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Identification of RFLP markers closely linked to the H1 gene conferring resistance to Globodera rostochiensis in potato

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Resistance to the root cyst nematode Globodera rostochiensis is an agronomic trait that is at present incorporated into most new potato varieties. Major dominant genes are available that originate from wild and cultivated Solanum species closely
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  Theor Appl Genet (1993) 85:541-544 9 Springer-Verlag 1993 Identification of RFLP markers closely linked to the HI gene conferring resistance to Globodera rostochiensis in potato C. Gebhardt 1 D. Mugniery 2 E. Ritter 3 E Salamini 1 and E. Bonnel 4 1 Max-Planck-Institut fiir Zfichtungsforschung, Carl-von-Linn~-Weg 10, D-5000 K61n 30, FRG 2 INRA, Domaine de la Motte au Vicomte, B.P. 29, F-35650, Le Rheu, France 3 Centro de Investigacion y Mejora Agraria (CIMA) Granja Modelo de Arkaute (Alava), Apdo. 46-01080 Vitoria-Gasteiz, Spain 4 GERMICOPA, 8, rue Olivier de Serres - B.P. 85, F-29103, Quimper Cedex, France Received August 30, 1992; Accepted September 18, 1992 Communicated by G. Wenzel Summary. Resistance to the root cyst nematode Glo- bodera rostochiensis is an agronomic trait that is at pres- ent incorporated into most new potato varieties. Major dominant genes are available that srcinate from wild and cultivated Solanum species closely related to the cul- tivated European potato Solanum tuberosum ssp. tubero- sum). One of those genes, H1, from S. tuberosurn ssp. andigena, was mapped to a distal position on potato chromosome V using restriction fragment length poly- morphism (RFLP) markers. The HI locus segregates in- dependently from Grol, a second dominant gene pre- sumably from S. spegazzinii that confers resistance to G. rostochiensis and which has been mapped to chromo- some VII. One marker, CPll3, was linked without re- combination to the H1 locus. Key words: Potato - G. rostochiensis - RFLP - Marker Introduction In potato the gene HI confers complete resistance to the root cyst nematode Globodera rostochiensis (Woll.) Behrens, pathotypes Rol and Ro4 (Kort et al. 1977). The gene was discovered in the Commonwealth Potato Col- lection (CPC) in 5 accessions (out of about 1300 tested) of Solanum tuberosum ssp. andigena (Ellenby 1954). Ge- netic analysis using selfed seeds of accession CPC 1673 revealed the presence of a single dominant gene named HI (Toxopeus and Huijsman 1953; Huijsman 1955). The germ plasm of CPC 1673 was subsequently incorporated into S. tuberosum ssp. tuberosum breeding lines and into many European varieties, among those cvs 'Granola', Correspondence to: C. Gebhardt 'Maris Piper' and others widely cultivated (Ross 1986). The availability of HI, together with a few other genes srcinating from S. vernei and S. spegazzinii, was suffi- cient to solve the problem of infection caused by patho- type Rol of G. rostochiensis (Ross 1986). The genetic relationship among the available genes conferring resistance to G. rostochiensis are to a large extent unknown. Moreover, the identity of resistance genes fom different sources is often unclear, as identifica- tion is based on their activity against different pathotypes of the nematode, and these pathotypes are themselves difficult to define and subject to change. Recently, we have mapped a major dominant gene conferring resis- tance to G. rostochiensis to chromosome VII of potato (Barone et al. 1990). This locus was named Grol and, based on pedigree information and resistance tests with different G. rostochiensis pathotypes, should correspond to the Fb gene of S. spegazzinii first described by Ross (1962). In the present article we report the genomic posi- tion of H1 as mapped in a genetic background where HI can be traced back to the founder clone CPC 1673. Materials and methods The HI gene was mapped in the progeny of a cross between the dihaploid line Amaryl H5 carrying H1 and a 2n Solanum phureja line (L85DS3.1) susceptible to the nematode. Amaryl H5 was obtained at the INRA Station de Recherches sur la Pomme de Terre et les Plantes A Bulbe, INRA K6raiber, Ploudaniel 29260 Lesneven, France, from the 4n variety 'Amaryl' via partheno- genesis after pollination with S. phureja (Hermsen and Verde- nius 1973). 'Amaryl' had been selected from the cross 'Saskia' x (CPC 1673-20 x Furore) (Joosten 1988). CPC 1673-20 was a resistant plant (genotype HHhh) srcinating from the selfing of S. tuberosum ssp. andigena accession CPC 1673 (Toxo- peus and Huijsman 1953). Fifty-three tetraploid varieties were obtained as described in G6rg et al. (1992). Twenty varieties were selected either for their  542 resistance to pathotypes Rol and Ro4 of G. rostochiensis (beschreibende Sortenliste Kartoffeln 1989) or for containing the HI gene (Ross 1986). These varieties were: 'Accent', 'Arnika', 'Barbara', 'Certo', 'Clarissa', 'Darwina', 'Granola', 'Ilse', 'Indira', 'Katja', 'Margit', 'Monza', 'Nora', 'Optima', 'Pamir', 'Panda', 'Pirola', 'Ponto', 'Producent', and 'Quarta'. Thirty-three susceptible varieties were also selected: 'Achat', 'Astrid', 'Atica', 'Carola', 'Cosima', 'Clivia', 'Datura', 'Desir6e', 'Erna', 'Erntestolz', 'Fausta', 'Forelle', 'Grandifolia', 'Grata', 'Hansa', 'Hela', 'Ilona', 'Irmgard', 'Isna', 'Isola', 'Linda', 'Luna', 'Mentor', 'Moni', 'Ostara', 'Palma', 'Prima', 'Saphir', 'Saskia', 'Selma', 'Sieglinde', 'Sommerstfirke' and 'Uila'. Resistance to the G. rostochiensis Rol population Ecosse was tested in a petri dish according to the method of Mugni6ry and Person (1976). Cuttings from plants of the first tuber gener- ation (Mugni6ry and Balandras 1986) were inoculated with five juveniles per root. A root system showing the presence of ne- matode females was rated as susceptible. Resistant genotypes showing the absence of cysts were transferred into pots with infested soil and examined after 3 months to confirm their resis- tance rating. Between 0.3 and 0.5 g freeze-dried leaf material per geno- type was used for extracting total genomic DNA. DNA extrac- tion, restriction digests, electrophoresis, blotting and hybridiza- tion methods have been described previously (Gebhardt et al. 1989). Linkage analysis was performed as in previous investiga- tions (Ritter etal. 1990; Barone et al. 1990). RFLP markers selected to cover all potato chromosomes (Gebhardt et al. 1991; unpublished results from this laboratory) were hybridized to filters with TaqI- or RsaI-restricted DNA of the parents and F~ progeny. Results One hundred and eleven hybrid lines of the cross Amaryl H5 x S. phureja were tested for resistance to G. ros- tochiensis, pathotype Rol. Amaryl H5 was totally resis- tant to Rol, while the S. phureja parent was susceptible. Among the seedlings, 58 were found to be resistant and 53 susceptible which supports the hypothesis that the H1 gene is heterozygous in Amaryl H5 and segregates in the cross in a 1:1 ratio (P>0.5). Out of the 111 hybrid lines scored for resistance, for practical reasons only 91 lines, 44 resistant and 47 suscep- tible, were screened for RFLP alleles that cosegregated with the resistance trait. Linkage was detected with six out of ten informative markers of potato chromosome V: GP22, GP265, GP270, CPll3, GP78 and GP188. One marker, CPll3, was linked with zero percent recombina- tion to the HI locus. The linkage map of potato chromo- some V, including the position of the H locus as deduced from recombination values measured in the resistant par- ent Amaryl H5, is shown in Fig. 1. No distortion of segregation ratios was observed for chromosome V in the cross studied. The length of the linkage map of chromo- some V of Amaryl H5 was 93 centiMorgans, which is a 20% increase over its length of 78 centiMorgans reported in the RFLP map of Gebhardt et al. (1991). Fifty-three tetraploid German potato varieties were surveyed for the presence of the RFLP allele that had been found to be totally linked in coupling to H1 in lg.4 3.4 27.1 6.7 8.1 10.8 10.0 GP I8 GP21 GP291 GP 17 a) GP 188 GP265 c) GP78 2.5 CPI13 HI 2.0 GP270 4.0 GP265 a) GP22 Fig. 1. Linkage map of potato chromosome V as deduced from recombination fi'equencies in Amaryl H5 carrying the HI gene. Map distances arc given in centiMorgans (Kosambi 1944). GP markers are derived from genomic DNA; CP markers from the cDNA of potato. Small letters in parenthesis indicate that more than one locus was mapped with the same marker probe. The cDNA marker CPI 13 is linked with zero percent recombination to the HI locus Amaryl H5. Their names are listed in the Materials and methods. Twenty varieties are described as being resis- tant to pathotypes Rol and Ro4 of G. rostochiensis in the Beschreibende Sortenliste Kartoffeln (1989) and in Ross (1986). The RFLP allele indicative of the H1 gene in Amaryl H5 was, however, not present in the germ plasm surveyed (data not shown). There was also no alternative RFLP allele detectable (with the same restriction enzyme and probe) by which resistant and susceptible varieties could be separated. Discussion The potential of RFLPs for marker-based selection was recognized 10 years ago (Beckmann and Soller 1983; Tanksley 1983). A prerequisite for using RFLP markers is that ones have to be identified that are closely linked to agronomically relevant genes. Resistance to the root 'cyst nematodes G. rostochiensis and G. pallida are con- sidered to be important traits in breeding programs. In this article we report a mapping experiment that identi- fies four RFLP markers linked, within 10 centiMorgans, to the HI gene conferring resistance to G. rostochiensis. One marker, CPII3, was shown to be linked to the HI locus without recombination, based on an Fa population of 91 genotypes. The HI locus maps at the end of potato  543 chromosome V and, therefore, segregates independently from the nematode resistance locus Grol, which has been mapped onto chromosome VII (Barone et al. 1990). HI is also genetically unrelated to the Mi locus that induces resistance to the root knot nematode Meloidogyne incog- nita; this locus is located on chromosome VI of the homeologous tomato genome (Klein-Lankhorst et al. 1991; Messeguer et al. 1991). Two other resistance loci have also been allocated to chromosome V: the Rx2 lo- cus conferring extreme resistance to potato virus X (Rit- ter et al. 1991) and RI conferring race-specific resistance to Phytophthora infestans (Leonards-Schippers etal. 1992). Both of these latter loci map close to the marker locus GP2I and, therefore, are located in a segment of chromosome V that is 63 centiMorgans away from CPll3 (Fig. t). If we also take Pto, a Pseudomonas resis- tance gene identified on tomato chromosome V (Martin et al. 1991), into consideration and if we assume homeol- ogy between the potato and tomato genome for resis- tance loci, chromosome V appears to be particularly rich in disease resistance loci. There may be several reasons for the absence of the RFLP allele linked to HI in Amaryl H5 in a series of resistant potato varieties having reported pathotype specificity of H1. First, and this seems the most likely explanation, the source of resistance in the varieties test- ed was not the same as in Amaryl H5. Genetic evidence, for example, indicated that resistant germ pIasm derived from S. vernei in some cases contains the same genetic locus as that present in S. tuberosum ssp. andigena CPC 1673 (Scurrah et al. 1973). S. vernei, in addition to CPC 1673, has also been widely used in potato breeding for introducing resistance to G. rostochiensis. Second, in the S. tuberosum ssp. andigena germ plasm from which the HI gene srcinated, more than one HI allele was present in a polymorphic genetic background. Third, during ex- tended backcross breeding the linkage between H1 and closely linked marker sequences have been broken. To be able to use maker CPll3 for selecting H1 genotypes, it is therefore necessary- as in human genetics - to determine the informative allele or the marker phase in the germ plasm employed in crosses. A positive and important outcome of this study is that the selection of Grol, Itl genotypes is now feasible based on the RFLP markers flanking both loci. This should result in phenotypes having highly durable resis- tance to most, if not all pathotypes of G. rostochiensis. Acknowledgements. The authors thank H. Thelen for technical assistance and M. Pasemann for preparing the manuscript. The project was supported by the Bundesministerium ffir Forschung und Technologic (BMFT) under Project no. 1.10. References Barone A, Ritter E, Schachtschabel U, Debener T, Salamini F, Gebhardt C (1990) Localization by restriction fragment length polymorphism mapping in potato of a major domi- nant gene conferring resistance to the potato cyst nematode Globodera rostochiensis. Mol Gen Genet 224:177-182 Beckman JS, Soller M (1983) Restriction fragment length poly- morphisms in genetic improvement: methodologies, map- ping and costs. Theor Appl Genet 67:35-43 Beschreibende Sortenliste Kartoffeln (1989) Bundessortenamt (ed). Verlag Alfred Strohte, Frankfurt Ellenby C (1954) Tuber-forming species and varieties of the ge- nus Solanum tested for resistance to the potato root eelworm Heterodera rostochiensis Wollenweber. Euphytica 3:195-202 Gebhardt C, Ritter E, Debener T, Schachtschabel U, Walke- meier B, Uhrig H, Salamini F (1989) RFLP-analysis and linkage mapping in Solarium tuberosum. Theor Appl Genet 78:65-75 Gebhardt C, Ritter E, Barone A, Debener T, Walkemeier B, Schachtschabel U, Kaufmann H, Thompson RD, Bonierbale MW, Ganal MW, Tanksley SD, Salamini F (1991) RFLP maps of potato and their alignment with the homoeologous tomato genome. Theor Appl Genet 83:49-57 G6rg R, Schachtschabel U, Ritter E, Salamini F, Gebhardt C (1992) Discrimination among 136 tetraploid potato varieties by fingerprints using highly polymorpbic DNA markers. Crop Sci 32:815-819 Hermsen JGTh, Verdenius J (1973) Selection from Solanum tuberosum group phureja of genotypes combining high-fre- quency haploid induction with homozygosity for embryo spot. Euphytica 22:244-259 Huijsman CA (1955) Breeding for resistance to the potato root eelworm. II. Data on the inheritance of resistance in andi- genum-tuberosum crosses obtained in 1954. Euphytica 4:133-140 Joosten A (1988) Geniteurslijst voor aardappelrassen. Com- missie ter bervordering van het kweken en het oderzoek van nieuwe aardappelrassen (C.O.A.), Postbus 32, NL-6700 AA Wageningen, The Netherlands Klein-Lankhorst R, Rietveld P, Machiesls B, Verkerk R, Weide R, Gebhardt C, Koornneef M, Zabel P (1991) RFLP markers lii~ked to the root knot nematode resistance gene Mi in tomato. Theor Appl Genet 81:661- 667 Kort J, Ross H, Rumpenhorst H J, Stone SR (1977) An interna- tional scheme for identifying and classifying pathotypes of potato cyst nematodes Globodera rostochiensis and G. palli- da. Nematologica 23:333-339 Kosambi DD (1944) The estimation of map distances from re- combination values. Ann Eugen 12:172-175 Leonards-Schippers C, Gieffers W, Salamini F, Gebhardt C (1992) The R1 gene conferring race-specific resistance to Phytophthora infestans in potato is located on potato chro- mosome V. Mol Gen Genet 233:278-283 Martin GB, Williams JGK, Tanksley SD (1991) Rapid identifi- cation of markers linked to a Pseudomonas resistance gene in tomato by using random primers and near-isogenic lines. Proc Natl Acad Sci USA 88:2336-2340 Messeguer R, Ganal M, de Vicente MC, Young ND, Bolkan H, Tanksley SD (1991) High resolution RFLP map around the root knot nematode resistance gene Mi) in tomato. Theor Appl Genet 82:529-536 Mugni~ry D, Balandras C (1986) Mise au point d'un test de resistance pr6core des pomme de terre ~ Globodera sp. Pota- to Res 29:45-53 Mugni~ry D, Person F (1976) M~thode d61evage de quelques n6matodes fi Kystes du genre Heterodera. Sci Agron Rennes, pp 217-222 Ritter E, Gebhardt C, Salamini F (1990) Estimation of recombi- nation frequencies and construction of RFLP linkage maps  544 in plants from crosses between heterozygous parents. Genet- ics 125:645-654 Ritter E, Debener T, Barone A, Salamine F, Gebhardt C (1991) RFLP mapping on potato chromosomes of two genes con- trolling extreme resistance to potato virus X (PVX). Mol Gen Genet 227:81-85 Ross H (1962) fQber die Vererbung der Resistenz gegen den Kartoffelnematoden Heterodera rostochiensis Wolf.) in Kreuzungen yon Solarium famatinae Bitt. et Wittm. mit Solarium tuberosum L. und mit S. chacoense Bitt. Der Zuechter 32: 74- 80 Ross H (1986). Potato breeding - problems and perspectives. J Plant Breed [Suppl 13] Scurrah MM de, Plaisted RL, Harrison ML (1973) Resistance to the potato nematode Heterodera rostoehiensis Woll. in clones derived from Solanurn vernei. Am Potato J 50:9-18 Tanksley SD (1983) Molecular markers in plant breeding. Plant Mol Biol Rep 1:3-8 Toxopeus H J, Huijsman CA (1953) Breeding for resistance to potato root eelworm. I. Preliminary data concerning the inheritance and the nature of resistance. Euphytica 2:180- 186
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