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Localization by restriction fragment length polymorphism mapping in potato of a major dominant gene conferring resistance to the potato cyst nematode Globodera rostocbiensis

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A major dominant locus conferring resistance against several pathotypes of the root cyst nematode Globodera rostochiensis was mapped on the linkage map of potato using restriction fragment length polymorphism (RFLP) markers. The assessment of
  Mol Gen Genet (1990) 224:177-182 © Springer-Verlag 1990 Localization by restriction fragment length polymorphism mapping in potato of a major dominant gene conferring resistance to the potato cyst nematode lobodera rostochiensis Amalia Barone , Enrique Ritter, Undine Schachtschabel, Thomas Debener, Francesco Salamini and Christiane Gehhardt Max-Planck-Institut fiir Ziichtungsforschung, Carl-von-Linn&Weg 10, W-5000 Kgln 30, Federal Republic of Germany Received May 18, 1990 Summary. A major dominant locus conferring resistance against several pathotypes of the root cyst nematode Globodera rostochiensis was mapped on the linkage map of potato using restriction fragment length polymor- phism (RFLP) markers. The assessment of resistance versus susceptibility of the plants in the experimental population considered was based on an in vivo (pot) and an in vitro (petri dish) test. By linkage to nine RFLP markers the resistance locus Grol was assigned to the potato linkage group IX which is homologous to the tomato linkage group 7. Deviations from the additivity of recombination frequencies between Grol and its neighbouring markers in the pot test led to the detection of a few phenotypic misclassifications of small plants with poor root systems that limited the observation of cysts on susceptible roots. Pooled data from both tests provided better estimates of recombination frequencies in the linkage interval defined by the markers flanking the resistance locus. Key words: Restriction fragment length polymorphism - Potato - Globodera rostochiensis - Resistance - SoIa- num spegazzinii Introduction GIobodera rostochiensis (Woll.) Behrens and G. pallida (Stone) Behrens are two cyst-forming nematode species, which parasitize the roots of the potato and other species of the Solanaceae, and cause considerable yield reduc- tion. In both Globodera species several pathotypes exist (Kort et al. 1977). Whereas in potato the available genet- ic resistances to G. pallida are incomplete and based on polygenic systems (Dellaert et al. 1988; Ross 1986), dominant major genes srcinating from different Sola- num species are known which confer complete resistance * Present address: Department of Agronomy and Plant Genetics, University of Naples, 1-80055 Portici, Naples, Italy Offprint requests to: C. Gebhardt to different pathotypes of G. rostochiensis. These genes have been incorporated into many potato cultivars (Ross 1986); however, none of them has been genetically mapped, neither in relation to each other nor to any other marker. A new class of molecular markers, termed RFLPs (restriction fragment length polymorphism) allows the construction of dense genetic maps. The RFLP markers can be used to map the position of major genes in the genome, as well as genetic loci affecting polygenically inherited traits (Tanksley et al. 1989). This offers the prospect of applying marker-based selection schemes in breeding programmes. Based on the known chromosom- al location of the Fusarium oxysporum resistance gene I2 in tomato, the use of RFLPs mapping to the same chromosome led to the identification of a closely linked RFLP marker (Sarfatti et al. 1989). Resistance alleles can now be selected using DNA patterns, thereby avoid- ing the possibility of phenotypic misclassification of the resistance trait. With similar information concerning the position on the genetic map of maize, McMullen and Louie (1989) used RFLPs to map more precisely a resis- tance gene to maize dwarf mosaic virus (MDMV). In the two cited cases the genetic analysis was based on nearly isogenic lines (NILs). Availability of NILs also facilitated the detection of RFLP markers closely linked to the Tm-2a locus conferring resistance to tobacco mo- saic virus (Young et al. 1988), while the use of addition lines helped to identify RFLP markers linked to a resis- tance gene against Heterodera schachtii in sugar beet (Jung et al. 1990). The complete RFLP map was not essential for the approach used by the latter two groups. For the cultivated tetraploid potato neither a classi- cal genetic map, isogenic lines nor addition lines are available. Recently, a RFLP map was constructed com- prising at present ca. 300 RFLP loci (Gebhardt et al. 1989; unpublished results from the same laboratory). In this paper we describe the first application of this RFLP map to localize a major resistance gene in a popu- lation derived from a cross between a parent heterozy- gous for the resistance gene and a susceptible parent. Both parents were diploid and highly heterozygous.  178 Materials and methods Plant material. Two diploid potato clones, H82.337/49 and H80.696/4, from the collection at the Max-Planck- Institut fiir Zfichtungsforschung were crossed to produce an F1 population. Clone H80.696/4 (in the text referred to as PR) was an interspecific hybrid between S. spegaz- zinii and S. tuberosum ssp. tuberosum. It is resistant to G. rostochiensis, pathotypes Rol and Ro5. The S. tubero- sum ssp. tuberosum clone H82.337/49 (referred to as Ps) is susceptible. At random, 124 F1 seeds were chosen to constitute the segregating offspring. Seedlings were planted in pots and finally 100 genotypes propagated in the greenhouse. Leaves and shoots were harvested and freeze dried for DNA extraction and RFLP analysis. First generation tubers were used for inoculation with G. rostochiensis. Resistance tests for Globodera rostochiensis. Two differ- ent methods were used: 1. Pot test. In spring, tubers (three replications per geno- type) were grown in 11 cm pots in soil obtained from fields (Bordenau, Hannover) highly contaminated with cysts of G. rostochiensis pathotype Rol. The root sys- tems were checked 7-8 weeks after germination for the presence of cysts, indicating that the plants were suscep- tible. The cysts were not washed out and only a visual evaluation of the peripheral root system was performed. A genotype was scored as resistant if none of the three replicates had cysts and as susceptible if at least one of the three replicates showed cysts. 2. Petri dish test. A modified resistance test described by Mugni~ry and Balandras (1986) was applied. Tubers were washed in water, 70 ethanol and water again. Small tuber pieces with at least one eye were placed on 2 agar-agar in petri dishes and incubated at 20 ° C in the dark for 4-5 days. The developing roots were inoculated with 3-5 freshly hatched Rol larvae per root (cysts were provided by Dr. Mugni6ry, INRA, F-35650 Le Rheu, France). After incubation for 3-4 weeks at 20°C in the dark, newly formed cysts were visible on susceptible roots. RFLP analysis. DNA extraction, restriction digests, elec- trophoresis, blotting and hybridization procedures were as described previously (Gebhardt et al. 1989). Only CsC1 purified DNA was used and three restriction en- zymes, TaqI, RsaI and AluI were employed. Probes. The inserts of 32 genomic and 25 cDNA potato clones mapping to the 12 linkage groups of potato (Geb- hardt et al. 1989; this laboratory, unpublished results) were used as probes. In addition, the two genomic toma- to clones TG20 and TG61 were used which were pro- vided by S.D. Tanksley (Cornell University, Ithaca, New York, USA). Linkage analysis. Data analysis, linkage tests and two- point estimates of recombination frequencies were per- formed as described by Gebhardt et al. (1989) and Ritter et al. (1990). In the linkage analysis, resistance was treat- ed as a single segregating restriction fragment. The order of linked loci was determined by multipoint estimates computed with algorithms given in Morton et al. (1986) and Lander and Green (1987). The EM algorithm was used for handling missing data (Little and Rubin 1987; Lander and Green 1987). Results Segregation of the nematode resistance gene In a set of 38 diploid potato clones used for RFLP analy- sis one genotype, an interspecific hybrid between S. tu- berosum ssp. tuberosum and S. spegazzinii (H80.696/4, line 40, Gebhardt et al. 1989), was known to be resistant against the G. rostochiensis pathotypes Rol and Ro5 (J. Hesselbach and H. Hemme, unpublished results). F1 seeds were available from a cross between this resistant clone and the susceptible clone H82.337/49. One hundred individual genotypes were grown from the F1 seeds and further propagated by tubers. Around 25 of the plants had a small growth habit, low vigour and did not consistently produce tubers. Eighty-three geno- types were classified by the pot test for resistance to G. rostochiensis, pathotype Rol. The segregation in 40 resistant and 43 susceptible plants fitted the 1:1 ratio with a X 2 value of 0.108, as expected for the segregation of a trait controlled by a single dominant gene, with PR being heterozygous for the resistance allele and Ps homozygous susceptible. In the pot test, PR and Ps plants were always found to be resistant and susceptible, re- spectively. RFLP analysis Total genomic DNA was extracted from the 100 avail- able F1 genotypes, digested either with TaqI, RsaI or AluI, size separated by electrophoresis in denaturing polyacrylamide gels and electroblotted onto nylon mem- branes. The filters were hybridized against a set of marker probes selected by two criteria. First, the markers should be evenly spaced on their linkage groups covering with a minimum number a maximum of the map length. Second, the markers should reveal heterozy- gous restriction fragments specific for the resistant par- ent in order to be useful in linkage detection. Markers revealing fragments heterozygous in both parents (and therefore segregating 3:1 in the progeny of the cross) would be less efficient due to the higher standard error of the estimate of the recombination frequency, whereas fragments segregating due to a heterozygous state of the susceptible parent would be useless for detection of linkage with the resistance allele. Based on these criteria, informative markers were identified, by scoring the RFLP patterns available for 38 diploid potato clones including the two parents of the F1 population on which this work is based (Gebhardt et al. 1989). Hybridizations with 57 potato probes and 1 tomato probe (TG20, see Materials and methods), permitted the  PPF1 SRRSSRSRRSR? S?RSRSRR??R SRS??SSSSSS?RSS? ?RR??RRR 179 832 ......... 700 -- ~ I -- I -- i lilfl ~ OI I # a b 525 ~ ~'~ ~ ~m~ ,-, ~ -- o~ ~ '~m -,'~ ~ 447 ~ ~ " ""~ ~ ,~ .,. i~ ~ila, ,,m~ ~ aiD" Fig. 1. Consegregation of the restriction fragment ength polymor- phism marker CP56 with the nematode resistance ocus GroI. Ge- nomic DNA was digested with AluI, separated in a 4 denaturing polyacrylamide gel, transferred to a nylon membrane and hybrid- ized to the 32P-labelled probe CP56 (480 bp). S, susceptible; R, resistant; ?, resistance status not determined; Ps, susceptible parent mip e H82.337/49; PR, resistant parent H80.696/4; F1, progeny ofPs x PR. The fragment identifition s given on the right, the fragment sizes in bases on the left. Presence of fragment c is linked o susceptibili- ty, absence to resistance, with the exception of recombinants indi- cated by the asterisks evaluation of 161 segregating fragments, 73 from the resistant parent PR, 55 from the susceptible parent Ps and 33 from both. The segregation of 50 fragments de- viated significantly (P<5 ) from the expected ratios (1:1 for a heterozygous fragment present in only one parent and 3:1 for a heterozygous fragment present in both parents, see also Ritter et al. 1990), 34 of which were inherited from PR, 12 from both parents and only four from Ps. Cosegregation between the nematode resistance phe- notype as determined in the pot test and a particular RFLP was first detected with the marker CP56 from linkage group IX (numbering of Gebhardt et al. 1989). This is illustrated in Fig. 1. Fragment c segregating from the resistant parent PR was linked to the resistance locus : its presence was linked to susceptibility and its absence to resistance, with the exception of two recombinants, The other segregating fragments a, d and e were compo- nents of the same genetic locus. The complete pattern of five fragments including the non-segregating fragment b can be explained by segregation of four alleles each one composed of two fragments, with Ps having the allel- ic constitution (ad)/(be) and PR carrying (bd)/(bc) (see Gebhardt et al. 1989 for details on RFLP mapping using 4 bp recognition enzymes and Ritter et al. 1990 for a discussion on fragment configurations). Available markers mapping to the same chromosomal region as CP56 were tested. Linkage was detected to eight further probes mapping to linkage group IX (Fig. 2, probes GP219, CPI03, GP127, TG20, CP51, GP77, GP32, GP27), four of which detected additional loci (indicated in Fig. 2 by the bracketed small letters) on other linkage groups. One of these markers, TG20, was a reference probe for tomato chromosome 7 and has been mapped to a homologous position in potato (Bonierbale et al. 1988). Together with the second probe TG61 from toma- to chromosome 7, also mapping to potato linkage group IX in the cross used for map construction (Fig. 2A), TG20 aligned the potato linkage group IX of Gebhardt et al. (1989) to the homologous tomato/potato chromo- some 7 of Bonierbale et al. (1988). Recombination frequencies between the G. rosto- chiensis resistance locus, indicated by the symbol Grol, and linked RFLP marker fragments were determined with two-point estimates, and the order of the loci was deduced from multipoint estimations. Missing values were treated with the EM algorithm. The highest lod scores were obtained for the linkage order shown in Fig. 2B. The recombination frequencies between five out of the ten loci considered are given in Table 1 (half matrix above diagonal) together with the number of the equiva- lent recombinants (half matrix below diagonal). The values obtained with the segregation data for Groi in the pot test are shown in Table 1A. Deviations were observed from the additivity of recombination frequen- cies between Grol and neighbouring RFLP markers. Considering similar issues, Bailey (1961) summarizes the case of three loci A, B and C with the order ABC and no interference. The recombination fraction between A and C, Y~ + 2, should follow Trow's formula: Y1+2 =Y1 +Y2 - 2Y1Y2 where Y1 is the recombination frequency between A and B and Yz that between B and C. With small recombina- tion frequencies, the term 2Y1Y z can be neglected and Y~ + 2 should be the sum of Y1 and Y2. Small deviations from the additivity of the recombination frequencies be- tween consecutive loci can be caused genetically by dou- ble crossover events, or experimentally by unbalanced numbers of progeny scored for individual markers (miss- ing data).  180 GP84(b)--GP219 --CPI03(a) GPI27(b)- TG20(a) CP51(c) CP56 - -GP77 GP32 GP27 CP55(b) TG61 cP54(c) - -cP43(b) CP52 CP43(a) B -GP219 -CPI03(a) SP127(b) TG20(a) Grol CP56 -CPSl(c) .GP77 GP32 :GP27 -CP43(b) CP52 5 cM Fig. 2A and B. Extended linkage group IX (A) of the potato map (Gebhardt et al. 1989) and (B) as derived from the progeny of the cross between H82.337/9 x H80.696/4, including the nematode resistance locus Grol. Linkage group IX corresponds to linkage group 7 in the tomato map (Bonierbale et al. 1988). Distances are in cM. Linkage order was obtained with multipoint estimates. GP markers are of genomic and CP markers of cDNA srcin in potato. Bracketed letters indicate that more than one locus was detected with the same probe. TG20 and TG61 are reference markers of tomato Whereas the recombination frequencies between RFLP loci showed good agreement with the expected additivity, the distance Y I + Y2 between the loci CP51(c) and TG20(a) encompassing Grol was considerably larger than the distance Y~ + 2 (Y1 = 6.3, Y2 = 14.6, Y1 + Y2=20.9 versus Y~+2=8.4, see Table 1A), indicating a possible overestimation of the recombination frequen- cies between Grol and CP51(c) and TG20(a), respective- ly. These deviations could be due to misclassification of genotypes with respect to nematode resistance in the pot test. Plants with susceptible genotypes but poor root systems may have been scored as resistant, based on the visual examination of their roots. Furthermore, four of the putative recombinants among Grol CP51(c) and TG20(a) would have srcinated from a double recombi- nation event, which is rather unlikely considering the short intervals involved. In order to test the possibility of misclassification, 39 genotypes were reclassified by the petri dish test. Be- sides normally growing plants with resistant and suscep- tible genotypes, the apparent recombinants among Grol, CP51(c) and TG20(a) were included as well as the genotypes associated with poor growth, although several of those could not be retested because of lack of tubers. Two plants previously classified as resistant in the pot test were clearly susceptible in the petri dish test, one of which was one of the four double recombin- Table 1. Recombination frequencies (half matrix above diagonal) and number of recombinants (below diagonal) between restriction fragment length polymorphism markers and the nematode resis- tance locus Grol A. Pot test GP27 CP51(c) Grol TG20(a) GP219 GP27 - 6.3 13.6 16.5 20.8 CP51(c) 6 - 6.3 8.4 12.8 Grol 11 5 - 14.6 19.8 TG20(a) 16 8 12 - 5.2 GP219 20 12 16 5 - B. Combined results from pot and petri dish test GP27 CP51(c) Grol TG20(a) GP219 GP27 - 6.3 9.6 16.5 20.8 CP51(c) 6 - 2.9 8.4 12.8 Grol 7 2 - 8.2 13.9 TG20(a) 16 8 6 - 5:2 GP219 20 12 10 5 - ants. The other one, however, generated a new double recombinant. One plant classified as susceptible in the pot test was reproducibly resistant in the petri dish test, thereby removing one recombinant between Grol and TG20(a). It was treated as resistant because the scorings of the pot test were inconsistent in this case. One addi- tional genotype, unclassified in the pot test, was suscepti- ble in the petri dish test and was therefore considered as susceptible. In all other cases (36 out of 39) the results were consistent between pot test and petri dish test. Data were then combined from both tests, giving priority to the petri dish test in the three cases of contra- dictory results. Ten scorings of plants associated with poor growth were treated as missing values. These ten genotypes might have been misclassified but could not be retested. Three of the four apparent double recombin- ants were included here, thereby reducing, for example, the number of recombinants between Grol and CP51(c) from five to two (Table 1). The recombination frequen- cies were recalculated using 74 scorings for the nematode resistance (33 resistant, 41 susceptible) and are shown in Table 1 B. The sume of the intervals Grol-CP51 c) and Grol-TG20 a) was now more additive with respect to the interval CP51(c)-TG20(a) (Y1=2.9, Y2=8.2, Y 1 + Y2 = 11.1 versus Y 1 + 2 = 8.4, see Table 1 B). The order of the loci on chromosome IX, as obtained from multipoint estimates, was the same irrespective of the data set used. No discrepancies except quantitative differences between equivalent map distances were ob- served between the structure of linkage group IX in the cross Ps x PR and the cross srcinally used for linkage map construction (Gebhardt et al. 1989) as shown in Fig. 2. Discussion According to Ross (1986) several dominant genes for resistance against G. rostochiensis have been identified  181 in the accessions of S. tuberosum ssp. andigena, S. vernei and S. spegazzinii and incorporated by breeders into modern European potato varieties. The H1 gene srci- nated from the S. tuberosum ssp. andigena clone CPC 1673 and confers resistance against the pathotypes Rol and Ro4. Similar genes in terms of pathotype specificity were found in clones derived from S. vernei. In S. spegaz- zinii three major genes were described. The Fa gene, effective against Rol and Ro2 but not Ro5, the Fc gene, protecting against some pathotypes of both G. rosto- chiensis and G. pallida, and the Fb gene conferring resis- tance to Rol, Ro5 and, when associated with accompa- nying favourable minor genes, also to Ro2, Ro3 and Ro4. The resistant parent PR of the segregating population considered in this paper is an interspecific hybrid be- tween a diploid S. tuberosurn ssp. tuberosum and a S. spegazzinii clone, neither of which are now available. PR is, however, not only resistant to Rol and Ro5 but also to Ro2, Ro3 and Ro4 as classified by H.J. Rumpen- horst (Biologische Bundesanstalt, Mfinster/Westfalen, FRG). This suggests that the mapped nematode resis- tance locus Grol may correspond to the Fb gene which was first described by Ross (1962). The locus Grol was clearly mapped by linkage to nine RFLP markers to the potato linkage group IX which is homologous to the tomato chromosome 7 (Fig. 2; Gebhardt et al. 1989; Bonierbale et al. 1988). Its map position is different from that of the tomato resistance locus Mi, effective against the nematode Meloidogyne incognita, which is located on chromosome 6 (van Daelen 1989; Medina-Filho and Stevens 1980). It is not known how many of the nema- tode resistance genes described in the genus Solanum and characterized with respect to srcin and pathotype specificity correspond to independent genetic loci. Fol- lowing our mapping of the first resistance locus, it will be possible to characterize the other genes relative to it. The resistance locus GroI was mapped in progeny from a cross between heterozygous parent clones. The percentage of heterozygosity, as estimated by RFLP analysis (Gebhardt et al. 1989) was 68 for the resistant parent PR and 54 for the susceptible parent Ps. The higher heterozygosity of PR was reflected in the higher number of segregating restriction fragments compared to Ps- This was of advantage for mapping the resistance locus because a sufficient number of informative markers became available. It was, in fact, much easier to detect linkage for the nematode resistance than for resistance to potato virus X, which segregated from par- ent Ps in the same progeny (this laboratory, unpublished results). The interspecific srcin of parent PR offers an explanation for its high heterozygosity. In parent PR also, a higher frequency of distorted segregation ratios was observed compared to parent Ps- Distorted segrega- tion ratios are the result of the differential viability of gametes and/or zyogtes and have been observed in other intra- and interspecific potato crosses as well (Gebhardt et al. 1989; Bonierbale et al. 1988). The map position of GroI between its nearest neigh- bouring RFLP markers CP51(c) and TG20(a) was not influenced by the method of classification of the nema- tode resistance. However, distortions of the recombina- tion frequencies between Grol-CP51 c), Grol-TG20 a) and CP51(c)-TG20(a) were observed. This was probably the result of misclassification of plants with a poor root system, a situation which limits the observation of cysts. Overestimation of resistance genotypes in scoring experi- mental populations of potato genotypes has also been reported by Howard and Fuller (1975) and Mugni6ry and Balandras (1986). Evidently, the misclassification of even a single recombinant has a strong influence on the estimate of the recombination frequency between tightly linked genes. The petri dish test seemed more reliable than the pot test for the assessment of resistance genotypes. The most consistent results were obtained by combining the evaluations of both tests and not con- sidering the results of genotypes of extremely poor growth habit (Table I B). The order of the RFLP loci on linkage group IX was similar in the cross used in this study and in the one used srcinally for construction of the potato RFLP map (Fig. 2). The small differences found between map distances on both linkage groups are the consequence of the independent meiotic events from which the two progenies srcinated. The substantial agreement between the two chromosome maps demonstrates the suitability of our RFLP map for use in different genetic back- grounds, such as is present today in S. tuberosum breed- ing material heavily introgressed by DNA from foreign species. Acknowledgements. The authors thank D. Mugni6ry for his help in setting up the petri dish technique and for providing us with cyst material for inoculation; H.J. Rumpenhorst for performing resistance tests with line H80.696/4. S.D. Tanksley for providing the clones TG20 and TG61, B. Walkemeier for excellent technical assistance and M. Pasemann for typing the manuscript. This work was supported by the Bundesministerium ffir Forschung und Tech- nologie (BMFT) under Project no. BCT 03902-1.06. References Bailey NTJ (1961) Introduction to the mathematical theory of link- age. Clarendon Press, Oxford Bonierbale MW, Plaisted RL, Tanksley SD (1988) RFLP maps based on a common set of clones reveal modes of chromosomal evolution in potato and tomato. Genetics 120:1095-1103 Dellaert LMW, Vinke H, Meyer K (1988) The inheritance of resis- tance to the potato cyst nematode Globodera pallida PA3 in wild Solarium species with broad spectrum resistance. Euphytica 5:105-116 Gebhardt C, Ritter E, Debener T, Schachtschabel U, Walkemeier B, Uhrig H, Salamini F (1989) RFLP-analysis and linkage map- ping in Solanum tuberosum. Theor Appl Genet 78 : 65-75 Howard HW, Fuller JM (1975) Testing potatoes bred from Sola- num vernei for resistance to the white potato cyst nematode, Heterodera pallida. Ann Appl Biol 81:75-78 Jung C, Kleine M, Fischer F, Herrmann RG (1990) Analysis of DNA from a Beta procumbens chromosome fragment in sugar beet carrying a gene for nematode resistance. Theor Appl Gen- et, in press Kort J, Ross H, Rumpenhorst HJ, Stone AR (1977) An interna- tional scheme for identifying and classifying pathotypes of po- tato cyst nematodes Globodera rostchoiensis and G, pallida. Ne- matologica 23 : 333 339
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