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RFLP mapping on potato chromosomes of two genes controlling extreme resistance to potato virus X (PVX

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Two different chromosomal locations of major genes controlling extreme resistance to potato virus X (PVX) were found by restriction fragment length polymorphism (RFLP) analysis of two populations segregating for the resistance. The resistance geneRx1
  Mol Gen Genet (1991) 227:81-85 002689259100147P © Springer-Verlag 1991 RFLP mapping on potato chromosomes of two genes controlling extreme resistance to potato virus X PVX) Enrique Ritter 1, Thomas Debener 1, Amalia Barone 2, Francesco Salamini I and Christiane Gebhardt 1 1 Max-Planck-Institut /Jr Zfichtungsforschung, W-5000 K61n 30, FRG 2 Department of Agronomy and Plant Genetics, University of Naples, 1-80055 Portici, Naples, Italy Received November 10, 1990 Summary. Two different chromosomal locations of ma- jor genes controlling extreme resistance to potato virus X (PVX) were found by restriction fragment length poly- morphism (RFLP) analysis of two populations segregat- ing for the resistance. The resistance gene Rxl mapped to the distal end of chromosome XII, whereas Rx2 was located at an intermediate position on linkage group V in a region where reduced recombination and segrega- tion distortion have also been observed. These linkage anomalies were due to abnormal behaviour of the chro- mosome contributed by the resistant parent P34. The results presented were obtained using two different stra- tegies for mapping genes of unknown location. One ap- proach was the use of probes revealing polymorphic loci spread throughout the genome and resulted in the map- ping of Rxl. The second approach was based on the assumption of possible linkage between the resistance gene and clone-specific DNA fragments introduced from a wild potato species. Rx2 was mapped by adopting this strategy. Key words: Potato virus X - Resistance genes - RFLP - Solanum tuberosum - Genetic introgression Introduction Among potato viruses, virus X (PVX) may induce heavy yield losses. Types of resistance to infection by this virus have been described as (1) field resistance based on mi- nor genes and (2) localized hypersensitivity and extreme resistance due to single dominant major genes (reviewed in Ross 1986). Genes conferring a localized hypersensiti- vity reaction can be overcome by several virus strains, while extreme resistance protects against all important virus isolates from North America and Europe. Resis- tance genes, mainly derived from wild potato species, are present in several modern cultivars. Until recently the practical use by plant breeders of major genes active against virus infection has been, to Offprint requests to: R.G. Herrmann some extent, hindered by the poor state of potato genet- ics. This is due to the tetraploidy of the cultivated varie- ties of Solanurn tuberosurn but also to the low vitality and fertility of their diploid derivatives. Recently a new molecular marker class, termed restriction fragment length polymorphisms (RFLPs), has become available and provides a useful tool for constructing dense genetic chromosomal maps for the species (Bonierbale et al. 1988; Gebhardt et al. 1989). The availability of chromo- some-specific RFLP probes has rendered attractive the mapping of major genes affecting morpho-physiological or resistance traits. Different strategies have been devel- oped in various plants of agricultural significance for the mapping of resistance genes via RFLP markers. Nearly isogenic lines (NILs) have facilitated the mapping of gene I2 conferring resistance to Fusarium oxysporum race 2 in tomato (Sarfatti et al. 1989), a resistance to maize dwarf mosaic virus strain A (McMullen and Louie 1989) and gene Tm-2a conferring resistance to tobacco mosaic virus (Young et al. 1988). Addition lines have permitted the detection of RFLP markers linked to a resistance to Heterodera schachtii in sugar beet (Jung et al. 1990). Recently Barone et al. (1990) have mapped the gene Grol controlling resistance to Globodera rosto- chochiensis in an F1 population after crossing diploid potato lines. The population used by Barone et al. (1990) for map- ping the GroI gene also segregated according to Mende- lian ratios for extreme resistance to PVX. For the same trait a second segregating F1 population was available that srcinated from a resistant parent different in srcin from the one used in the previous cross. This paper de- scribes the experiments that revealed that in the two crosses the genes conferring resistance to PVX were dif- ferent and mapped at two different positions on the RFLP map of potato. Materials and methods Plant material. A set of 38 diploid potato clones (Geb- hardt et al. 1989) from the collection of the Max-Planck- Institut ffir Zfichtungsforschung was screened for ex-  82 treme resistance to PVX. The resistant line H82.337/49 (P18) was crossed with the susceptible line H80.696/4 (P40) to produce an F1 population. The offspring segre- gated for PVX resistance, indicating that P18 was het- erozygous for the resistance allele. The test population consisted of 123 Fa seedlings which were tested for resis- tance to PVX. Leaves and shoots from 100 genotypes were harvested and freeze-dried to be used for DNA extraction and RFLP analysis (see Barone et al. 1990). A second diploid line, H77.409/13 (P34), also character- ized by extreme resistance to PVX in the heterozygous state, was crossed with the susceptible line H82.309/5 (PI6) to produce a second segregating F1 population of 110 seedlings which were grown in pots and tested for resistance. From this population DNA was extracted from 21 resistant and 23 susceptible plants for RFLP analysis. The two F~ populations considered will be des- ignated as F1840 and F3416 respectively. Tests for resistance to PVJ . Four different PVX isolates, srcinating from different potato varieties, and the strain PVX BS from the tobacco variety Xanthi were used to screen the srcinal set of 38 potato clones. Only the strain PVX BS was used to classify the two segregating populations. All virus isolates were provided by H.-L. Weidemann, Biologische Bundesanstalt, Braunschweig. The isolates were propagated on tobacco Samsun NN. Resistance to PVX was testedessentially as described by Cockerham (1970). Sap as the source of inoculum was prepared from two ground leaves of systemically infected tobacco plants. The sap was diluted with 50 ml 0.05 M phosphate buffer, pH 7. The solution was smoothly rubbed with a small sponge on potato leaves previously dusted with Carborundum (400 mesh). After 1 rain the leaves were rinsed with water and the proce- dure was repeated 1 and 2 weeks later. Potato plants were considered resistant when they showed localized micro-necrosis or no response at all to the infection. Susceptible plants showed a pronounced mottling typical of virus reproduction in the plant. The classification of plants as resistant was confirmed by a serological test (ELISA, Biochemica Test Combina- tion, Potato virus X; Boehringer/Mannheim). The me- chanical inoculations were performed directly on F1 seedlings and on their tuber descendants. The infection of the srcinal set of clones and of the two segregating populations was carried out twice by grafting potato scions on plants of tomato cv. Estrella EZ previously infected by mechanical inoculation with PVX. Potato genotypes were classified as susceptible if viruses were detected by the ELISA test in at least one of all assays. RFLP analysis. DNA extraction, restriction digests, elec- trophoresis, blotting and hybridization procedures were as described by Gebhardt et al. (1989). Probes. To screen the population F1840 the inserts of 32 genomic and 25 cDNA clones of potato were used as probes. In addition, the genomic tomato clone TG68 provided by S.D. Tanksley (Cornell University, Ithaca, N.Y., USA) was also utilised. The 58 markers map to the 12 linkage groups of potato (Gebhardt et al. 1989; C. Gebhardt et al., in preparation). Population F3416 was tested with only seven potato probes. Data and linkage analysis. Data analysis, linkage tests, estimation of recombination frequencies and determina- tion of the linear order of linked loci, including multi- point linkage and the EM algorithm for handling miss- ing data, were performed as described in Gebhardt et al. (1989), Ritter et al. (1990) and Barone et al. (1990). Re- sistance was treated in our linkage analysis as an extra RFLP fragment, being present in resistant and absent in susceptible plants. Results Segregation of P VJ resistance On screening the set of 38 diploid potato clones for PVX resistance, only lines 18 and 34 showed extreme resis- tance. F1 seeds of crosses of these resistant genotypes with susceptible genotypes were available. In the F 1 from cross F1840, 53 resistant and 64 susceptible plants were found (6 out of 123 could not be classified). This fitted the segregation ratio of 1 : 1 (Z2L. 1 = J.03; n.s.) expected when a genotype heterozygous for a single dominant gene is crossed to a homozygous recessive. From the 100 plants selected for RFLP analysis, 6 did not produce tubers and could not be exposed to all virus tests. They have been omitted from our analysis. The second Ft, F3416, segregated 31 resistant and 79 susceptible plants. This segregation ratio (Z2~:1=20.95; significant, P< 0.001) did not fit the type of inheritance found for the previous cross (see Discussion for interpretations). RFLP analysis Marker selection and characteristics of the polymor- phisms found for the cross F1840 have been described elsewhere (Barone etal. 1990). Probes distributed throughout the genome were screened first. After having detected linkage with a specific marker, neighbouring markers identified from the RFLP map were tested for tighter or looser linkage. To align our linkage data with those of other groups, tomato probes were mapped to potato linkage groups allowing an alignment of potato chromosomes to the homeologous chromosomes of to- mato (C. Gebhardt et al., in preparation). In the cross F1840 linkage was established between the resistance gene and seven markers mapping to linkage group XII (Fig. 1 A, new nomenclature, previously X in Gebhardt et al. 1989). These included the loci GP91 c), CPI06, CPll4, GP34, CPIO3 b) and CP60. One linked locus was revealed by the genomic tomato probe TG68 from tomato chromosome XII. All loci located in the cross F1840 by a multipoint linkage test occupied positions with the same order as established in the potato map of C. Gebhardt et al. (in preparation). The resistance gene Rxl was mapped to the most distal region of chro- mosome XII, 3.3 cM from the marker CP60 (Fig. 1A).  83 A GP81 GPgd b) GP99 a) ~Pl14 CPSO CPl3~ a) GPI22 cp2o b) GP91(c~ CPIOB TG68 GP21 GP213 GPI b) GP85 a) GPT8 CP31 GP28 b) Rx2 OPIT ~ GPIB8 GP22 5 cM xl Fig. IA and B. The positions of the loci Rxl and Rx2 controlling extreme resistance to potato virus X (PVX) on chromosome XII (A) and V (B) of potato. Chromosome numbers are according to the tomato nomenclature (Bonierbale et al. 1988). Chromosome XII is equivalent to linkage group X, and chromosome V to linkage group V in a previous potato restriction fragment length polymor- phism (RFLP) map (Gebhardt et al. 1989). The chromosome map in A was obtained from cross F1840, that in B from cross F3416. Linkage orders were inferred from multipoint estimates. Distances are given in centimorgans (Kosambi units, Kosambi 1944) based on the recombination frequencies between RFLP fragments of the resistant parents. Potato markers are of genomic (GP) and of cDNA srcin (CP). Letters in parentheses indicate that more than one locus was detected with the same probe. TG68 is a reference marker of tomato. Probes revealing linkage with the resistance genes in the crosses F1840 and F3416 are underlined. A few addi- tional markers (given in lighter letters) of the potato RFLP map are included at their approximate positions in order to outline the total structure of the chromosomes For the cross F3416 a different strategy was adopted: probes having a higher probability of being linked to the resistance gene were initially selected. The RFLP analysis of the set of 38 clones of S. tuberosum (described in Gebhardt et al. 1989) had indicated 12 fragments, cor- responding to loci at several positions in the genome, that were specifically present only in clone 34, the resis- tant parent of the cross F3416. The loci associated with these fragments were considered to be candidates for linkage with the resistance trait because they would indi- cate chromosomal segments introduced into the S. tuber- osum genome from wild Solanum species that carried a PVX resistance gene (Debener et al. 1991). Following this strategy, we first considered the 12 probes men- tioned above for the linkage studies. The first probe tested, GP21 on chromosome V (homeologous to toma- to chromosome V, C. Gebhardt et al., in preparation), provided evidence of linkage, showing a recombination frequency of 4.5 with the resistance gene Rx2. As shown in Fig. 2, only two recombinants were found among the susceptible plants analysed with GP21. Loci GPI7 a) and GP188, also mapping on chromosome V (Fig. 1 B), showed linkage with Rx2 with recombination frequencies of 6.8 and 9.1 respectively. Two addi- tional loci mapping to the same region, GP213 and GP28 b), were not suitable for this cross: Probe GP213 indeed revealed a polymorphism but only the susceptible parent was heterozygous for the fragment, and the re- striction fragment determining the locus GP28 b) was missing in F3416. Two markers from the chromosomal region encompassing Rxl (on linkage group XII) as well as GP21 were informative for the resistant parent in both populations and could, therefore, confirm the inde- pendence of Rxl and Rx2: neither CP60 nor GP34 showed linkage with Rx2 (recombination frequencies of 48 and 50 respectively). Conversely, GP21 was not linked to Rxl. The linear order of the RFLP loci tested was identical in F3416 and in the map of chromosome V developed from a different cross (C. Gebhardt et al. 1989, in prepa- ration). The linkage with three markers therefore allo- cated Rx2 to chromosome V between GP21 and GP17 a) (Fig. 1 B). The interval between the loci GP21 and GP188 revealed considerable differences in the ex- tent of recombination between genotypes: the interval length was 29.6 cM when measured between alleles of parent P16 and 13.6 cM between alleles of parent P34. In the cross used for constructing the RFLP map of potato, the distance between the same loci was 33.0 cM, a value similar to that measured in line 16. The recombi- nation frequency obtained for the same chromosomal region in the cross F1840 also indicated a distance of ca. 30 cM. The reduced recombination observed in the interval GP21-GP188 therefore seems to be specific for the chromosome V carried by line 34. We should point out that an influence of size of the sample of plants selected for RFLP analysis on the extent of recombina- tion can be excluded because the recombinant to non- recombinant ratio was not statistically significantly dif- ferent in resistant and susceptible plants. Discussion According to Cockerham (1970) two independent genes for extreme resistance to PVX exist: one can be traced back to the potato seedling USDA 41956, the first resis- tant genotype described (Schultz and Raleigh 1933) and to the S. andigena clone CPC 1673. The second was derived from S. acaule and is present in the clone MPI 44.1016/10 (Ross 1986). The potato clones MPI 44.1016/ 10 and CPC 1673 have frequently been used in potato breeding at the Max-Planck-Institut filr Ziichtungsfor- schung (H. Ross, personal communication). This ex- plains our observation that in the segregating popula-  84 P P F1 16 3Z \ i esistont Susceptible × X ~--C *--d t= a b t--Ct ~b Fig. 2. Southern blot of potato DNA probed with the RFLP marker GP21 showing cosegregation with the PVX resistance locus Rx2. The first two lanes on the left correspond to the susceptible (P16) and the resistant parent (P34), followed by resistant and susceptible genotypes of the segregating F1 progeny. Of the 21 resistant genotypes analysed, only 20 are shown. The four segregat- ing RFLP alleles are given on the right. Alleles a and b each consist of two cosegregating restriction fragments and are derived from P34. Alleles c and d are derived from P16. Allele a is specific for P34 and is linked in repulsion with the resistance locus Rx2. The two recombinants are indicated by (x) tions two different locations for genes controlling ex- treme resistance to PVX were found: one on the distal end of chromosome XII and the other in an intermediate position on chromosome V. Due to the complex crossing programme followed at the institute it was, however, impossible to assess unambiguously the source of resis- tance from the pedigrees of the resistant lines PI8 and P34. The pedigree of line 34 included the resistant clone MPI 44.10/6/10 (H. Uhrig, unpublished results): it is then likely that the resistance gene of line 34 Rx2) is descended from S. acaule. The second resistance gene Rxl, present in P18, could have been inherited from CPC 1673 and would correspond to the resistance gene from S. andigena. Two different approaches for gene mapping are pre- sented in this paper. With population F1840 we adopted the strategy of systematically screening the genome with probes choosen according to their map position. As soon as linkage was detected with a particular locus, neigh- bouring markers were tested until the gene was located in an interval between two consecutive markers, or, as in the case of Rxl, at the distal end of one chromosome. The following considerations help to define the necessary number of probes: a total genomic length of 1000 cM would require 25 markers to detect linkage within a range of 20 cM. One further marker is necessary for orienting the interval on the chromosome and, depend- ing on the marker density of the map and on the varia- tion of marker interval lengths in different populations, a few further probes are needed to determine the position of the gene with the best possible accuracy. The second approach was successful with population F3416. It was based on the assumption that the resis- tance gene had been introduced from a evolutionarily distant Solarium species together with several 'exotic' RFLP alleles (Debener et al. 1991). Whereas a large part of the genome of this species must have been eliminated during further breeding, one or more DNA fragments descending from the wild species and linked to the resis- tance gene should have been conserved in the genome of line P34. Although exotic RFLP alleles are common to many wild species widely used in potato breeding (Debener et al. 1991) and several such DNA fragments were present outside chromosome V in the genome of line P34, linkage to Rx2 was detected with GP21, the first such probe tested. It must be mentioned however that, contrary to our expectations, Rx2 was found to be linked in repulsion with the exotic fragment revealed by probe GP21 (allele a in Fig. 2). The simplest explana- tion for this finding is that an Rx2-bearing ancestor was used intensively during potato breeding at the Max- Planck-Institut. During the generations of breeding, the linkage in coupling between the resistance gene Rx2 and the wild species allele revealed by GP21 was broken by crossing over but both recombinant segments of chro- mosome V were retained in the germplasm pool. Both parents of P34 therefore carried the complementary re- combinant segments.  85 While segregation of the resistance gene in F1840 agrees with the ratio of 1:1 expected for a genotype heterozygous for a single dominant gene crossed to a homozygous recessive, the segregation found in F3416 shows significant deviations from this ratio and resem- bles more a 1 : 3 ratio (resistant versus susceptible). This could occur as a result of the existence of a complemen- tary effect of two independent genes both contributing to the expression of extreme resistance. Assuming the two gene hypothesis, however, in the fraction of suscep- tible genotypes, independent segregation of the allelic fragments revealed by probe GP21 would be expected. This was not the case because only two recombinants were found among the susceptible plants (Fig. 2): within the set of 21 resistant and 23 susceptible plants the RFLP allele linked to Rx2 was present 23 times and absent 21 times. This distribution among susceptible and resis- tant plants can be obtained by chance only with a proba- bility lower than 5x 10 -13. It can be concluded that the extreme resistance of P34 is due to a single dominant gene located in a chromosomal region with distorted segregation. This finding is not uncommon. While map- ping RFLPs in different progenies of potato in the chro- mosomal region surrounding Rx2, aberrant segregation ratios were also observed (C. Gebhardt et al., in prepara- tion). Other abnormalities, like the reduced recombination found between the loci GP2I and GP188 for alleles of P34 as compared with alleles of P16, could result from the presence of chromosomal regions descended from wild species (C. Gebhardt et al., in preparation; Debener et al. 1991). The occurrence of segregation distortion would also explain the segregation ratios found in tetra- ploid potato crosses by Stevenson et al. (1939), who sug- gested a complementary effect of two genes controlling extreme resistance to PVX. This hypothesis was rejected later by Mills (1965) and Cockerham (1970). Acknowledgements. The authors thank H.-L. Weidemann for pro- viding the virus X isolates, H. Uhrig and H. Ross for their efforts in investigating the pedigrees of the resistant parental potato lines, and S.D. Tanksley for providing the tomato marker TG68. This work was supported by the Bundesministerium fiJr Forschung und Technologie (BMFT) under Project no. BCT 03902-1.06. 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 dominant gene conferring resistance to the potato cyst nematode Globodera rostochiensis. Mol Gen Genet 224:177-182 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 Cockerham G (1970) Genetical studies on resistance to potato vi- ruses X and Y. Heredity 25: 309-348 Debener T, Salamini F, Gebhardt C (1991) Germplasm introgres- sions from wild species into potato Solanum tuberosum ssp. tuberosum) breeding lines can be detected by RFLPs (Restric- tion Fragment Length Polymorphisms). Plant Breeding, in press 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 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 Kosambi DD 0944) The estimation of map distances from recom- bination Values. Ann Eugen 12:172-175 McMullen MD, Louie R (1989) The linkage of molecular markers to a gene controllig the symptom response in maize to Maize Dwarf Mosaic Virus. Mol Plant-Microbe Interact 2:309-314 Mills WR (1965) Inheritance of immunity of potato virus X. Am Potato J 42: 294-295 (Abstract) Ritter E, Gebhardt C, Salamini F (1990) Estimation of recombina- tion frequencies and construction of RFLP linkage maps in plants from crosses between heterozygous parents. Genetics 125:645--654 Ross H (1986) Potato breeding: problems and perspectives. J Plant Breeding 37 [Suppl] Sarfatti M, Katan J, Fluhr R, Zamir D (1989) An RFLP marker in tomato linked to the Fusarium oxysporum resistance gene I2. Theor Appl Genet 78 : 755-759 Schultz ES, Raleigh WP (1933) Resistance of potato to latent mosa- ic. Phytopathology 23:32 (Abstract) Stevenson F J, Schultz ES, Clark CF (1939) Inheritance of immuni- ty from virus X (latent mosaic) in the potato. Phytopathology 29: 362-365 Yound ND, Zamir D, Ganal MW, Tanksley SD (1988) Use of isogenic lines and simultaneous probing to identify DNA markers tightly linked to the Tm-2a gene in tomato. Genetics 120:57 585 Communicated by R.G. Herrmann
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