Identification of a QTL conferring seedling and adult plant resistance to eyespot on chromosome 5A of Cappelle Desprez

Eyespot is an economically important fungal disease of wheat and other cereals caused by two fungal species: Oculimacula yallundae and Oculimacula acuformis. However, only two eyespot resistance genes have been characterised and molecular markers made available to plant breeders. These resistances are Pch1, introduced into wheat from the relative Aegilops ventricosa, and Pch2, originally identified in the cultivar Cappelle Desprez (CD). There are drawbacks associated with both resistances; Pch1 is linked to deleterious traits carried on the Ae. ventricosa introgression and Pch2 has been shown to have limited effectiveness. An additional resistance has been reported on chromosome 5A of CD that confers resistance to eyespot in adult plants. In the present study, we demonstrate that resistance on this chromosome is effective against both O. yallundae and O. acuformis eyespot pathogens and confers resistance at both seedling and adult plant stages. This resistance was mapped in both seedling bioassays and field trials in a 5A recombinant population derived from a cross between CD and a CD single chromosome substitution line carrying 5A from the susceptible line Bezostaya. The resistance was also mapped using seedling bioassays in a 5A recombinant population derived from a cross between the susceptible line Chinese Spring (CS) and a single chromosome substitution line carrying 5A from CD. A single major QTL on the long arm of chromosome 5A was detected in all experiments. Furthermore, the SSR marker Xgwm639 was found to be closely associated with the resistance and could be used for marker-assisted selection of the eyespot resistance by plant breeders.     

The development of PCR-based markers for the selection of eyespot resistance genes Pch1 and Pch2

Two eyespot resistance genes (Pch1 and Pch2) have been characterised in wheat. The potent resistance gene Pch1, transferred from Aegilops ventricosa, is located on the distal end of the long arm of chromosome 7D (7DL). Pch2 derives from the variety Cappelle Desprez and is located at the distal end of chromosome 7AL. The RFLP marker Xpsr121 and the endopeptidase isozyme allele Ep-D1b, are very closely linked to Pch1, probably due to reduced recombination in the region of the introgressed A. ventricosa segment. Pch2 is less closely linked to these markers but is thought to be closer to Xpsr121 than to Ep-A1b. In the present study simple sequence repeat (SSR) markers were integrated into the genetic map of a single chromosome (7D) recombinant (RVPM) population segregating for Pch1. Sequence-tagged-site (STS)-based assays were developed for Xpsp121 and a 7DL wheat EST containing a SSR. SSR markers Xwmc14 and Xbarc97 and the Xpsr121-derived marker co-segregated with Pch1 in the RVPM population. A single chromosome (7A) recombinant population segregating for Pch2 was screened for eyespot resistance and mapped using SSRs. QTL interval mapping closely associated Pch2 with the SSR marker Xwmc525.     

The eyespot resistance genes <Emphasis Type="Italic">Pch1</Emphasis> and <Emphasis Type="Italic">Pch2</Emphasis> of wheat are not homoeoloci

Key message

Phenotyping and mapping data reveal that chromosome intervals containing eyespot resistance genes Pch1 and Pch2 on 7D and 7A, respectively, do not overlap, and thus, these genes are not homoeloci.

Abstract

Eyespot is a stem-base fungal disease of cereals growing in temperate regions. Two main resistances are currently available for use in wheat. Pch1 is a potent single major gene transferred to wheat from Aegilops ventricosa and located on the distal end of chromosome 7D. Pch2, a moderate resistance deriving from Cappelle Desprez, is located at the end of 7AL. The relative positions of Pch1 and Pch2 on 7D and 7A, respectively, suggest that they are homoeoloci. A single seed decent recombinant F7 population was used to refine the position of Pch2 on 7A. New markers designed to 7D also allowed the position of Pch1 to be further defined. We exploited the syntenic relationship between Brachypodium distachyon and wheat to develop 7A and 7D specific KASP markers tagging inter-varietal and interspecific SNPs and allow the comparison of the relative positions of Pch1 and Pch2 on 7D and 7A. Together, phenotyping and mapping data reveal that the intervals containing Pch1 and Pch2 do not overlap, and thus, they cannot be considered homoeloci. Using this information, we analysed two durum wheat lines carrying Pch1 on 7A to determine whether the Ae.ventricosa introgression extended into the region associated with Pch2. This identified that the introgression is distal to Pch2 on 7A, providing further evidence that the genes are not homoeoloci. However, it is feasible to use this material to pyramid Pch1 and Pch2 on 7A in a tetraploid background and also to increase the copy number of Pch1 in combination with Pch2 in a hexaploid background.

     

Eyespot resistance gene Pch-1 in H-93 wheat lines. Evidence of linkage to markers of chromosome group 7 and resolution from the endopeptidase locus Ep-D1b

Summary Gene Pch1, which confers resistance to eyespot disease (Pseudocercosporella herpotrichoides Fron), has been located on chromosome 7D in the H-93 wheat-Aegilops ventricosa transfer lines using isozyme markers and DNA probes corresponding to group 7 chromosomes. Previous experiments had failed to ascertain this location. The lack of segregation of the resistance trait in progeny from reciprocal crosses between lines H-93-70 and VPM1 indicates that their respective resistance factors are allelic. Line H-93-51 carries the endopeptidase allele Ep-D1b but is susceptible to eyespot, which indicates that resistance to eyespot is not a product of the Ep-D locus, as had been proposed in a previous hypohesis.     

Exploiting co-linearity among grass species to map the <Emphasis Type="Italic">Aegilops ventricosa-</Emphasis>derived <Emphasis Type="Italic">Pch1</Emphasis> eyespot resistance in wheat and establish its relationship to <Emphasis Type="Italic">Pch2</Emphasis>

Introgressions into wheat from related species have been widely used as a source of agronomically beneficial traits. One such example is the introduction of the potent eyespot resistance gene Pch1 from the wild relative Aegilops ventricosa onto chromosome 7DL of wheat. In common with genes carried on many other such introgressions, the use of Pch1 in commercial wheat varieties has been hindered by linkage drag with yield-limiting traits. Attempts to break this linkage have been frustrated by a lack of co-dominant PCR markers suitable for identifying heterozygotes in F₂ populations. We developed conserved orthologous sequence (COS) markers, utilising the Brachypodium distachyon (Brachypodium) genome sequence, to provide co-dominant markers in the Pch1 region. These were supplemented with previously developed sequence-tagged site (STS) markers and simple sequence repeat (SSR) markers. Markers were applied to a panel of varieties and to a BC₆ F₂ population, segregating between wheat and Ae. ventricosa over the distal portion of 7DL, to identify recombinants in the region of Pch1. By exploiting co-linearity between wheat chromosome 7D, Brachypodium chromosome 1, rice chromosome 6 and sorghum chromosome 10, Pch1 was located to an interval between the flanking markers Xwg7S and Xcos7-9. Furthermore candidate gene regions were identified in Brachypodium (364 Kb), rice (178 Kb) and sorghum (315 Kb) as a prelude to the map-based cloning of the gene. In addition, using homoeologue transferable markers, we obtained evidence that the eyespot resistances Pch1 and Pch2 on chromosomes 7D and 7A, respectively, are potentially homoeoloci. It is anticipated that the COS marker methodology could be used for the identification of recombinants in other introgressions into wheat from wild relatives. This would assist the mapping of genes of interest and the breaking of deleterious linkages to enable greater use of these introgressions in commercial varieties.     

Identification of an RFLP interval containing Pch2 on chromosome 7AL in wheat.

The gene Pch2 in 'Cappelle Desprez' is one of two genes found in hexaploid wheat known to confer resistance to eyespot disease. This study was conducted to develop an RFLP linkage map of the distal portion of wheat chromosome 7AL, and to locate and identify markers closely associated with Pch2 for use in marker-assisted selection. Ten loci in addition to Pch2 were mapped on chromosome 7AL, using segregation data from 102 homozygous chromosome 7A recombinant substitution lines derived from 'Chinese Spring' x 'Chinese Spring' ('Cappelle Desprez' 7A). The Pch2 locus was bracketed by two RFLP markers, one 11.0 cM distal to Xcdo347 and the other 18.8 cM proximal to Xwg380. The position of Pch2 on chromosome 7AL is similar to that of Pch1 on chromosome 7DL, suggesting that these resistance genes are homoeoloci. Although no single marker was closely linked to Pch2, simultaneous selection of the flanking RFLP markers Xcdo347 and Xwg380 could be used for selecting Pch2, since double recombination occurred in only 3% of the recombinant population. The use of the flanking RFLP markers to select for Pch2, in combination with previously identified Pch1-linked markers, would facilitate the development of cultivars carrying two genes for resistance to eyespot.     

Resistance to eyespot of wheat, caused by Tapesia yallundae, derived from Thinopyrum intermedium homoeologous group 4 chromosome

Thinopyrum intermedium was identified previously as resistant to Tapesia yallundae, cause of eyespot of wheat. Using GUS-transformed isolates of T. yallundae as inoculum, we determined that wheat lines carrying Th. intermedium chromosome 4Ai#2 or the short arm of chromosome 4Ai#2 were as resistant to the pathogen as the eyespot-resistant wheat- Th. ponticum chromosome substitution line SS767 (PI 611939) and winter wheat cultivar Madsen, which carries gene Pch1 for eyespot resistance. Chromosome 4E from Th. elongatum and chromosome 4J from Th. bessarabicum did not confer resistance to T. yallundae. Genome-specific PCR primers confirmed the presence of Thinopyrum chromatin in these wheat- Thinopyrum lines. Genomic in situ hybridization using an St genomic probe from Pseudoroegneria strigosa demonstrated that chromosome 4Ai#2 belongs to the J^s genome of Thinopyrum. The eyespot resistance in the wheat- Th. intermedium lines is thus controlled by the short arm of this J^s chromosome. This is the first report of resistance to T. yallundae controlled by a J^s genome chromosome of Th. intermedium.     

Effects of Chromosome Substitutions on Copper Toxicity Tolerance in Wheat Seedlings

Substitution of wheat (Triticum aestivum L.) chromosomes 7A, 1D, 3A, 3B, 3D, 4A and 4D of cultivar Cappelle Desprez by their homologues of cultivar Bezostaya-1 increased the seedling tolerance to high concentrations of copper (1 M CuSO₄ 5 H₂O). Substitution of chromosome 1A had negative effects on seedling tolerance.     

Genome-wide association mapping of resistance to eyespot disease (<Emphasis Type="Italic">Pseudocercosporella herpotrichoides</Emphasis>) in European winter wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) and fine-mapping of <Emphasis Type="Italic">Pch1</Emphasis>

Key message

Genotypes with recombination events in the Triticum ventricosum introgression on chromosome 7D allowed to fine-map resistance gene Pch1, the main source of eyespot resistance in European winter wheat cultivars.

Abstract

Eyespot (also called Strawbreaker) is a common and serious fungal disease of winter wheat caused by the necrotrophic fungi Oculimacula yallundae and Oculimacula acuformis (former name Pseudocercosporella herpotrichoides). A genome-wide association study (GWAS) for eyespot was performed with 732 microsatellite markers (SSR) and 7761 mapped SNP markers derived from the 90 K iSELECT wheat array using a panel of 168 European winter wheat varieties as well as three spring wheat varieties and phenotypic evaluation of eyespot in field tests in three environments. Best linear unbiased estimations (BLUEs) were calculated across all trials and ranged from 1.20 (most resistant) to 5.73 (most susceptible) with an average value of 4.24 and a heritability of H ² = 0.91. A total of 108 SSR and 235 SNP marker–trait associations (MTAs) were identified by considering associations with a ?log₁₀ (P value) ≥3.0. Significant MTAs for eyespot-score BLUEs were found on chromosomes 1D, 2A, 2D, 3D, 5A, 5D, 6A, 7A and 7D for the SSR markers and chromosomes 1B, 2A, 2B, 2D, 3B and 7D for the SNP markers. For 18 varieties (10.5%), a highly resistant phenotype was detected that was linked to the presence of the resistance gene Pch1 on chromosome 7D. The identification of genotypes with recombination events in the introgressed genomic segment from Triticum ventricosum harboring the Pch1 resistance gene on chromosome 7DL allowed the fine-mapping of this gene using additional SNP markers and a potential candidate gene Traes_7DL_973A33763 coding for a CC-NBS-LRR class protein was identified.

     

Isolation of a chromosomally engineered durum wheat line carrying the Aegilops ventricosa pchl gene for resistance to eyespot.

The chromosome 7Dv of Aegilops ventricosa (syn. Triticum ventricosum, 2n = 4x = 28, genome DvDvMvMv) carries the gene Pch1 for resistance to eyespot. This gene has previously been transferred to chromosome 7D of bread wheat, T. aestivum (2n = 6x = 42, genome AABBDD). To (1) enhance the level of resistance of bread wheat by increasing the copy number of Pch1, and (2) create eyespot-resistant triticales, meiotically stable Pch1-carrying durum lines were selected from the backcross progenies of a cross between Ae. ventricosa and T. durum cv. Creso ph1c (2n = 4x = 28, genome AABB). The Pch1 transfer, likely resulting from homoeologous recombination, was located at the distal position on the long arm of chromosome 7A. The 7A microsatellite marker Xgwm 698 was found closely linked in repulsion to the introgression in the resistant recombination lines, and the endopeptidase allele located on chromosome 7A of cv. Creso ph1c was lost.     

Dissecting of the FHB resistance QTL on the short arm of wheat chromosome 2D using a comparative genomic approach: from QTL to candidate gene

Colinearity in gene content and order between rice and closely related cereal crops has been a powerful tool for gene identification. Using a comparative genomic approach, we have identified the rice genomic region syntenous to the region of the short arm of wheat chromosome 2D, on which quantitative trait loci (QTLs) for Fusarium head blight (FHB) resistance and for controlling accumulation of the mycotoxin deoxynivalenol (DON) are closely located. Utilizing markers known to reside near the FHB resistance QTL and data from several wheat genetic maps, we have limited the syntenous region to 6.8 Mb of the short arm of rice chromosome 4. From the 6.8-Mb sequence of rice chromosome 4, we found three putative rice genes that could have a role in detoxification of mycotoxins. DNA sequences of these putative rice genes were used in BLAST searches to identify wheat expressed sequence tags (ESTs) exhibiting significant similarity. Combined data from expression analysis and gene mapping of wheat homologues and results of analysis of DON accumulation using doubled haploid populations revealed that a putative gene for multidrug resistance-associated protein (MRP) is a possible candidate for the FHB resistance and/or DON accumulation controlling QTLs on wheat chromosome 2DS and can be used as a molecular marker to eliminate the susceptible allele when the Chinese wheat variety Sumai 3 is used as a resistance source. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Genetic analysis of durable resistance to yellow rust in bread wheat

Yellow rust, caused by Puccinia striiformis, is one of the most damaging diseases affecting bread wheat in temperate regions. Although resistance to yellow rust is frequently overcome by new virulent races, a durable form of resistance in the French bread wheat Camp Rémy (CR) has remained effective since its introduction in 1980. We used 217 F₇ recombinant inbred lines (RILs) derived from the cross between CR and the susceptible cultivar Récital to identify and map quantitative trait loci (QTLs) involved in durable yellow rust resistance. Six significant QTLs that were stable over a 4-year period were detected. Two QTLs, denoted QYr.inra-2DS and QYr.inra-5BL.2, were located on the short arm of chromosome 2D and the long arm of chromosome 5B, respectively. Each explained on average 25–35% of the observed phenotypic variation and were probably inherited from Cappelle Desprez, a parent of CR that confers durable adult plant resistance to yellow rust. QYr.inra-2DS probably corresponds to the Yr16 gene. The most consistent QTL, designated QYr.inra-2BL, was located on the centromeric region of chromosome 2B and explained 61% of the phenotypic variation in 2003. This QTL was responsible for seedling-stage resistance and may correspond to a cluster of genes, including Yr7. The remaining QTLs were mapped to the short arm of chromosome 2B (R²=22–70%) and to the long arm of chromosomes 2A (R²=0.20–0.40) and 5B (R²=0.18–0.26). This specific combination of seedling and adult plant resistance genes found in CR and CD may constitute the key to their durable resistance against yellow rust.     

CO2Effects on Phasic Development, Leaf Number and Rate of Leaf Appearance in Wheat

It has been predicted that the concentration of CO₂in the aircould double during the 21st century. Though it is recognizedthat CO₂-doubling could increase yield through its effects onplant photosynthesis and stomatal behaviour, it is unclear whetherCO₂-doubling will change phasic development in wheat. A phytotronstudy was conducted with two contrasting cultivars of wheat,Condor (spring) and Cappelle Desprez (winter), to determinewhether development is affected by a season-long exposure to360 and 720 ppmv CO₂. Plants were vernalized for 50 d (8/4 °C,8 h photoperiod) before their exposure to the CO₂treatments. There were significant differences between cultivars in theduration of different phenophases as well as in the final numberof leaves. However, CO₂concentration had no effect in eithercultivar on the duration of the early developmental phase toterminal spikelet initiation, or on the final number of leaves,though CO₂-doubling did slightly increase the later phase fromterminal spikelet initiation to heading in Cappelle Desprez.Condor and Cappelle Desprez also differed markedly in the dynamicsof leaf appearance. While the former had a constant rate ofleaf appearance throughout development, the latter had a fastrate initially (between leaves 1 and 7), similar to that ofCondor, which was followed by a slower rate after the appearanceof leaf 7. Overall, CO₂-doubling did not significantly affectthe rates of leaf appearance nor the shape of the relationship.Phyllochron for the first seven leaves was the same for bothCO₂concentrations. However, the change in phyllochron associatedwith CO₂-doubling for leaves 7–12 in Cappelle Desprez,although quite small (4%), accounts for part of the slightlyincreased duration of the phase from terminal spikelet initiationto heading under high CO₂concentration in that cultivar. Weconclude that CO₂concentration does not influence developmentin wheat to a degree relevant to agronomy. Carbon dioxide; climatic change; development; leaf number; phyllochron     

Mapping QTL for resistance to eyespot of wheat in Aegilops longissima

Eyespot is an economically important disease of wheat caused by the soilborne fungi Oculimacula yallundae and O. acuformis. These pathogens infect and colonize the stem base, which results in lodging of diseased plants and reduced grain yield. Disease resistant cultivars are the most desirable control method, but resistance genes are limited in the wheat gene pool. Some accessions of the wheat wild relative Aegilops longissima are resistant to eyespot, but nothing is known about the genetic control of resistance. A recombinant inbred line population was developed from the cross PI 542196 (R) × PI 330486 (S) to map the resistance genes and better understand resistance in Ae. longissima. A genetic linkage map of the S(l) genome was constructed with 169 wheat microsatellite markers covering 1261.3 cM in 7 groups. F(5) lines (189) were tested for reaction to O. yallundae and four QTL were detected in chromosomes 1S(l), 3S(l), 5S(l), and 7S(l). These QTL explained 44 % of the total phenotypic variation in reaction to eyespot based on GUS scores and 63 % for visual disease ratings. These results demonstrate that genetic control of O. yallundae resistance in Ae. longissima is polygenic. This is the first report of multiple QTL conferring resistance to eyespot in Ae. longissima. Markers cfd6, wmc597, wmc415, and cfd2 are tightly linked to Q.Pch.wsu-1S ( l ), Q.Pch.wsu-3S ( l ), Q.Pch.wsu-5S ( l ), and Q.Pch.wsu-7S ( l ), respectively. These markers may be useful in marker-assisted selection for transferring resistance genes to wheat to increase the effectiveness of resistance and broaden the genetic diversity of eyespot resistance.     

Fine mapping and targeted SNP survey using rice-wheat gene colinearity in the region of the <Emphasis Type="Italic">Bo1</Emphasis> boron toxicity tolerance locus of bread wheat

Toxicity due to high levels of soil boron (B) represents a significant limitation to cereal production in some regions, and the Bo1 gene provides a major source of B toxicity tolerance in bread wheat (Triticum aestivum L.). A novel approach was used to develop primers to amplify and sequence gene fragments specifically from the Bo1 region of the hexaploid wheat genome. Single-nucleotide polymorphisms (SNPs) identified were then used to generate markers close to Bo1 on the distal end of chromosome 7BL. In the 16 gene fragments totaling 19.6 kb, SNPs were observed between the two cultivars Cranbrook and Halberd at a low frequency (one every 613 bp). Furthermore, SNPs were distributed unevenly, being limited to only two genes. In contrast, RFLP provided a much greater number of genetic markers, with every tested gene identifying polymorphism. Bo1 previously known only as a QTL was located as a discrete Mendelian locus. In total, 28 new RFLP, PCR and SSR markers were added to the existing map. The 1.8 cM Bo1 interval of wheat corresponds to a 227 kb section of rice chromosome 6L encoding 21 predicted proteins with no homology to any known B transporters. The co-dominant PCR marker AWW5L7 co-segregated with Bo1 and was highly predictive of B tolerance status within a set of 94 Australian bread wheat cultivars and breeding lines. The markers and rice colinearity described here represent tools that will assist B tolerance breeding and the positional cloning of Bo1. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.