not only between the NILs for the Pm3 locus, but also among NILs possessing different alleles of the Pm3 locus. The location of the marker Xwhs179 was confirmed to be on homoeologous chromosome group 1 with the help of nullitetrasomic wheat lines. The linkage relationship between this probe and the Pm3 locus was estimated with double haploid lines derived from a cross between wheat cvs Club and Chul (Pm3b). The genetic distance was determined to be 3.3±1.9 cM.     

Localization of the Laevigatum powdery mildew resistance gene to barley chromosome 2 by the use of RFLP markers

Summary The powdery mildew disease resistance gene Ml(La) was found to belong to a locus on barely chromosome 2. We suggest that this locus be designated MlLa. Linkage analysis was carried out on 72 chromosome-doubled, spring-type progeny lines from a cross between the winter var Vogelsanger Gold and the spring var Alf. A map of chromosome 2 spanning 119cM and flanked by two peroxidase gene loci was constructed. In addition to the Laevigatum resistance locus the map includes nine RFLP markers, the two peroxidase gene loci and the six-row locus in barley.     

RFLP markers linked to the root knot nematode resistance gene Mi in tomato

Summary The Mi gene originating from the wild tomato species Lycopersicon peruvianum confers resistance to all major root knot nematodes (Meloidogyne spp.). This single dominant gene is located on chromosome 6 and is very closely linked to the acid phosphatase-1 (Aps-1) locus. Resistance to nematodes has been introgressed into various cultivars of the cultivated tomato (L. esculentum), in many cultivars along with the linked L. peruvianum Aps-1 ¹ allele. By using a pair of nearly isogenic lines differing in a small chromosomal region containing the Mi and Aps-1 loci, we have identified two RFLP markers, GP79 and H6A2c2, which are located in the introgressed L. peruvianum region. Analysis of a test panel of 51 L. esculentum genotypes of various origins indicated that GP79 is very tightly linked to the Mi gene and allows both homozygous and heterozygous nematode-resistant genotypes to be distinguished from susceptible genotypes, irrespective of their Aps-1 alleles. Marker H6A2c2 is linked to the Aps-1 locus and is capable of discriminating between the L. peruvianum Aps-1 ¹ allele and the L. esculentum Aps-1 ³ and Aps-1 ⁺ alleles. In combination, these RFLP markers may provide a powerful tool in breeding tomatoes for nematode resistance.     

Mapping of resistance to the potato cyst nematode Globodera rostochiensis from the wild potato species Solanum vernei

A population of diploid potato (Solanum tuberosum) was used for the genetic analysis and mapping of a locus for resistance to the potato cyst nematode Globodera rostochiensis, introgressed from the wild potato species Solanum vernei. Resistance tests of 108 genotypes of a F₁ population revealed the presence of a single locus with a dominant allele for resistance to G. rostochiensis pathotype Ro1. This locus, designated GroV1, was located on chromosome 5 with RFLP markers. Fine-mapping was performed with RAPD and SCAR markers. The GroV1 locus was found in the same region of the potato genome as the S. tuberosum ssp. andigena H1 nematode resistance locus. Both resistance loci could not excluded to be allelic. The identification of markers flanking the GroV1 locus offers a valuable strategy for marker-assisted selection for introgression of this nematode resistance.Abbreviations BSA bulked segregant analysis - RAPD random-amplified polymorphic DNA - RFLP restriction fragment length polymorphism - SCAR sequence-characterized amplified region     

Divergence and allelomorphic relationship of a soybean virus resistance gene based on tightly linked DNA microsatellite and RFLP markers

The use of genetically diverse resistance sources is important in breeding for durable disease resistance. Detection and evaluation of resistance genes by conventional inheritance experiments, however, often require laborious screening and genetic testing. In the present study, a marker-assisted screening for resistance sources was initiated in soybean [Glycine max (L.) Merr] using one DNA microsatellite and two RFLP markers tightly linked to a soybean mosaic virus (SMV) resistance gene (Rsv1). The three marker loci were used to screen 67 diverse soybean cultivars, breeding lines, and plant introductions. Five variants were found at the microsatellite locus (HSP176L), and the two RFLP loci (pA186 and pK644a) near Rsv1 show a remarkably higher level of restriction polymorphism than Rsv1-independent RFLP loci. Several specific variants at the three marker loci were found to be correlated with virus resistance, among which HSP176L-2 can be detected by PCR, thus may be useful for germplasm screening. The grouping of the 67 accessions according to their multilocus marker variants agrees with the available pedigree information. When all, or most, of the cultivars within a given group with the same Rsv1-linked marker variant are resistant, their SMV resistance is most likely conferred by Rsv1. These putatively Rsv1-carrying groups contain a total of 38 SMV-resistant lines including six differential cultivars that are known to carry Rsv1. The remaining seven resistant accessions (Columbia, Holladay, Peking, Virginia, FFR-471, PI 507403, and PI 556949) do not carry resistance marker variants, and at least some of them could be sources of resistance genes independent of Rsv1.     

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     

Identification and mapping of pm2026: a recessive powdery mildew resistance gene in an einkorn (Triticum monococcum L.) accession

Triticum monococcum accession TA2026 showed resistance to wheat powdery mildew. To identify the resistance gene and transfer it to common wheat, genetic analysis and molecular mapping were conducted using an F₂ population and derived F₃ families from the cross of TA2026 × M389. The results indicated that TA2026 possessed a recessive powdery mildew resistance gene. This gene was mapped to the terminal portion of chromosome 5A^mL and flanked by SSR marker loci Xcfd39 and Xgwm126. Eight RFLP markers previously mapped to the terminal chromosome 5A^mL were converted into STS markers. Three loci, detected by MAG1491, MAG1493 and MAG1494, the STS markers derived from RFLP probes CDO1312, PSR164 and PSR1201, respectively, were linked to this resistance gene with Xmag1493 only 0.9 cM apart from it. In addition, the STS marker MAG2170 developed from the tentative consensus wheat cDNA encoding the Mlo-like protein identified a locus co-segregating with Xmag1493. This is the first recessive powdery mildew resistance gene identified on chromosome 5A^m, and is temporarily designated pm2026. We have successfully transferred it to a tetraploid background, and this resistance stock will now be used as the bridge parent for its transfer to common wheat.     

Genetic analysis of the obligate parasitic barley powdery mildew fungus based on RFLP and virulence loci

Summary Genome organization of the biotrophic barley powdery mildew fungus was studied using restriction fragment length polymorphism (RFLP). Genomic DNA clones containing either low-or multiple-copy sequences appeared to be the best RFLP markers, as they frequently revealed polymorphisms that could be readily detected. A total of 31 loci were identified using 11 genomic DNA clones as probes. Linkage analysis of the 31 RFLP loci and five virulence loci resulted in the construction of seven groups of linked loci. Two of these contained both RFLP markers and virulence genes. RFLP markers were found to be very efficient in characterizing mildew isolates, as only three markers were necessary to differentiate 28 isolates. The DNA of the barley powdery mildew fungus appeared to contain a considerable number of repetitive sequences dispersed throughout the genome.     

Linkages between restriction fragment length,isozyme, and morphological markers in lentil

Summary A genetic linkage map of lentil comprising 333 centimorgans (cM) was constructed from 20 restriction fragment length, 8 isozyme, and 6 morphological markers segregating in a single interspecific cross (Lens culinaris × L. orientalis). Because the genotypes at marker loci were determined for about 66 F₂ plants, linkages are only reported for estimates of recombination less than 30 cM. Probes for identification of restriction fragment length polymorphisms (RFLPs) were isolated from a cDNA and EcoRI and PstI partial genomic libraries of lentil. The cDNA library gave the highest frequency of relatively low-copy-number probes. The cDNAs were about twice as efficient, relative to random genomic fragments, in RFLP detection per probe. Nine markers showed significant deviations from the expected F₂ ratios and tended to show a predominance of alleles from the cultigen. Assuming a genome size of 10 Morgans, 50% of the lentil genome could be linked within 10 cM of the 34 markers and the map is of sufficient size to attempt mapping of quantitative trait loci.     

Genetic characterization of powdery mildew resistance in U.S. hard winter wheat

Powdery mildew significantly affects grain yield and end-use quality of winter wheat in the southern Great Plains. Employing resistance resources in locally adapted cultivars is the most effective means to control powdery mildew. Two types of powdery mildew resistance exist in wheat cultivars, i.e., qualitative and quantitative. Qualitative resistance is controlled by major genes, is race-specific, is not durable, and is effective in seedlings and in adult plants. Quantitative resistance is controlled by minor genes, is non-race-specific, is durable, and is predominantly effective in adult plants. In this study, we found that the segregation of powdery mildew resistance in a population of recombinant inbred lines developed from a cross between the susceptible cultivar Jagger and the resistant cultivar 2174 was controlled by a major QTL on the short arm of chromosome 1A and modified by four minor QTLs on chromosomes 1B, 3B, 4A, and 6D. The major QTL was mapped to the genomic region where the Pm3 gene resides. Using specific PCR markers for seven Pm3 alleles, 2174 was found to carry the Pm3a allele. Pm3a explained 61% of the total phenotypic variation in disease reaction observed among seedlings inoculated in the greenhouse and adult plants grown in the field and subjected to natural disease pressure. The resistant Pm3a allele was present among 4 of 31 cultivars currently being produced in the southern Great Plains. The genetic effects of several minor loci varied with different developmental stages and environments. Molecular markers associated with these genetic loci would facilitate incorporating genetic resistance to powdery mildew into improved winter wheat cultivars.     

Geographic distribution of alleles at the Ga2 locus for segregation distortion in barley

Summary A distorted segregation of esterase alleles at the complex loci, Est1, Est2 and Est4, was found in an F₂ population. This distortion is typical for cross combinations between the Ga2Ga2 and ga2ga2 genotypes responsible for segregation distortion, since the Ga2 locus is linked with the complex loci encoding the esterase isozymes. The segregation of esterase isozyme patterns in F₂ populations between 473 varieties of barley and a tester of ga2ga2 genotype was examined, and the genotypes inducing segregation distortion were detected. Varieties with a ga2ga2 genotype are widely distributed throughout the world, whereas Ga2Ga2 varieties are found only in eastern and southern regions of Asia, from Japan to North India, with a low frequency. In varieties collected from these regions, some associations were detected between alleles at the Ga2 locus and esterase isozyme patterns. Additionally, most of the Ga2 barley varieties are naked and possess a BtBtbt2bt2 genotype for a non-brittle rachis.     

Large deletions within the first intron in <Emphasis Type="Italic">VRN-1</Emphasis> are associated with spring growth habit in barley and wheat

The broad adaptability of wheat and barley is in part attributable to their flexible growth habit, in that spring forms have recurrently evolved from the ancestral winter growth habit. In diploid wheat and barley growth habit is determined by allelic variation at the VRN-1 and/or VRN-2 loci, whereas in the polyploid wheat species it is determined primarily by allelic variation at VRN-1. Dominant Vrn-A1 alleles for spring growth habit are frequently associated with mutations in the promoter region in diploid wheat and in the A genome of common wheat. However, several dominant Vrn-A1, Vrn-B1, Vrn-D1 (common wheat) and Vrn-H1 (barley) alleles show no polymorphisms in the promoter region relative to their respective recessive alleles. In this study, we sequenced the complete VRN-1 gene from these accessions and found that all of them have large deletions within the first intron, which overlap in a 4-kb region. Furthermore, a 2.8-kb segment within the 4-kb region showed high sequence conservation among the different recessive alleles. PCR markers for these deletions showed that similar deletions were present in all the accessions with known Vrn-B1 and Vrn-D1 alleles, and in 51 hexaploid spring wheat accessions previously shown to have no polymorphisms in the VRN-A1 promoter region. Twenty-four tetraploid wheat accessions had a similar deletion in VRN-A1 intron 1. We hypothesize that the 2.8-kb conserved region includes regulatory elements important for the vernalization requirement. Epistatic interactions between VRN-H2 and the VRN-H1 allele with the intron 1 deletion suggest that the deleted region may include a recognition site for the flowering repression mediated by the product of the VRN-H2 gene of barley.     

A high-resolution map of the vicinity of the R1 locus on chromosome V of potato based on RFLP and AFLP markers

The R1 allele confers on potato a race-specific resistance to Phytophthora infestans. The corresponding genetic locus maps on chromosome V in a region in which several other resistance genes are also located. As part of a strategy for cloning R1, a high-resolution genetic map was constructed for the segment of chromosome V that is bordered by the RFLP loci GP21 and GP179 and includes the R1 locus. Bulked segregant analysis and markers based on amplified fragment length polymorphisms (AFLP markers) were used to select molecular markers closely linked to R1. Twenty-nine of approximately 3200 informative AFLP loci displayed linkage to the R1 locus. Based on the genotypic analysis of 461 gametes, eight loci mapped within the GP21–GP179 interval. Two of those could not be seperated from R1 by recombination. For genotyping large numbers of plants with respect to the flanking markers GP21 and GP179 PCR based assays were also developed which allowed marker-assisted selection of plants with genotypes Rr and rr and of recombinant plants.     

Abundance, variability and chromosomal location of microsatellites in wheat

The potential of microsatellite sequences as genetic markers in hexaploid wheat (Triticum aestivum) was investigated with respect to their abundance, variability, chromosomal location and usefulness in related species. By screening a lambda phage library, the total number of (GA)_n blocks was estimated to be 3.6 x 10⁴ and the number of (GT)_n blocks to be 2.3 x 10⁴ per haploid wheat genome. This results in an average distance of approximately 270 kb between these two microsatellite types combined. Based on sequence analysis data from 70 isolated microsatellites, it was found that wheat microsatellites are relatively long containing up to 40 dinucleotide repeats. Of the tested primer pairs, 36% resulted in fragments with a size corresponding to the expected length of the sequenced microsatellite clone. The variability of 15 microsatellite markers was investigated on 18 wheat accessions. Significantly, more variation was detected with the microsatellite markers than with RFLP markers with, on average, 4.6 different alleles per microsatellite. The 15 PCR-amplified microsatellites were further localized on chromosome arms using cytogenetic stocks of Chinese Spring. Finally, the primers for the 15 wheat microsatellites were used for PCR amplification with rye (Secale cereale) and barley accessions (Hordeum vulgare, H. spontaneum). Amplified fragments were observed for ten primer pairs with barley DNA and for nine primer pairs with rye DNA as template. A microsatellite was found by dot blot analysis in the PCR products of barley and rye DNA for only one primer pair.     

An RFLP marker for rb in pea

Summary We have located an RFLP marker, corresponding to the locus Vc-5, which is linked to the r _b locus. We also show that the heterogeneity at the Vc-5 locus is less among r _br_b lines than among pea genotypes as a whole. The relevance of this RFLP is discussed in relation to the construction of the double recessive rr r _br_b genotypes.     

A resistance gene analog useful for targeting disease resistance genes against different pathogens on group 1S chromosomes of barley,wheat and rye

Comparative sequence analysis of the resistance gene analog (RGA) marker locus aACT/CAA (originally found to be tightly linked to the multiallelic barley Mla cluster) from genomes of barley, wheat and rye revealed a high level of relatedness among one another and showed high similarity to a various number of NBS-LRR disease resistance proteins. Using the sequence-specific polymerase chain reaction (PCR), RGA marker aACT/CAA was mapped on group 1S chromosomes of the Triticeae and was associated with disease resistance loci. In barley and rye, the marker showed linkage to orthologous powdery mildew resistance genes Mla1 and Pm17, respectively, while in wheat linkage with a QTL against fusarium head blight (FHB) disease was determined. The use of RGA clones for R gene mapping and their role in the expression of qualitative and quantitative resistance is discussed.     

Development of PCR assays diagnostic for RFLP marker alleles closely linked to allelesGro1 andH1, conferring resistance to the root cyst nematodeGlobodera rostochiensis in potato

The use of RFLPs for marker-assisted selection schemes in potato breeding is hampered by the fact that RFLP technology requires good laboratory facilities, technical skills and high financial input. Marker technology based on the polymerase chain reaction (PCR) would facilitate the application of marker-assisted selection. PCR assays have been developed that are diagnostic for RFLP alleles at two marker loci,CP56 andCP113, which are closely linked in coupling to the nematode resistance allelesGro1 on chromosome VII andH1 on chromosome V of potato. By comparing DNA sequences among different marker alleles, point mutations were identified based on which allele-specific oligonucleotides were designed. Using allele-specific oligonucleotides as primers in PCR reactions, single-marker alleles were amplified by which the inheritance ofGro1 andH1 could be followed in crosses of diploid potato genotypes containing the genetically characterizedGro1 orH1 resistance allele. When tested in 136 unrelated tetraploid potato varieties, the marker allele indicative ofGro1 was not correlated with the presence of nematode resistance. The marker allele indicative for theH1 resistance allele was correlated with nematode resistance. It was, however, found in four varieties only of the 136 tested.     

Identification of Quantitative Trait Loci Influencing Wood Specific Gravity in an Outbred Pedigree of Loblolly Pine

We report the identification of quantitative trait loci (QTL) influencing wood specific gravity (WSG) in an outbred pedigree of loblolly pine (Pinus taeda L.). QTL mapping in an outcrossing species is complicated by the presence of multiple alleles (>2) at QTL and marker loci. Multiple alleles at QTL allow the examination of interaction among alleles at QTL (deviation from additive gene action). Restriction fragment length polymorphism (RFLP) marker genotypes and wood specific gravity phenotypes were determined for 177 progeny. Two RFLP linkage maps were constructed, representing maternal and paternal parent gamete segregations as inferred from diploid progeny RFLP genotypes. RFLP loci segregating for multiple alleles were vital for aligning the two maps. Each RFLP locus was assayed for cosegregation with WSG QTL using analysis of variance (ANOVA). Five regions of the genome contained one or more RFLP loci showing differences in mean WSG at or below the P = 0.05 level for progeny as grouped by RFLP genotype. One region contained a marker locus (S6a) whose QTL-associated effects were highly significant (P > 0.0002). Marker S6a segregated for multiple alleles, a prerequisite for determining the number of alleles segregating at the linked QTL and analyzing the interactions among QTL alleles. The QTL associated with marker S6a appeared to be segregating for multiple alleles which interacted with each other and with environments. No evidence for digenic epistasis was found among the five QTL.