Mapping a Gene Conferring Resistance to Pseudocercosporella Herpotrichoides on Chromosome 4V of Dasypyrum Villosum in a Wheat Background

1 Mapping a gene conferring resistance to Pseudocercosporella herpotrichoides on chromosome 4V of Dasypyrum villosum in a wheat background

Ahmet Yildirim, Stephen S. Jones, and Timothy D. Murray

Abstract: The objectives of this study were to map and tag the previously undescribed eyespot resistance gene PchDv on

chromosome 4V of Dasypyrum villosum in a wheat background. The 82 F2 plants used for mapping were produced from a cross between a susceptible wheat ‘Yangmai–5’ (4V(4D)) substitution line and a resistant wheat ‘Chinese Spring’ disomic

addition line of chromosome 4V of D. villosum. Segregation for resistance and susceptibility among F2 plants was 3:1, indicating that resistance was controlled by a single dominant gene. PchDv mapped to the distal part of chromosome 4V and was bracketed by two RFLP markers, Xcdo949 and Xbcd588, in a 33-cM interval. This distance could not be reduced, owing to a lack of polymorphic loci in this region. Theoretically, double recombination in this region occurs in 3.3% of the individuals; therefore, 96.7% of the selected genotypes would have PchDv, with simultaneous selection for both flanking

markers. Double recombination between the flanking markers was observed in 2 out of 82 (2.4%) F2 individuals. Key words: eyespot, Haynaldia villosa, Triticum aestivum, RFLP, wild wheat.

Résumé : L’objectif de cette étude était de déterminer l’emplacement chromosomique et de marquer un gène de résistance au piétin-verse qui n’avait pas encore été décrit, PchDv, sur le chromosome 4V du Dasypyrum villosum dans un fond génétique

de blé. Les 82 plantes F2 employées pour la cartographie ont été produites par croisement entre une lignée de substitution sensible du blé ‘Yangmai–5’ (4V(4D)) et une lignée d’addition résistante disomique du blé ‘Chinese Spring’ portant le

chromosome 4V du D. villosum. Un ratio de ségrégation de 3 résistantes : 1 sensible parmi les plantes F2 a révélé que la résistance était sous le contrôle d’un seul gène dominant. Le gène PchDv a été assigné à un intervalle de 33 cM sur le partie distale du chromosome 4V entre les marqueurs RFLP Xcdo949 et Xbcd588. La distance n’a pu être réduite davantage en raison de l’absence de loci polymorphes dans cette région. Théoriquement, des doubles recombinaisons dans cette région surviennent dans 3,3% des individus, ainsi 96,7% des génotypes sélectionnés à l’aide des deux marqueurs posséderont le gène

PchDv. Parmi les 82 plantes F2 examinées, deux double recombinants (2,4%) ont été observés. Mots clés : piétin-verse, Haynaldia villosa, Triticum aestivum, RFLP, blé sauvage. [Traduit par la Rédaction]

Introduction Aegilops ventricosa (Dosba and Dossinault 1973) and is used extensively in breeding programs because of tightly linked Eyespot, caused by the facultative parasite Pseudocerco- isozyme (McMillin et al. 1986) and RFLP markers (Chao et al. sporella herpotrichoides (Fron.) Deighton, is an economically 1989). Pch1 alone does not provide complete resistance to the important disease of wheat in the U.S. Pacific Northwest and disease, but is more effective than the second gene, Pch2. Pch1 many other winter wheat growing areas of the world (Johnson occurs on the long arm of chromosome 7D and, recently, Pch2 1992; Wiese 1991). Previously, there were only two resistance was mapped to the long arm of chromosome 7A (de la Peña genes known for eyespot. The first, Pch1, was transferred from et al. 1997). Occurrence of new pathotypes of the pathogen increases the possibility of circumventing existing resistance Corresponding Editor: J.P. Gustafson. (Jones et al. 1995). Dasypyrum villosum (2n = 14, genome VV), a distant rela- Received May 28, 1997. Accepted September 16, 1997. tive of wheat, was identified as being highly resistant to eye- A. Yildirim1 and S.S. Jones.2 Department of Crop and Soil spot, and a resistance gene(s) was located in a chromosome 4V Sciences, Washington State University, Pullman, WA addition line (Murray et al. 1994). Subsequently, it was deter- 99164–6420, U.S.A. mined that a disomic wheat – D. villosum 4V(4D) substitu- T.D. Murray. Department of Plant Pathology, Washington tion line was susceptible to eyespot (Yildirim et al. 1997; State University, Pullman, WA 99164–6430, U.S.A. A. Yildirim, unpublished data). The existence of the suscepti- 1 Present address: Gaziosmanpasa University Ziraat Fakültesé, ble 4V substitution line makes it possible to map the resistance 60110 Tokat, Turkey. gene in a wheat background, which is advantageous, because 2 Author to whom all correspondence should be addressed the chromosome is homozygous in a disomic state. Dasypyrum (e-mail: [email protected]). villosum is a cross-pollinating diploid species (Frederiksen

Fig. 1. C-banded somatic chromosomes of CS + 4V (a) and ‘Yangmai–5’ (4V(4D)) (b). Arrows point to D. villosum chromosome 4V.

1991) and, theoretically, the genetic constitution is mostly het- prevent disease. In fact, ‘Rendezvous’, which reportedly pos- erozygous, thus complicating standard F2 mapping protocols. sesses both genes (Hollins et al. 1988), and ‘Madsen’ and In addition, inbreeding depression and self-incompatability ‘Hyak’, which are the only resistant cultivars in the U.S. (Allan make the production of homozygous lines difficult. Moreover, et al. 1989, 1990), still sustain yield losses when disease is the level of resistance in the disomic addition line of chromo- severe (Jones et al. 1995). Such losses demonstrate the need some 4V in a ‘Chinese Spring’ background was shown to be for cultivars with improved eyespot resistance, produced either equal to D. villosum itself (Murray et al. 1994). The level of by identifying new sources of resistance or through pyramid- resistance in a hexaploid background conferred by a gene on ing resistance genes in the same cultivar. a D. villosum chromosome shows that this gene has potential The objective of this study was to map PchDv on chromo- in wheat improvement programs. some 4V, with the ultimate goal of the rapid and successful The success and accuracy of a map strongly depend on the transfer of the gene into a stable wheat background. precise identification of the individual genotypes in the mapping population. Previously, the mapping of eyespot resistance Materials and methods genes and the development of cultivars with this resistance was slowed by an inability to recognize and select for resistant Genetic stocks genotypes with certainty. Field evaluations based on visual A disomic chromosome 4V addition line of D. villosum in a ‘Chinese Spring’ background (CS + 4V) (2n = 44) (Hyde 1953; Sears 1953) scores did not unambiguosly differentiate between highly re- was obtained from the Wheat Genetics Resource Center, Kansas State sistant, resistant, and susceptible genotypes (Jones et al. 1995). University, Manhattan, Kans. An experimental wheat line with chro- Recently, an improved technique for evaluating resistance was mosome 4V substituted for chromosome 4D in the cultivar ‘Yangmai–5’ described that uses a GUS (β-glucuronidase) transformed (4V(4D)) (2n = 42) was provided by Dr. P.D. Chen, Cytogenetics strain of P. herpotrichoides (de la Peña and Murray 1994). In Institute, Nanjing Agricultural University, Nanjing, China. The lines this technique, disease development in 4- to 8-week-old seed- VPM–1 (resistant breeding line) and ‘Chinese Spring’ (susceptible) lings is scored on the basis of the amount of GUS. The amount were used as controls for eyespot reaction. of fungal growth on young seedlings, and thus disease severity, is directly related to the amount of GUS activity. Therefore, Mapping population A genetic map of chromosome 4V was constructed using a population the method clearly differentiates among highly resistant, resis- of 82 F2 plants produced from a cross between the resistant disomic tant, and susceptible genotypes. CS + 4V addition line and the susceptible ‘Yangmai–5’ (4V(4D)) Identification and transfer of new genes for eyespot resis- substitution line. Initially, 98 F2 plants were produced, but only 82 of tance is important, since neither Pch1 nor Pch2 completely them were used in the construction of the map, owing to insufficient

Fig. 2. Southern blot of EcoRV-, XbaI-, and SalI-digested genomic Fig. 3. In situ hybridization pattern of MI in pollen mother cells of + + DNA of ‘Chinese Spring’ (lanes 1), CS 4V (lanes 2), and F1 hybrid between CS 4V and ‘Yangmai–5’ (4V(4D)) hybridized ‘Yangmai–5’ (4V(4D)) (lanes 3), using p380. with D. villosum specific probe p380. The arrow indicates paired 4V chromosomes and the open arrowhead indicates the univalent 4D chromosome.

amounts of F2 DNA and (or) delayed germination. Chromosome 4V from both parents was expected to pair in F1 hybrids, producing a trisomic individual with 2n = 43 chromosomes. Homologous recombination between the two 4V chromosomes was required for mapping. Chromosome 4D from the addition line would be univalent in the hybrids, and in most cases was expected to be lost in the next generation. Aneuploid lines of ‘Chinese Spring’ (Sears 1954) were used to verify chromosomes involved in polymorphic loci.

Eyespot reaction and statistical analysis A genetically modified strain of P. herpotrichoides, with growth rate and pathogenicity similar to its parental strain but expressing GUS, MAPMAKER computer program was used to determine recombination was used to identify resistant plants. Seedlings were inoculated at the frequencies and locus order (Lander et al. 1987). two-leaf stage and the disease reaction was scored as described previously by Yildirim et al. (1995). GUS values of F2 plants were dif- C-banding ferentiated by Fisher’s least significant difference (LSD) (P = 0.05), The presence of chromosome 4V in the parents was confirmed by based on the standard error of mean differences of the parents (Little C-banding. Mitotic chromosomes were prepared for C-banding ac- and Hills 1978). F2 plants with GUS values that exceeded the mean of cording to Cai et al. (1996). C-banding was performed as described the resistant parent (CS + 4V) value plus the LSD value were consid- by Gill et al. (1991a). ered susceptible. In situ hybridization Isolation of genomic DNA and Southern hybridization The pairing behaviour of 4V chromosomes in F1 plants was checked by in situ hybridization (ISH). Anthers of F plants at meiotic meta- Prior to disease evaluation, leaves were harvested from each F2 plant 1 phase I (MI) were collected and fixed in ethanol – acetic acid (3:1) at for DNA extraction. Genomic DNA isolation, from individual F2 plants and single-enzyme digests of genomic DNA with restriction room temperature for 2–3 days, or at 4°C for longer periods of time endonucleases BamHI, DraI, EcoRI, EcoRV, HindIII, PstI, XbaI, or (1–8 weeks). Anthers at MI were squashed in 45% acetic acid after XhoI, and Southern transfers were performed according to Cadle et al. staining in 2% acetocarmine for 10 min. Cover glasses were removed (1994). The DNA blots were prehybridized for at least 6 h at 65°C in by freezing the slides at –80°C. A highly repetitive D. villosum specific probe, p380 (obtained from C. De Pace, Department of Agricul- a mixture of 7% SDS, 0.25 M Na2HPO4 (pH 7.2), and 1 mM EDTA (pH 8.0) (Church and Gilbert 1984). After hybridization with tural Biology and Chemistry, University of Tuscia, Viterbo, Italy), [32P]dCTP labelled probe and washing, blots were exposed to x-ray was labelled with biotin-16-dUTP by nick translation (Enzo Diagnos- film (Eastman Kodak Co., Rochester, N.Y.) for 5–10 days at –80ºC, tics Nick Translation Kit). Probe hybridization and signal detection depending upon the intensity of the signals. were performed according to Friebe et al. (1993).

Detection of polymorphism and linkage analysis Results Seventy-seven single-copy cDNA and genomic clones containing homologous sequences on the long arm of group 4 chromosomes of Genetic constitution of the parents, meiotic pairing, and wheat were used to detect polymorphism between the parents. Probes F2 plants that revealed polymorphism were used for segregation analysis. The The cross between CS + 4V (2n = 44) and ‘Yangmai–5’

+ Fig. 4. Frequency distribution of eyespot reactions of 82 F2 plants derived from a cross between CS 4V and ‘Yangmai–5’(4V(4D)) inoculated with a genetically modified strain of P. herpotrichoides expressing GUS. The severity of the eyespot reactions was determined using a GUS assay and was expressed as nanomoles of methylumbelliferone (MU) per plant. Mean GUS activities for CS + 4V and ‘Yangmai–5’ (4V(4D)) were 99.5 and 460.2 nmol MU/plant, respectively. (LSD = 96.4; P = 0.05).

(4V(4D)) (2n = 42) is complex in terms of the fate of the 4D phic with one or more of the 5–8 restriction enzymes. The and 4V chromosomes. The presence of chromosome 4V in remaining clones did not hybridize to chromosome 4V, were both parents was confirmed by C-banding analysis (Fig. 1) and polymorphic on other chromosomes, or were not polymorphic by Southern hybridization with the D. villosum specific probe for any locus. An RFLP linkage map of the long arm of p380 (Fig. 2). F1 plants had 43 chromosomes as expected; in chromosome 4V, consisting of 13 loci and including PchDv, all 100 meiotic cells observed, ISH with p380 revealed that the was constructed (Fig. 5). The PchDv locus mapped to the distal 4V chromosomes paired normally, whereas chromosome 4D part of chromosome 4VL and was bracketed by RFLP markers from the addition line was present as a univalent (Fig. 3). Most Xcdo949 and Xbcd588 in a 33-centimorgan (cM) interval. Seg- χ2 of the unpaired 4D chromosomes in the F2 were expected to be regation data for each RFLP locus was analyzed using the lost; in fact, on the basis of Southern hybridization, 65% of the test (P = 0.05) and showed a good fit to a 1:1 ratio. F2 individuals lost chromosome 4D. Southern hybridization of F plants with the D. villosum specific clone verified the pres- 2 Discussion ence of chromosome 4V in each F2 individual. A new eyespot resistance gene, derived from D. villosum, was Disease reaction mapped to the distal part of the long arm of chromosome 4V The frequency distribution for eyespot reactions of the 82 in a wheat background. This gene, designated PchDv (R.A. F2 plants based on GUS scores was skewed towards resistance McIntosh, personal communication), was located in a 33-cM (Fig. 4). Sixty-five F2 plants were resistant and 17 were suscepti- interval bracketed by RFLP markers Xcdo949 and Xbcd588. ble. The ratio between resistant and susceptible plants was 3:1 Xcdo949 was 16 cM from PchDv and is the most distal probe (P = 0.05), supporting the hypothesis that resistance was con- mapped on chromosome 4DL (Nelson et al. 1995); Xbcd588, trolled by a single dominant gene. The mean GUS values for one of the most distal probes mapped on chromosome 4AL the resistant parent, CS + 4V, and the resistant check, VPM–1, (Nelson et al. 1995), was 17 cM from PchDv. This region were 99.5 ± 2.3 nmol methylumbelliferone (MU)/plant and showed very low polymorphism. The rarity of polymorphic 86.6 ± 7.3 nmol MU/plant, respectively. The values for the markers in this region is also evident in the barley chromosome 4 susceptible parent, ‘Yangmai–5’ (4V(4D)), and the susceptible map of Kleinhofs et al. (1993) and in the wheat chromosome 4 check, ‘Chinese Spring’, were 460.2 ± 60.1 nmol MU/plant maps of Gill et al. (1991b), Hart et al. (1993), Gale et al. and 392.8 ± 90.9 nmol MU/plant, respectively. The LSD value (1995), and Nelson et al. (1995). for the parental means was 96.4 nmol MU/plant (P = 0.05). Although the distance between the flanking markers and PchDv is large, simultaneous selection for both flanking RFLP Map construction and linkage analysis markers could still provide an efficient selection of PchDv, Twelve clones of 77 chromosome group 4L RFLP (restriction because the theoretical chance of recovering the gene would fragment length polymorphism) probes tested were polymor- be 96.7%. Transfer of PchDv to wheat would also be more

Fig. 5. Genetic linkage map of the long arm of chromosome 4V of map, these distances were 73 and 37.3 cM, respectively. + × D. villosum constructed from 82 F2 plants derived from CS 4V Xpsr104 and Xpsr163 mapped to the short arm of chromosome ‘Yangmai–5’ (4V(4D)). Map distances and locus order were 4A, but mapped to the long arms of chromosomes 4B and 4D, determined using MAPMAKER 3.0 (Lander et al. 1987). with the same distance (2.7 cM), but in reverse order (Gale et al. 1995). We have also mapped these loci on the long arm of chromosome 4V in an order that was consistent with that on chromosomes 4BL and 4DL. These results are in agreement with a previously reported pericentric inversion on chromosome 4AL of wheat relative to chromosomes 4Am, 4B, and 4D (Naranjo 1990; Devos et al. 1995). The mapping population, and the approach of using this type of cross to map a non-wheat gene, made the cytological characterization of both 4V chromosomes extremely important. Mitotic C-banding patterns of the parents in this study revealed 4V chromosomes in both parents (Fig. 1). In addition, ISH with p380 verified that chromosome 4V was pairing normally and that the extra univalent was a wheat chromosome (Fig. 3). These results eliminated any doubts about recombination events on 4V chromosomes, an element needed for a successful map. This technique has proven useful for mapping homozygous 4V chromosomes from different sources and has eliminated the need to deal with the heterozygosity of D. villosum at the diploid level. Mapping PchDv on chromosome 4V in a wheat background will provide a more efficient ma- nipulation of the gene through molecular assisted selection techniques.

Acknowledgments Special thanks are extended to Drs. B.S. Gill and P.D. Chen for providing CS + 4V and ‘Yangmai–5’ (4V(4D)) seeds, and to A. Kleinhofs, C. De Pace, M.D. Gale, M.E. Sorrells, and P. Leroy for providing the clones used in this study.

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