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Tight physical linkage of the nematode resistance gene gpa2 and the virus resistance gene rx on a single segment introgressed from the wild species solanum tuberosum subsp. andigena cpc 1673 into cultivated potato

MPMI Vol. 12, No. 3, 1999, pp. 197–206. Publication no. M-1999-0111-01R. 1999 The American Phytopathological Society Tight Physical Linkage of the Nematode
Resistance Gene Gpa2 and the Virus Resistance Gene
Rx on a Single Segment Introgressed from
the Wild Species Solanum tuberosum subsp. andigena
CPC 1673 into Cultivated Potato

Jeroen Rouppe van der Voort,1 Konstantin Kanyuka,2 Edwin van der Vossen,3 Abdelhafid
Bendahmane,2 Paul Mooijman,2,3 René Klein-Lankhorst,3 Willem Stiekema,3 David Baulcombe,2
and Jaap Bakker1

1The Graduate School of Experimental Plant Sciences, Wageningen Agricultural University, Department of Nematology, P.O. Box 8123, 6700 ES Wageningen, The Netherlands; 2The Sainsbury Laboratory,Norwich Research Park, Colney, Norwich NR4 7UH, U.K.; 3DLO-Centre for Plant Breeding andReproduction Research (CPRO-DLO), P.O. Box 16, 6700 AA, Wageningen, The Netherlands Accepted 12 November 1998.
Comparative mapping studies within several plant genera
characterized by a hypersensitive response (HR; Staskawicz et have shown that genes for resistance to taxonomically di-
al. 1995; Crute and Pink 1996; Hammond-Kosack and Jones verse pathogens are often co-localized on the maps of these
1996). Two distinct genetic arrangements have been found genotypes. Here we describe the genetic analysis of such a
among R loci: either single loci, many different alleles of gene cluster on chromosome 12 of potato. This cluster
which can occur in the germplasm (Bent et al. 1994; Mindri- harbors the resistance loci Gpa2 and Rx, which confer re-
nos et al. 1994; Lawrence et al. 1995; Grant et al. 1995), or sistance to the potato cyst nematode Globodera pallida and
different genes that occur at compound loci forming R-gene potato virus X (PVX), respectively. By screening two map-
clusters (Jones et al. 1994; Martin et al. 1993, 1994; Dixon et ping populations with 10 CAPS (cleaved amplified poly-
al. 1996; Anderson et al. 1997; Parniske et al. 1997; Song et morphic sequence) markers derived from AFLP
al. 1997). The occurrence of different R genes on the same (amplified fragment length polymorphism) markers and
genomic location in different species of a plant genus has been the ends of overlapping bacterial artificial chromosome
ascribed to the common origin of R gene families (Baker et al.
(BAC) clones, it is demonstrated that Gpa2 and Rx are
1997). This is exemplified by the recent characterization of tightly linked. From a recombination fraction of 1/3386,
the Cf-4/9 locus of tomato. The R genes Cf-4 and Cf-9 origi- found after screening two different mapping populations,
nate from the wild species Lycopersicon hirsutum and L. a genetic distance of 0.02 cM between Gpa2 and Rx was
pimpinellifolium, respectively, and confer resistance to differ- calculated. The physical length of this genetic interval does
ent races of the fungal pathogen Cladosporium fulvum. Se- not exceed 300 kb of DNA as determined by the total size
quencing of the entire region in both the wild and the culti- of overlapping BAC clones in the Rx/Gpa2 contig. Evi-
vated species revealed several (active) R gene homologs, dence is provided that both genes have been introgressed
indicating that frequent sequence exchange between these from a single wild species clone, Solanum tuberosum
tandemly repeated genes created novel R gene specificities subsp. andigena CPC 1673. This suggests that these genes
(Thomas et al. 1997; Parniske et al. 1997). Similar evolution- underlying the disease resistance specificities to potato vi-
ary processes and subsequent diversification in different plant rus X and potato cyst nematodes have a common evolu-
species may also underlie the generation of novel disease re- sistance specificities against different pathogen species. Forexample, a complex locus is known on chromosome 5 of po-tato. This cluster comprises at least five R loci: R1, which be- Most plant resistance genes (R genes) are dominantly in- stows resistance to Phytophthora infestans (Leonards- herited and are often involved in resistance processes that are Schippers et al. 1992); Nb, conferring an HR type of resis-tance, and Rx2, conferring extreme resistance to potato virusX (De Jong et al. 1997; Ritter et al. 1991); and Gpa and Grp1, Corresponding author: Jeroen Rouppe van der Voort conferring resistance to the potato cyst nematode (Kreike et Telephone: +31 317 485085; Fax: +31 317 484254 al. 1994; Rouppe van der Voort et al. 1998). All these loci originate from different genetic backgrounds.
Jeroen Rouppe van der Voort, Konstantin Kanyuka, Edwin van der The occurrence of R-gene-like sequences at compound R Vossen, and Abdelhafid Bendahmane contributed equally to the re- loci has recently been demonstrated for a number of plant spe- Vol. 12, No. 3, 1999 / 197
cies (Martin et al. 1993, 1994; Dixon et al. 1996; Leister et al.
population Pa2-D383 able to develop on the roots of the tested 1996; Anderson et al. 1997; Parniske et al. 1997; Song et al.
genotypes and resistance to PVXCP4 (Fig. 1). On the basis of 1997). However, it is impossible to predict from the DNA se- the G. pallida Pa2-D383 test the S1-Cara and F1SH×RH quence whether these genes are functional to different patho- genotypes could be assigned to a resistant class (mean number gen species. At present, only a few examples exist that shed of cysts 37 ± 7 [SD]) or a susceptible class (mean 715 ± 62 light on the physical linkage of R genes mapped at complex [SD]). These plants showed either absence or presence of a loci that respond to unrelated (pathogen) signals. The genes hybridization signal on dot blots of total RNA of the same encoding Pto and Fen in tomato, which induce hypersensitive- plants infected with PVX CP4. Resistance tests with G. pallida like necrosis upon recognition of the bacterial avirulence gene population Pa3-Rook (results not presented) showed that cv.
product AvrPto and the chemical compound Fenthion, respec- Cara is susceptible to this nematode population, indicating the tively (Martin et al. 1994; Tang et al. 1996; Scofield et al.
specificity of the G. pallida resistance. The results of the Pa2- 1996), are localized several tens of kilobases from each other D383 and PVX CP4 tests on this selected set of plant genotypes (Salmeron et al. 1996; Rommens et al. 1995). In lettuce, the indicated that Gpa2 and Rx cosegregate in both mapping Dm6 locus to downy mildew and the root aphid resistance lo- populations with a maximum probability of P = 0.016 (= cus Ra are genetically linked and originate from the same (1/2)6) that the observed linkage could be explained by Lactuca serriola accession (Crute and Dunn 1980; Crute chance. The S1-Cara recombinants were previously used to 1992). However, from the size of the genetic interval, these localize Rx in a 1.21 cM interval between markers IPM3 and loci may still lie 6 Mb apart (Anderson et al. 1996). A remark- IPM5 (Bendahmane et al. 1997). Cosegregation of Gpa2 with able case is the Mi gene in tomato. This gene exhibits a pleio- Rx suggested that Gpa2 also resides in this region (Fig. 2A).
trophic effect and confers resistance to different species of theroot knot nematode (Meloidogyne spp.) and the potato aphid Fine mapping of Gpa2.
Macrosiphum euphorbiae (Milligan et al. 1998; Rossi et al.
Fine mapping of the Gpa2 locus was carried out with cleaved amplified polymorphic sequence (CAPS; Konieczny Here, the identification of a novel R gene cluster in potato is and Ausubel 1993) markers that were originally developed for described. Gpa2, which bestows specific resistance to a small the fine mapping of the Rx locus (Bendahmane et al. 1997; set of populations of the potato cyst nematode Globodera pal- Kanyuka et al., in press). These CAPS markers are derived lida (Rouppe van der Voort et al. 1997) and Rx, which confers from the Rx flanking amplified fragment length polymorphism extreme resistance to potato virus X (PVX; Ritter et al. 1991; (AFLP) markers PM3, PM4, and PM5 (Bendahmane et al.
Bendahmane et al. 1997), were previously mapped on the 1997) and from the end sequences of BAC (bacterial artificial short arm of chromosome 12. We now provide evidence that chromosome) clones, previously isolated from a BAC library both genes are physically tightly linked and that they reside on prepared from cv. Cara, that map to the GP34-IPM5 interval an introgression segment that was introduced into a number of (Kanyuka et al., in press). In addition, two other CAPS mark- European potato cultivars from a single wild potato clone, ers, IPM4a and IPM4c, were developed via a low-stringency Solanum tuberosum subsp. andigena CPC 1673.
PCR (polymerase chain reaction) screening assay (Kanyuka etal., in press). CAPS marker IPM4 was designated IPM4a as digestion of low-stringency IPM4 products identified not onlythe original IPM4 locus but also new DNA fragments that are Cosegregation of Gpa2 and Rx resistance.
polymorphic between PVX-resistant and PVX-susceptible The resistance loci Gpa2 and Rx were previously mapped in genotypes (Kanyuka et al., in press). One of these marker al- separate mapping populations to the same 6 cM genetic inter- leles was designated IPM4c. The relative positions of the val on the distal end of chromosome 12, between the RFLP CAPS markers are depicted in Figure 2B.
(restriction fragment length polymorphism) markers GP34 and To identify recombination events in the IPM3-IPM5 region, CT79 (Rouppe van der Voort et al. 1997; Bendahmane et al.
a total of 2,788 S1-Cara genotypes were screened for the pres- 1997). Linkage of Gpa2 and Rx was assumed because the re- ence of markers IPM3 and IPM5. In addition, 598 genotypes sistance donors of both mapping populations, i.e., the diploid from population F1SH×RH were subjected to a GP34-IPM5 genotype SH83 and the tetraploid cv. Cara, harbor introgres- marker screening as marker IPM3 is not informative in this sions from the same wild potato clone, Solanum tuberosum population. This resulted in the identification of 20 IPM3- subsp. andigena CPC 1673. To determine the degree of link- IPM5 recombinants in the S1-Cara population and 9 GP34- age between both genes, segregation of Gpa2 and Rx was IPM5 recombinants in the F1SH×RH population. To map followed in a selected set of progeny of population F1SH×RH these recombination events more precisely, a second marker and S1-Cara.
screening was carried out with all the markers available in the Gpa2 and Rx resistance tests were performed on the paren- IPM3-IPM5 region (Figs. 2B and 3). Subsequent G. pallida tal genotypes Cara, SH83, and RH89, four S1 genotypes that Pa2-D383 tests on these genotypes showed that Gpa2 is lo- are recombined in a 1.21 cM interval between markers GP34 calized between markers IPM4c and 111R. Interestingly, and IPM5 (Bendahmane et al. 1997), and two F1 genotypes among the 2,788 S1-Cara genotypes and 598 F1SH×RH that harbor cross-over events in a 6 cM interval between genotypes tested, only one genotype, S1-761, was identified in markers GP34 and CT79 (Rouppe van der Voort et al. 1997).
which a recombination event had occurred between Gpa2 and These progeny clones exhibit the closest recombination events Rx. On the other side of Gpa2, genotype S1-B811 was used to to Gpa2 and Rx and were previously used to border the Gpa2 identify marker 111R as a flanking marker for the Gpa2 inter- and Rx intervals. The results of the resistance tests showed a val. The marker order deduced from the analysis of the clear correlation between the number of cysts of G. pallida F1SH×RH population corresponds to that found in population 198 / Molecular Plant-Microbe Interactions
S1-Cara. Therefore, estimates of recombination frequencies potato genotype SH83. This library consists of 60,000 clones deduced from both populations were merged to obtain an es- with an average insert size of 100 kb (data not shown), repre- timate of the average recombination value for each marker senting between six and seven haploid genome equivalents of interval (Table 1). The graphical genotypes (Young and Tanks- potato. PCR screening of this SH83 BAC library with markers ley 1989) shown in Figure 2B display the boundaries of the Rx 45L and 77R yielded a BAC clone of 110 kb (SHBAC43) that and Gpa2 intervals. Since there is only one recombinant found spans the gap between BAC77 and BAC45, resulting in a between Gpa2 and Rx the average genetic distance between contiguous physical map of the Gpa2 locus (Fig. 2C).
Gpa2 and Rx is estimated to be 0.02 cM.
Previously, it was shown that BAC77 spans the Rx gene (Kanyuka et al., in press). By adding the separate insert sizes Physical linkage of Rx and Gpa2.
of the individual BAC clones in the Rx/Gpa2 region (Fig. 2C), The preparation of a BAC library from cv. Cara and the the maximum physical size of the Rx/Gpa2 interval was de- subsequent identification and isolation of Cara BAC clones termined to be 300 kb. This figure combined with the calcu- that map to the IPM3-IPM5 Rx interval has been described in lated genetic distance of 0.04 cM for the interval between Kanyuka et al. (in press). Figure 2C depicts the relative posi- markers 77L and 111R results in a ratio of 7.5 Mb/cM for this tions of four previously identified Cara BAC clones (BAC77, BAC45, BAC221, and BAC111) that map to the 0.04 cMIPM4c-111R genetic interval harboring the Gpa2 locus. As Origin of Gpa2 and Rx resistance.
these BAC clones do not fully cover the Gpa2 locus and addi- To address the question of whether Gpa2 and Rx were tional PCR screening of the Cara BAC library with CAPS jointly introgressed from the same wild species clone, a subset markers 45L and 77R (see Figure 2C) failed to yield a Cara of the progeny of S1-Cara and F1SH×RH as well as two po- BAC clone that spanned the remaining gap between BAC77 and tato clones derived from a selfing of the original wild clone BAC45, a second BAC library was prepared from the diploid (abbreviated as CPC 1673-a and CPC 1673-b) and nine potato Fig. 1. Cosegregation of Gpa2 and Rx resistance demonstrated on the parental clones Cara, SH83, and RH89 as well as on four offspring genotypes of
population S1-Cara (S1-152, S1-421, S1-160, S1-1189) and two genotypes of population F1SH×RH (F1-27, F1-140). A, Cysts recovered from the roots
of one replicate of each plant. Magnification ×5. B, Hybridization signal on dot blots of total RNA of the same set of plants infected with PVXCP4.
Vol. 12, No. 3, 1999 / 199
cultivars harboring introgressions from clone Solanum tubero- The CAPS marker profiles of the analyzed potato geno- sum subsp. andigena CPC 1673 (hereinafter referred to as types harboring introgressions from CPC 1673 were highly CPC 1673) were evaluated for the presence of common DNA similar to those of the selfed CPC 1673 genotypes. All marker alleles in the GP34-IPM5 region of chromosome 12.
CAPS marker alleles in coupling phase to Rx appeared to As the primers for these CAPS markers may amplify various be of CPC 1673 origin (Fig. 3; Table 2). Among the seven sequences scattered all over the genome, only the DNA CPC 1673 cultivars tested, a remarkably high number of dif- fragments linked in coupling phase to Rx were scored.
ferences in the size of introgressed region of 0.9 cM were Fig. 2. A, Relative position of the Rx/Gpa2 interval on potato chromosome 12. B, Graphical genotypes showing differences in the size of the CPC1673-
derived segments. Presented genotypes border respective intervals of Gpa2 and Rx. Introgression segments indicated by thick bars. Marker interval size
in B is not on scale. C, Physical map of the Rx/Gpa2 region. Open rectangles represent bacterial artificial chromosome (BAC) clones derived from the
S1-781 BAC library. Gray shaded rectangle indicates BAC clone SHBAC43 isolated from the BAC library of genotype SH83. Estimated sizes of the
BAC clones are given between parentheses.
200 / Molecular Plant-Microbe Interactions
observed. A total of five different haplotypes of the CPC 1673-derived chromosomal segment was identified. Thesedifferences in introgression segment lengths led unambigu- The hypothesis that both Gpa2 and Rx may have originated ously to the same marker order. Hence, it is concluded that from S. tuberosum subsp. andigena CPC 1673 was tested by Gpa2 and Rx originate from the same wild species clone and screening two different mapping populations for nematode were simultaneously introgressed into domesticated potato and virus resistance. This screening was aided by a wealth of DNA markers available in the Rx region (Bendahmane et al.
Fig. 3. CAPS (cleaved amplified polymorphic sequence) marker analysis on a set of S1-Cara and F1SH×RH progeny as well as a series of Solanum
subsp. andigena CPC 1673-derived potato clones. A, Marker 77L bracketing left border of the Rx interval by clone SH83. B, Marker IPM4c
bracketing right border of Rx interval and left border of Gpa2 interval. C, Marker 77R bracketing left border of Gpa2 interval. See also Figure 2.
Vol. 12, No. 3, 1999 / 201
1997; Kanyuka et al., in press). Evidence was found that Gpa2 CPC 1673 clone in their pedigree (e.g., cvs. Multa and Sat- and Rx indeed derive from the same wild species clone since urna from CPC 1673-1 and Alcmaria and Amaryl from CPC all the marker alleles that flank Gpa2 and Rx were identified 1673-20) contained introgression segments of different in selfings of the original CPC 1673 clone. In addition, these lengths. The finding that a considerable level of variation ex- marker alleles also occurred in the Pa2-D383 and PVXCP4 re- ists between the sizes of the introgressed segments provides sistant cultivars that harbor introgressions from CPC 1673.
an additional source of "rare recombinants" within a selected Tight genetic linkage of both genes was demonstrated after region of wild origin.
segregation analysis in 3,386 offspring derived from two Significant differences in recombination frequencies were populations. For the IPM3-IPM5 region, 20 recombinant observed between marker intervals GP34-77L and 73L-218R genotypes were identified, of which only one clone showed and the intervals in the region 77L-73L. Given the physical recombination between Gpa2 and Rx. Compared with this re- sizes of the intervals GP34-IPM3 (110 kb; Kanyuka et al., in combination fraction, the number of introgression segment press), 77L-111R (<300 kb), and 73L-IPM5 (about 150 kb; length differences among the CPC 1673 cultivars tested was Kanyuka et al., in press) it is concluded that the recombination remarkably high. The CPC 1673 cultivars tested lacked differ- frequency in the 77L-111R interval is significantly reduced.
ent parts of the IPM3-IPM5 region despite the fact that four The reduced recombination frequency in the 77L-111R inter- out of five of these clones were probably selected for PVX val, a region that is of wild species origin, may be the result of resistance. This may be explained by the relatively early use a high level of sequence heterogeneity between the homolo- of S. tuberosum subsp. andigena CPC 1673 in breeding pro- gous chromosomes within this interval. This assumption is grams that aimed to introgress resistance into cultivated So- based on the model that sequence homology between two lanum spp. Selection based on agronomically important traits DNA strands initiates reciprocal genetic exchange during originating from S. tuberosum may have severely reduced the meiosis I (Borts and Haber 1987; Priebe et al. 1994). As a re- fraction of CPC 1673-derived DNA in concurrent hybrids.
sult, a reduction in the recombination frequency will be en- This explanation may also be applicable to the high level of countered in regions of heterogeneous origin (containing in- allelic variation found at an RFLP locus near the H1 gene trogressions of foreign DNA), compared with regions that (Niewöhner et al. 1995). From this study, it appeared that only have a common origin. The suppressive effect of introgres- four out of 87 potato clones, known to be resistant to the Ro1 sions on recombination frequencies has been shown, e.g., in pathotype of the potato cyst nematode G. rostochiensis, harbor barley (Görg et al. 1993) and in tomato (Rick 1969; Liharska a molecular marker allele that is tightly linked to the resis- et al. 1996).
tance locus H1. It is noted that the recombination events in the The identification of two R genes that originate from a sin- IPM3-IPM5 interval occurred after introgression into culti- gle wild species clone is very fortunate since the original S. vated potato because the cultivars that had the same selfed tuberosum subsp. andigena CPC 1673 clone has been selected Table 1. Number of recombinants, average recombination frequency, and standard error per marker interval
No. of recombinants per cross
Recombinants in S1-Cara (no.) Recombinants in F1SH×RH (no.) Recombination (%) ± standard error z Ten recombinants were found among 1,720 genotypes tested in the interval GP34-IPM3 (Bendahmane et al. 1997). Six recombinants were identified in the interval IPM3-77L among the 2,788 genotypes of S1-Cara. Recombination frequencies followed by a different letter differ significantly in a X2 testat P < 0.05.
Table 2. Potato clones with Solanum tuberosum subsp. andigena CPC1673 in their pedigree (with the exception of clone RH89) tested on the presence
of chromosome 12 specific CAPS allelesv
v Presence or absence of a CAPS (cleaved amplified polymorphic sequence) marker band that cosegregates with resistance in populations S1-Cara and F1SH×RH is indicated by "+" or "–", respectively. The order of the presented CAPS markers corresponds to the marker order on chromosome 12. ND= not determined.
w As determined by cyst counts on at least three replicates.
x As determined by a slot blot assay on at least three replicates.
y Data from Arntzen et al. (1994).
z Data from Anonymous (1991), "R" indicates extreme resistance.
202 / Molecular Plant-Microbe Interactions
neither for Gpa2 nor for Rx. Clone S. tuberosum subsp. andi- mapping the Gpa2 gene (Rouppe van der Voort et al. 1997) gena CPC 1673 was collected in the late 1940s in the Andes and consisted of a total of 598 genotypes. The resistant parent region in South America during a search for nematode resis- SH83-92-488 and the susceptible parent RH89-039-16 will be tance (Ellenby 1952). Only five out of more than 1,200 lines referred to as SH83 and RH89, respectively.
belonging to more than 60 species harbored nematode resis- Seed from a selfed offspring of Solanum tuberosum subsp.
tance. The resistance in CPC 1673 appeared to be conferred andigena CPC 1673 encoded CPC 3520 was obtained from by a single gene, H (later called the H1 gene), that was dis- the Commonwealth Potato Collection, Scottish Crop Research covered by screening populations derived from selfings of the Institute, Dundee, UK. The cultivars Multa, Alcmaria, Ama- original CPC 1673 clone (Toxopeus and Huijsman 1953; ryl, Marijke, and Saturna, having S. tuberosum subsp. andi- Huijsman 1955). H1 confers resistance to G. rostochiensis, for gena CPC 1673 in their pedigree, were derived from the col- which a gene-for-gene relationship has been established lection of the Centre for Plant Breeding and Reproduction (Janssen et al. 1991), and the gene has been mapped on chro- Research (CPRO-DLO) Wageningen, The Netherlands. It is mosome 5 (Gebhardt et al. 1993; Pineda et al. 1993). The oc- noted that the background of parent SH83 contains cultivar currence of a second nematode R gene at an unlinked position on the genome of one and the same wild species clone mayindicate that both H1 and Gpa2 may have a common ancestor, like the L6 and M genes in flax (Anderson et al. 1997).
The PVX resistance test was carried out with a cDNA of the Studies on Rx resistance have shown that it can be consid- PVXCP4 isolate (Goulden et al. 1993). Potato plants were ered mechanistically different from HR types of plant resis- graft-inoculated with scions of Lycopersicon esculentum cvs.
tance responses. The Rx-mediated response has been de- Ailsa Craig or Money Maker systemically infected with scribed as "extreme resistance." It is not associated with a PVXCP4. Dot blots were prepared from total RNA isolated localized necrotic response at the site of infection (Adams et from newly formed potato shoots 3 to 4 weeks post inocula- al. 1986; Köhm et al. 1993) but mediates inhibition of virus tion (Bendahmane et al. 1997). Extreme PVX resistance or replication after having been triggered by the coat protein of susceptibility was determined by the presence or absence of a PVX (elicitor of Rx resistance; Bendahmane et al. 1995).
hybridization signal on dot blots probed with 32P-labeled Nevertheless, Rx could still be involved in a mechanism that cDNA of PVX CP4 (Chapman et al. 1992). Three replicates per suppresses the viability of plant cells as plants with an Rx genotype were assayed.
background could not be transformed with the coat protein For the Gpa2 test G. pallida population Pa2-D383 was used gene of an Rx-avirulent PVX strain (Gilbert et al. 1998). A (Rouppe van der Voort et al. 1997). The nematode resistance common feature in virus and nematode pathogenesis is that assays were performed on plants derived from in vitro stocks, both types of pathogen are able to interfere with the plant cell stem cuttings, or tubers. In vitro plants were transferred from metabolism. Cyst nematode juveniles penetrate the roots and MS (Murashige-Skoog) medium containing 3% saccharose to migrate to the vascular cylinder, where they initiate the redif- a mixture of silversand and sandy loam under a moist chamber ferentiation of a procambial cell into a feeding site for 1 week. Two to 4 weeks after planting, plants showing (Williamson and Hussey 1996). Although the mechanism of vigorous growth were inoculated with nematodes. Assays Gpa2-mediated resistance has not yet been thoroughly inves- were further performed as described for stem cuttings and tu- tigated, the severe reduction in cyst number indicates that the bers (Rouppe van der Voort et al. 1997). Population Pa3-Rook nutrient flow through the nematode feeding site may be re- with virulence characteristics different from those of Pa2- duced in Gpa2 plants. Poor feeding site development can be D383 (Bakker et al. 1992) was used to confirm the specificity due to a localized necrosis near or at the syncytium, as dem- of Gpa2 resistance in tested plants. Virus and nematode resis- onstrated for the H1 gene (Rice et al. 1985). In view of their tance tests were applied on the parental genotypes as well as tight physical linkage, Gpa2 and Rx may share common com- the S1-Cara and F1SH×RH genotypes that showed a recombi- ponents within their signal-transduction pathways, as is the nation event in the GP34-IPM5 marker interval.
case for Pto and Fen. The Pto- and Fen-mediated responsesboth depend on Prf, a third and unrelated gene that occurs in Construction of the potato BAC libraries
the Pto/Fen gene cluster (Salmeron et al. 1996). Whether the and BAC library screening.
poor feeding-site development in the Gpa2-resistance re- The preparation of the Cara BAC library consisting of sponse is related to either an Rx-like response or to localized 160,000 clones is described in detail in Kanyuka et al. (in necrosis near or at the syncytium, as in the case of, e.g., the press). The SH83 BAC library was prepared essentially as H1 gene (Rice et al. 1985), remains to be investigated.
described in that paper with the following modifications.
High-molecular-weight potato DNA was prepared in agarose MATERIALS AND METHODS
plugs from potato nuclei as described in Liu et al. (1994). Halfof each plug (approximately 10 µg of DNA) was digested with 10 U of HindIII restriction enzyme for 1 h. Size selection was The tetraploid (2n = 4x = 48) progeny of selfed Cara (S1- carried out in two steps. Partially digested S. tuberosum DNA Cara) was initially constructed for fine mapping of Rx was initially subjected to CHEF (clamped homogeneous elec- (Bendahmane et al. 1997). This population was extended to a tric field) electrophoresis at 4°C in 0.5× TBE (Tris-borate- total of 2,788 genotypes. In addition, a diploid mapping EDTA) with a linear increasing pulse time of 60 to 90 s and a population (2n = 2x = 24) derived from a cross between the field strength of 6 V/cm for 18 h. After electrophoresis, lanes diploid clones SH83-92-488 and RH89-039-16 was investi- containing concatemers of bacteriophage lambda as a standard gated. This population, encoded F1SH×RH, has been used for for molecular weight (Bio-Rad, Richmond, CA) were re- Vol. 12, No. 3, 1999 / 203
Table 3. Primer sequences and thermal cycling conditions for CAPS markers in the Rx/Gpa2 intervalz
Polymerase chain reaction conditions
50°C 15 s 35 cycles 56°C 15 s 35 cycles 57°C 15 s 35 cycles 56°C 15 s 35 cycles 60°C 15 s 35 cycles 56°C 15 s 35 cycles z Information on additional CAPS (cleaved amplified polymorphic sequence) markers in the Rx/Gpa2 interval is given in Bendahmane et al. (1997).
moved and stained with ethidium bromide to locate the region DNAs were isolated by inverse PCR (Ochman et al. 1990) on of the gel containing potato DNA fragments ranging from 100 BAC DNA following the procedures described by Woo et al.
to 150 kb in size. This region was excised from the gel with a (1994) and Kanyuka et al. (in press). Sequences of the clones glass coverslip and subjected to a second size selection step in were determined (Sanger et al. 1977) and used to design PCR a 1% SeaPlaque (low-melting point) agarose gel (FMC, primers. The DNA sequences of the PCR primers, the corre- Rockland, ME). CHEF electrophoresis was carried out for 10 sponding thermal cycling conditions, and the appropriate re- h at 4°C with a field strength of 4 V/cm and a constant pulse striction endonuclease to identify a segregating allele in potato time of 5 s. The compression zone containing DNA fragments genomic DNA are presented in Table 3. Information on CAPS of 100 kb was excised from the gel and dialyzed against 50 ml markers that are used in the present study but not mentioned of TE (Tris-EDTA) for 2 h at 4°C. Dialyzed agarose slices in Table 3 can be found in Bendahmane et al. (1997).
were then melted at 70°C for 5 min and digested with 1 unit ofGELASE (Epicentre Technologies, Madison, WI) per 100 mg Analysis of recombination.
of agarose gel for 1 h at 45°C. Ligation of the size-selected Estimates of recombination frequencies and their standard DNA to HindIII-digested and dephosphorylated pBeloBAC11 errors were calculated with the aid of the program LINKAGE- (kindly provided by H. Shizuya, University of Southern Cali- 1 (Suiter et al. 1983) by choosing the appropriate genetic fornia, Los Angeles; Shizuya et al. 1992) and subsequent model for each cross. Data for the nonrecombinant class of transformation of ElectroMAX Escherichia coli DH10B com- genotypes were set for either a 3:1 segregation ratio for the petent cells (Life Technologies, Breda, The Netherlands) with tetraploid population S1-Cara (i.e., simplex-simplex marker the ligated DNA was carried as described in Kanyuka et al. (in configuration) or a 1:1 segregation ratio for population press), with the Bio-Rad Gene Pulser for electroporation. Set- F1SH×RH. Only strongly skewed segregation ratios will no- tings on the Bio-Rad Gene Pulser were as recommended for tably influence estimates of recombination frequencies (Säll E. coli by the manufacturer. Approximately 60,000 white and Nilsson 1994; Manly 1994). A chi-square test was used to colonies were picked individually into 384-well microtiter test for pairwise differences in recombination frequencies plates containing LB freezing buffer (36 mM K2HPO4, 13.2 between the marker intervals. The chi-square test criterion mM KH2PO4, 1.7 mM citrate, 0.4 mM MgSO4, 6.8 mM was determined from the recombinant and nonrecombinant (NH4)2SO4, 4.4% vol/vol glycerol, 12.5 µg of chloramphenicol classes for each marker interval. Differences (rejection of the per ml in Luria-Bertani [LB] medium), grown at 37°C for 24 null hypothesis) were significant when the test criterion was h and stored at –80°C. PCR screening of the BAC libraries greater than the X2[.05] value.
was carried out as described in Kanyuka et al. (in press).
Plant DNA marker screening.
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