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RFLP analysis and linkage mapping in Solanum tuberosum

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A morphologically and agronomically heterogeneous collection of 38 diploid potato lines was analysed for restriction fragment length polymorphisms (RFLPs) with 168 potato probes, including random genomic and cDNA sequences as well as characterized
  Theor Appl Genet (1989) 78:65-75 9 Springer-Verlag 1989 RFLP analysis and linkage mapping in olanum tuberosum C. Gebhardt, E. Ritter, T. Debener, U. Schachtschabel, B. Walkemeier, H. Uhrig and F. Salamini Max-Planck-Institut fiir Ziichtungsforschung, D-5000 K61n 30, FRG Received December 23, 1988; Accepted December 30, 1988 Communicated by G. Wenzel Summary. A morphologically and agronomically hetero- geneous collection of 38 diploid potato lines was ana- lysed for restriction fragment length polymorphisms (RFLPs) with 168 potato probes, including random genomic and cDNA sequences as well as characterized potato genes of known function. The use of four cutter restriction enzymes and a fragment separation range from 250 to 2,000 bases on denaturing polyacrylamide gels allowed the detection of RFLPs of a few nucleotides. With this system, 90% of all probes tested showed useful polymorphism, and 95% of those were polymorphic with two or all three enzymes used. On the average, 80% of the probes were informative in all pairwise comparisons of the 38 lines with a minimum of 49% and a maximum of 95%. The percentage of heterozygosity was deter- mined relative to each other for each line and indicated that direct segregation analysis in F1 populations should be feasible for most combinations. From a backcross involving one pair of the 38 lines, a RFLP linkage map with 141 loci was constructed, covering 690 cMorgan of the Solanum tuberosum genome. Key words: Solanum tuberosum - RFLP - Linkage map Introduction Besides direct sequence comparisons, restriction frag- ment length polymorphisms (RFLPs) are the most sensi- tive tool for the detection of DNA differences within or between species. The molecular basis of RFLPs is the loss or gain of a restriction site due to a point mutation within the enzymes recognition sequence, or a molecular event leading to insertion, deletion or inversion. Both situa- tions result in a length difference of genomic restriction fragments detectable on Southern blots. RFLPs are genetic markers with several advantages compared to conventional markers. They describe direct- ly the genotype instead of the phenotype and, therefore, are not influenced by the environment. The number of RFLP markers which can be mapped is only limited by the molecular differences existing between available genotypes; it is also proportional to the effort applied to detect them. RFLP studies and detailed RFLP linkage maps are recognized as important contributions of mo- lecular genetics to plant breeding (Beckmann and Soller 1986; Burr et al. 1983; Tanksley 1983) as well as to the fields of plant taxonomy and evolution (Song et al. 1988; Hosaka and Hanneman 1988 a, b). In recent years RFLP linkage maps have been devel- oped in several diploid plants, the most advanced being maize (Helentjaris et al. 1986; Helentjaris 1987), tomato (Bernatzky and Tanksley 1986a; Helentjaris et al. 1986; Zamir and Tanksley 1988), lettuce (Landry et al. 1987 b), rice (McCouch et al. 1988), pepper (Tanksley et al. 1988) and Arabidopsis (Chang et al. 1988). The cultivated po- tato Solanum tuberosum ssp. tuberosum) is a tetraploid with poorly developed cytogenetics. In fact, no linkage map is available for the species, although a few linkages among isoenzymes have been obtained by Douches and Quiros (1987; see also Desborough 1983). A comparative RFLP map between tomato and po- tato has been recently obtained from an interspecific cross involving the wild species S. phureja and S. cha- coense and a diploid S. tuberosum line (Bonierbale et al. 1988). However, it seemed desirable to us to produce a RFLP linkage map within a gene pool of diploid S. tuberosum, where many crosses of agronomic interest can be analysed with the aim of mapping disease resis- tance genes or quantitative trait loci. Therefore, we con- ducted a survey of RFLPs present in a collection of 38 diploid potato breeding lines, which were selected for  66 agronomic qualities over years at the Max-Planck- Institut ffir Zfichtungsforschung (review in Ross 1986). A RFLP linkage map has been obtained from a segregating backcross of one pair of diploid lines out of the 38 analysed. Materials and methods Plant material Thirty-eight diploid Solanum tuberosum lines from the collection of the Max-Planck-Institut ffir Zfichtungsforschung were se- lected for screening the probes. The line identifications together with their current running numbers are listed in the first two columns of Table 1. One F1 plant (H 86.0916/2) from a cross between lines 9 (H 81.691/1) and 16 (H 82.309/5) was the pollen parent in a backcross to line 16. Sixty-seven backcross lines were obtained and used for the linkage analysis. For DNA extraction, leaves and young shoots were har- vested from pot-grown plants in the greenhouse and stored at - 70 ~ Freeze-dried material was also used for DNA isolation. DNA-isolation Total genomic DNA was purified according to Saghai-Maroof et al. (1984), with the following modifications and additional purification steps: two times concentrated extraction buffer was used for fresh (stored frozen at -70 ~ as well as freeze-dried material. Further purification steps involved either one CsC1 gradient centrifugation step or a treatment with RNAse A (Boehringer Mannheim, 10 gg/ml, 30 min, 37 ~ followed by one phenol/chloroform and two chloroform extractions; con- taminating carbohydrates were partially removed by adjusting the final aqueous DNA-solution to 1 M NaC1, incubating 20 min at -20~ centrifuging 15 min at 4~ and discarding the gelatinous pellet. After a final ethanol precipitation, the DNA was resuspended in 10 mM TRIS HC1, 1 mM EDTA, pH 8.0. The concentration of CsCl-purified DNA was estimated by the absorbance at 260nm, otherwise by band-intensity on Ethidiumbromide-stained agarose gels. Restriction digests, electrophoresis and blotting Genomic DNA was restricted with 3-4 units/gg DNA of Taql, RsaI, HaeIII or AluI (Boehringer Mannheim) for 4 h or over- night, according to the supplier's instructions. Sample preparation, electrophoresis and blotting was es- sentially as described by Kreitman and Aquad6 (1986) with the following modifications: restriction digests containing 3-4 gg DNA were ethanol-precipitated and resuspended in 5 lal loading buffer (94 formamide, 10 mM EDTA, 0.05 xylene cyanol, 0.05 bromophenol blue). Undissolved material was pelleted by a short spin of the sample tubes. After denaturation (5 min, 95 ~ the sample tubes were transferred to ice and the super- natants were loaded into 3 mm wide slots of a 30 crn x 40 cm x 1 mm denaturing 4 polyacrylamide gel in 1 x TEB-buffer, 8 M urea. The running buffer was 1 x TEB (0.089 M TRIS-borate, 0.089 M boric acid, 2 mM EDTA). Electrophoresis was per- formed at 50 W constant power until the xylene cyanol dye reached the bottom of the gel. After electrophoresis, the gel was immediately transferred into a 30 cm x 40 cm electroblotting chamber (electrophoresis and electroblotting equipment manu- factured by the MPI workshop) and blotted in 23 1 0.4 x TEB- buffer for 1 h at 5 V/cm onto a nylon membrane (Amersham Hybond N). The DNA was covalently bound to the membrane by UV irradiation at 302 nm on a transilluminator for 5-10 min. Table 1. Characterization of 2nd potato lines by polymorphism and heterozygosity No. Line identi- Average (Min.-Max) Hetero- Relative fication informa- ( ) zygosity hetero- tive (LH) zygosity probes in index ( ) (RHI) 3 H80.577/1 79.3 (74.1 89.8) 62 0.370 5 H80.649/1 79.3 (74.1 90.5) 52 0.348 6 H80.695/12 85.7 (81.6-93.2) 60 0.381 7 H81.8/1 78.2 (72.8-88.4) 22 0.298 9 H81.691/1 78.6 (71.4 93.2) 57 0.350 10 H81.2045/10 87.0 (81.0 93.2) (1) (1) 11 H81.2062/1 81.7 (57.8-90.5) 63 0.381 12 H81.2077/11 82.2 (66.7-90.5) 55 0.364 13 H79.0134/44 84.9 (76.9-92.5) 58 0.355 15 H82.24/3 82.0 (74.8-89.8) 48 0.348 16 H82.309/5 77.8 (66.0-91.8) 59 0.350 17 H82.310/4 76.4 (49.0-90.5) 51 0.351 18 H82.337/49 75.9 (56.5-91.8) 54 0.342 19 H82.340/18 76.6 (68.0-91.2) 50 0.353 20 H82.350/6 76.2 (55.8-93.2) 50 0.319 23 H82.379/7 77.1 (71.4-91.8) 38 0.334 27 H81.802/7 78.0 (66.0-91.2) 57 0.341 28 H81.2074/2 81.5 (57.8-91.2) 63 0.363 29 H82.328/13 82.3 (77.6-91.8) 55 0.355 30 H82.350/7 76.3 (68.0-91.8) 39 0.334 31 H75.1207/7 79.6 (71.4 91.2) 54 0.357 32 H75.1208/13 81.1 (70.7-92.5) 55 0.360 33 H76.7/7 75.3 (55.8-90.5) 44 0.310 34 H77.409/13 81.8 (72.8-94.6) 53 0.352 35 H77.420/10 78.8 (71.4-92.5) 38 0.305 37 H80.572/5 80.0 (74.1-89.6) 59 0.352 38 H80.576/16 80.0 (71.4-90.5) 50 0.332 39 H79.0136/76 82.1 (78.2-89.8) 54 0.353 40 H80.696/4 86.5 (83.7-94.6) 68 0.378 41 H79.1506/I 80.7 (76.9-92.5) 40 0.309 44 H81.404/89 79.4 (72.8-92.5) 41 0.341 45 H82.310/10 76.3 (63.3-89.1) 59 0.357 46 H82.355/7 74.7 (66.0-89.8) 51 0.324 47 H82.364/19 77.8 (66.0 92.5) 47 0.323 48 H82.366/3 78.1 (69.4-94.6) 56 0.342 49 H82.368/3 77.0 (70.1-93.9) 50 0.327 50 H82.2032/1 86.1 (77.6-94.6) 50 0.357 51 H81.1506/60 80.4 (73.0-92.7) 50 0.330 Mean value 79.8 (49.0 94.6) (1) This line was heterogeneous either due to the mixing of plant materials or of DNAs or due to a ploidy level different from 2n. The calculation of the LH and RHI indexes was, therefore, omitted Probe preparation, hybridization and autoradiography Recombinant plasmids were purified according to Birnboim and Doly (1979). Inserts of cDNA and genomic clones, were isolated by electroelution from agarose gels and labelled with 32P-c~ dCTP (Amersham) using the random primer labelling method of Feinberg and Vogelstein (1983, 1984). For hybridization, one to four membranes were stacked on top of each other and placed around the inner wall of a plastic tube (30 cm • 3.5 cm in diameter, Macrolon), and sealed with a silicon plug with a syringe needle in the center for pressure exchange. Eight to ten milliliters per membrane (25 cm x 30 cm)  67 of 6 x SSC, 5 x Denhardt's solution, 0.5% SDS, 20 ~tg/ml dena- tured salmon sperm DNA were added to the tube for prehybri- dization and hybridization, respectively. The tubes were incubated at 65 ~ rotating around the long axis, in a hybridization oven (oven and tubes by Bachhofer, Reutlingen, FRG). Prehybridization and hybridization were performed overnight. Posthybridization washes were first in the tubes with 3 x 50ml 1 x SSC, 0.1% SDS, 65~ 15 min each, then with 2 x 31 (for up to 10 membranes) 5 mM Sodiumphos- phate, 1 mM EDTA, 0.2% SDS, pH 7.0, at room temperature for 30 min each or longer. The membranes were autoradio- graphed at -70~ from 3 to 10 days using Kodak X-OMAT AR5 film and one intensifier screen. Preparation of random eDNA clones Poly A + RNA was isolated from young shoots and leaves ac- cording to Bartels and Thompson (1983). Double-stranded eDNA was synthesized following Gubler and Hoffmann (1983), ligated to EcoRI linkers and cloned into the EcoRI site of the bluescribe vector (Vector Cloning Systems, San Diego/CA). Af- ter transformation of E. coli strain TG-2 (Hanahan 1983) with the recombinant DNA, a library of ca. 3,500 clones was ob- tained. Random cDNA probes were isolated from clones with inserts of a least 200 base pairs. The nomenclature used for eDNA RFLP markers is CPn(a), CPn(b) and so on, with n being an identification number and a, b etc indicating different loci within the same probe. Preparation of random genomic clones Total DNA from a 2n potato line was digested with PstI as described by Young et al. (1987). The fragments were separated on a 1% agarose gel. Fragments between 500 and 2,000 base pairs were cut out from the gel, electroeluted with a Biotrap (Schleicher and Schiill) and ligated into PstI-digested bluescribe vector. Clones from the library (2,500 clones) obtained after transformation of E. coli strain TG-2 were screened for highly repetitive sequences according to Landry and Michelmore (1985). Random genomic probes were isolated from clones giv- ing no hybridization signal with 32P-labelled total potato DNA. The nomenclature for genomic RFLP markers is GPn(a), GPn(b) etc. analogous to eDNA markers. Specified clones used as RFLP markers cDNA clones coding for 4-coumarate: CoA ligase (4CL) and phenylalanine ammonia-lyase (PAL) of potato were obtained from K.-H. Fritzemeier (MPI fiir Ziichtungsforschung, Co- logne; Fritzemeier et al. 1987). Two potato cDNA clones, pC 116 and pI 471, were provided by J. Taylor (ditto). pI 471 codes for a gene involved in the defense reaction of potato against Phytophthora infestans. A similar function is not confirmed for pC 116 which has been isolated in a similar context (J. Taylor and G. Strittmatter, unpublished results). The eDNA clone cod- ing for granule bound starch synthase Wx gene) of potato was obtained from M. Hergersberg, MPI, Cologne (Hergersberg 1988). M. Thangavelu, Plant Breeding Institute, Cambridge, provided the genomic clone 2PAc 59 (13.2 kb) containing an actin sequence of potato. A subcloned l.l-kb EcoRI/HindlII fragment was used as RFLP marker. The cDNA clone rbcS c (Eckes et al. 1985) and genomic clones coding for rbsS 1, rbcS 2a, rbcS 2b and rbcS 2c (Wolter et al. 1988) were obtained from F.P. Wolter, MPI, Cologne. S. Rosahl (ditto) provided the geno- mic clone pgT 5 containing a patatin gene (Rosahl et al. 1986). Coding sequences (pR-1 and pR-2) of glutamine synthetase of Phaseolus vulgaris (Gebhardt et al. 1986) were used as heterolo- gous probes for screening the potato cDNA library. Data and linkage analysis The presence and absence of specific restriction fragments were scored for each genotype on autoradiograms. Only clearly scor- able fragments were analysed and doubtful cases were excluded from further data processing. The potato lines described in this paper were highly heterozygous. The presence of a restriction fragment could, therefore, indicate either homo- or hetero- zygosity for that fragment. The state of homo- or heterozygosity of parental, F1 and backcross lines was inferred from segre- gation data. Linkage analysis and calculation of recombination frequencies were computed according to Bailey (1961). Each segregating fragment was first tested for distorted seg- regation ratio with the Z 2 test. Then for each pair of fragments (A and B), the fragment configuration was determined, depend- ing on whether the two fragments were present in both parents (configuration AB/AB) or in only one parent (Configuration AB/00). The mixed configurations AB/A0 and AB/0B were also analysed. From the phenotype distribution it was inferred whether A and B were in coupling or repulsion. Taking into account the fragment configuration and distorted segregation ratio (if present), linkage was tested for each pair of fragments with the X z test. Recombination frequencies were calculated using the "maximum likelihood" method. From the resulting matrix of recombination frequencies, linkage groups were deduced with the "nearest neighbour" method. Linkage sub- groups were obtained preferentially for the fragment configura- tions AB/00 (coupling and repulsion)of each backcross parent and AB/AB (coupling), because these configurations generally had the smallest standard errors. The linkage subgroups which were independently derived from both parents were united by allelic fragments (defined by total linkage in repulsion). Allelic fragments, and fragments showing 100% co-segregation within the same probe (example in Fig. 5) constituted one locus. Loci were arranged in linear order according to Haldane's formula. RFLP and linkage analysis were performed on an IBM- compatible PC with software developed by one of us (E. Ritter, unpublished results). Results and discussion The genetic material After a series of experiments with several diploid potato lines as representatives of the genetic material currently used in Europe to breed superior 4n varieties, a set of 38 was chosen for RFLP analysis (Table 1, columns 1 and 2). The criteria adopted in selecting the genotypes were large variability in morphological (e.g. flower colour, tuber shape, eye depth, skin colour) and agronomic char- acters (e.g. yield, starch content, disease resistance). The variability among the lines can partially be ascribed to several wild or cultivated Solanum species participating in various degrees to the pedigrees, the most important being S. acaule, S. spegazzinii, S. demissum, S. gourlayi, S. stenotomum, S. vernei, S. sparsipilum, S. stoloniferum and S. andigena. Lines 6 (hybrid between S. tuberosum and S. stenotonum) and 40 (hybrid between S. tuberosum and S. spegazzinii) contain the highest amount of germ- plasm from another Solanum species whereas, e.g., lines 13 and 39 are possibly pure tuberosum, (except for early and not well-defined introductions from S. demissum,  68 BH31*xBH5* HH140 x ~ --~44.1016/10 ~---- 149.54o/2 ~ BH91x BH171 BH1/,9 x BH31~x HH 346 HH439 x75.163/153 H77.01/27 H81.691/1 @ x H82.30915 @ t H69.1382/14 x HHZ,73 l T H26/.7 H2086 x USW5536.? BH165xBH5* t t [ t RH 109 xs. spg 8716 USWPH4 x USWH253 14 5o4n0a/2*l l / MittelfriJhe 144.1016/10 1 I ss::'~ Mittelfr(] he, S. ac{ Fig. 1. Pedigrees of the 2n potato lines 9 and 16 from which the segregating backcross population was obtained. Tetraploid genotypes are shown in boxes; * indicates the repeated use of the same genotype in the pedigree. Important varieties and species contributing to the lines 44.1016/10 and 49.540/2 are indicated at the bottom. Reduction of the ploidy level from 4n to 2n occurred via female parthenogenesis induced by S. phureja fertil- ization S. acaule and S. andigena into 4n varieties and breeding lines from which the diploids were later derived). The two pedigrees of lines 9 and 16 from which the backcross for linkage analysis was obtained are shown in Fig. 1, and their complexity is rather typical for most lines included in the set. In a similar manner as lines 9 and 16, certain 4n lines and varieties are common an- cestors of most of the diploid lines (Rudorf 1958: Ross and Jacobsen 1976) because of their good combining ability and valuable agronomic characters. The self- compatible line 51 was included in the set because it was used in transformation experiments by Knapp et al. (1988). Based on the pedigrees as far as known and on comparative RFLP analysis with 20 tetraploid potato varieties (Gebhardt et al. 1989), we concluded that the set of 38 diploid lines was a fairly good representation of the germplasm used in European potato breeding. RFLP analysis within 38 diploid potato lines From the historically narrow genetic basis of the Euro- pean potato (Simmonds 1976) and from the pedigree structure of the diploid gene pool, a high variability at the DNA level could not be expected a priori. Therefore, we adapted the four cutter filter hybridization technique of Kreitman and Aquad6 (1986) in order to improve the chances of detecting RFLPs in potato. Using four cutter restriction enzymes theoretically increases the length of sequence scored per probe by the increased number of sites (Kreitman and Aquad6 1986). Moreover, the sepa- ration range of DNA fragments between 250 and 2,000 bases allows the detection of fragment length differences as small as a few nucleotides. This is demonstrated in Fig. 2. Genomic DNA of the 38 diploid lines was di- gested with RsaI (Fig. 2a), HaeIII (Fig. 2b) and TaqI (Fig. 2 c), respectively. The fragments were separated on a denaturing 4% polyacrylamide gel and transferred to a nylon membrane by electroblotting. The three mem- branes were hybridized together against the random genomic probe GP24. The length of each hybridizing fragment was determined relative to molecular weight standards and is indicated in Fig. 2. The polymorphic pattern of the probe resulted in this case from length differences of 12-15 bases. With the experimental setup shown in Fig. 2 - except that HaeIII was replaced later on by AluI - 168 potato sequences were tested for RFLPs within the population of 38 2n lines. The probe sources were random genomic Pst clones of potato with inserts of 500-2,000 base pairs, random potato cDNA clones with inserts of at least 200 base pairs and several cloned potato genes with known or specified coding function ('Materials and methods'). Ex- amples of patterns revealed by the probes are shown in Figs. 3 and 4. Simple patterns as in Fig. 3 were caused by single copy sequences. In Fig. 3 a and b respectively, six and two allelic fragments were identified for which the homo- or heterozygous state could be determined. The percentage of heterozygosity given in Table 1 (column 5) was evaluated with this type of pattern. With one probe, null alleles were observed as shown in Fig. 3 c. This find- ing was also reported in other species (McCouch et al. 1988; Landry etal. 1987a) and suggested a polymor- phism due to insertion/deletion of DNA sequences. In contrast to the single copy probes of Fig. 3, a very complex polymorphic pattern was revealed by the anony- mous probe GP35 (Fig. 4). Whereas the most intensive hybridizing fragments showed hardly any variation, at least 23 polymorphic minor fragments were scored (indi- cated by arrows), taking into account only those for  69 Fig. 2a-c. Southern blots ofgeno- mic DNA of 38 potato lines (num- bered according to Table 1) hybrid- ized to the genomic probe GP24. 3-4 lag DNA were restricted with a RsaI, b HaelII and e TaqI respec- tively. The fragments were sepa- rated on a denaturing 4 polyacryl- amide gel and transferred to the nylon membrane by electroblot- ting. Fragment lengths were deter- mined relative to size markers (587, 540, 458, 434 and 267 bases, not shown) and are indicated to the left Fig. 3a-e. Examples of RFLP patterns obtained with single copy probes. Lines and experimental conditions as in Fig. 2. a TaqI di- gest, hybridized to the genomic se- quence GP79, b TaqI digest, hybridized to the cDNA sequence CP 57, c HaeIII digest, hybridized to the genomic sequence GP93. Allelic fragments are indicated by a?'I OWS
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