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Identification of 2n breeding lines and 4n varieties of potato ( Solanum tuberosum , ssp. tuberosum ) with RFLP-fingerprints

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The possibility of genotype identification with RFLP fingerprints was examined with 20 tetraploid potato varieties and 38 diploid potato lines. By using a sensitive detection system for small restriction fragment length differences and highly
  Theor Appl Genet (1989) 78:16-22 9 Springer-Verlag 1989 Identification of 2n breeding lines and 4n varieties of potato Solanum tuberosum, ssp. tuberosum) with RFLP-fingerprints C. Gebhardt C. Blomendahl U. Schachtschabel T. Debener F. Salamini and E. Ritter Max-Planck-Institut fiir Ziichtungsforschung, Abteilung Pflanzenziichung und Ertragsphysiologie, D-5000 K61n 30, FRG Received December 23, 1988; Accepted December 30, 1988 Communicated by G. Wenzel Summary. The possibility of genotype identification with RFLP fingerprints was examined with 20 tetraploid po- tato varieties and 38 diploid potato lines. By using a sensitive detection system for small restriction fragment length differences and highly variable potato sequences as probes, all genotypes (diploids and tetraploids) were dis- tinguished by a minimum of two probe/enzyme combina- tions. The best single probe/enzyme combination distin- guished 19 out of 20 4n varieties and 33 out of 38 2n lines. Intravarietal variability was very small compared to the intervarietal variability, and patterns obtained with dif- ferent DNA sources of the same genotype were identi- cal. Key words: RFLP - Potato - Fingerprint - Variety identi- fication Introduction The unequivocal identification of a genotype by DNA fingerprinting has been demonstrated in humans by Jeff- reys et al. (1985 a, 1985 b). Even closely related individu- als could be distinguished on the basis of their complex pattern of restriction fragments on Southern blots re- vealed by one or two hypervariable probes (mini- satellites). In plants, genotype-specific RFLP patterns (RFLP=restriction fragment length polymorphism) would have applications in the identification of breeding lines and varieties as well as in the characterization of germplasm resources (Beckmann and Soller 1986). RFLP-studies in maize (Evola et al. 1986; Helentjaris et al. 1985), oilseed (Figdore et al. 1988), lettuce (Landry et al. 1987), soybean (Apuya et al. 1988) and rice (Me- Couch et al. 1988) have shown that varieties can be dis- tinguished from each other by RFLPs. Such polymor- phisms have been widely used for the construction of linkage maps. An exception appears to be the tomato, in which intervarietal differences were rarely found (Helent- jaris et al. 1985). Depending on the existing variability in each species, an increasing number of probe-enzyme combinations would have to be used in order to distin- guish a large number of sometimes closely related variet- ies or lines. Probes equivalent in resolution to Jeffreys "minisatellites" have not been reported in plants. The cultivated European potato, Solanum tuberosum, ssp. tuberosum srcinates from a few introductions from the Andean region of South America (Simmonds 1976). Despite its narrow genetic base, a high degree of poly- morphism at the DNA level was detected within the sub- species (Gebhardt et al. 1989). Using highly polymorphic RFLP markers, together with a sensitive separation tech- nique for restriction fragments based on four cutter re- striction enzymes and denaturing polyacrylamide gels (Kreitman and Aquad6 1986), we could distinguish, with a minimum of two probe/enzyme combinations, 20 regis- tered potato varieties as well as 38 diploid breeding lines. Materials and methods Diploid breeding lines Thirty-eight diploid Solanum tuberosum lines from the collection of the Max-Planck-Institut fiir Ziichtungsforschung (MPI) were used in this study. The line identifications, together with the running numbers, are listed in Table 1. For DNA extraction, leaves and young shoots were harvested from pot grown plants in the greenhouse, frozen in liquid nitrogen and stored at -70 ~ until used. Tetraploid varieties Shoots of 20 registered varieties were harvested in August 1987 from plants growing in the field at Scharnhorst, the outstation  Table 1. List of 2n lines used Line no. Line iden- Line no. Line iden- tification 1 tification 3 H 80.577/1 30 H 82.350/7 5 H 80.649/1 31 H 75.1207/7 6 H 80.695/12 32 H 75.1208/13 7 H 81.8/1 33 H 76.7/7 9 H 81.691/1 34 H 77.409/13 10 H 81.2045/10 35 H 77.420/10 11 H 81.2062/1 37 H 80.572/5 12 H 81.2077/11 38 H 80.576/16 13 H 79.0134/44 39 H 79.0136/76 15 H 82.24/3 40 H 80.696/4 16 H 82.309/5 41 H 79.1506/1 17 H 82.310/4 44 H 81.404/89 18 H 82.337/49 45 H 82.310/10 19 H 82.340/18 46 H 82.355/7 20 H 82.350/6 47 H 82.364/19 23 H 82.379/7 48 H 82.366/3 27 H 81.802/7 49 H 82.368/3 28 H 81.2074/2 50 H 82.2032/1 29 H 82.328/13 51 H 81.1506/60 x The first two digits after the letter refer to the year of clone isolation of the MPI. The varieties were Amigo, Assia, Aula, Bintje, Bodenkraft, Darwina, Datura, Granola, Hansa, Indira, Isola, Nena, Ponto, Quarta, Ragna, Roxy, Saturna, Sira, Taiga and Valetta. Freezing and storage was as for 2n lines. Tubers derived from seed tuber stock plants were provided by the breeders of the varieties Assia (Uniplanta-Saatzucht, Niederarnbach, FRG), Datura (D. von Kameke, Grabau, FRG) and Hansa (Vereinigte Saatzuchten, Ebstorf, FRG). DNA was extracted from peeled, freeze-dried tubers which were stored at -20 ~ DNA from leaves and tubers was extracted according to Saghai-Maroofet al. (1984) and purified according to Gebhardt et al. (1989). The genomic DNA (ca. 5 per gel sample) was digested with the restriction enzymes TaqI and RsaI, respectively, using 3 enzyme units per txg DNA for 4 h or overnight, according to the supplier's instructions (Boehringer, Mannheim). The restriction fragments were size-separated on a denatur- ing 4% polyacrylamide gel and electrophoretically transferred onto a nylon membrane (Amersham, Hybond N), as described by Gebhardt et al. (1989). The inserts of two random genomic potato clones (GP1 and GP26, 870 bp and 940 bp, respectively) and one random potato cDNA-clone (CP6, 460 bp) were used as probes (Gebhardt et al. 1989). The inserts were labelled to high specific activities with the random primer method of Feinberg and Vogelstein (1983, 1984) using ct-(32p)-dCTP as the radioactive nucleotide (Amersham). Prehybridization, hybridization, posthybridiza- tion washes and auto-radiography were performed as described elsewhere (Gebhardt et al. 1989). Clearly identifiable restriction fragments of each probe/ enzyme combination were individually numbered. The presence or absence of each fragment was scored with 1 and 0, respective- ly in the 2n lines and 4n varieties. Those cases in which presence or absence of a fragment was questionable were scored with - 1. The matrix tables (genotypes • fragments) containing the values 1, 0 or -1 were computed using the programme package 17 WORDS & FIGURES (Lifetree Software). Data analysis was performed with an IBM-compatible PC and software developed by E. Ritter (unpublished results). Results During a search for RFLP markers suitable for linkage analysis in diploid potatoes (Gebhardt etal. 1989), probes were encountered which showed highly variable, although well-spaced, fragment patterns on genomic Southern blots within a set of 38 diploid potato lines. The question was addressed as to whether such hyper- variable sequences could also be used for the identi- fication of tetraploid potato varieties. Twenty registered varieties were selected ranging from Bintje (first regis- tered 1910), as an old pure tuberosum type variety, to modern varieties such as Assia (1980) or Valetta (1984), in which germplasm from several wild Solanum species was incorporated (Stegemann and Schnick 1985). Total genomic DNA was isolated from 2n lines and 4n varieties and restricted with TaqI and RsaI respectively. The fragments were size-separated on a denaturing 4% polyacrylamide gel and transferred to a nylon membrane by electroblotting. The filters were hybridized in turn against the labelled inserts of the two random genomic clones, GP1 and GP26, and the anonymous cDNA clone CP6. Comparison of 2n lines and 4n varieties The fragment pattern for one out of six probe/enzyme combinations, GP1/TaqI, is Shown in Fig. 1 A for 2n lines and in Fig. 1 B for 4n varieties. Out of 40 scored frag- ments in both populations, 38 were polymorphic (frag- ments 1-38 and 2 were homomorphic (fragments H1 and H2). Fragments were considered only if their pres- ence or absence could be assessed in most lines of each set. Twenty-seven fragments were common to both 2n and 4n populations, 10 being polymorphic only in the 2n lines and homomorphic in the varieties (fragments 8, 10, 12, 16, 20, 22, 24, 27, 28, 38), indicating that the varieties were more uniform with this probe compared to the diploid lines. Polymorphic fragments, present either in 2n lines or 4n varieties, were also detected (fragments 13, 19 and 32, 36, 37, respectively). However, they occurred in low frequencies or were weakly hybridizing fragments. The patterns of all six probe/enzyme combinations were analysed in a similar way, as demonstrated for GP1/TaqI, for common, 2n and 4n specific fragments, considering only polymorphic and homomorphic fragments scorable in both sets. The result is shown in Table 2. Ninety-four out of 111 scored fragments (85%) were present in both populations, 10 (9%) were detected only in diploids and 7 (6%) only in tetraploids. None of the specific fragments were very frequent or strongly hybridizing.  18 Fig. 1 A and B. Southern blot of 2n lines A and 4n varieties B. Total DNA was restricted with TaqI. Restriction fragments were separated on a denaturing 4% polyacrylamide gel, transferred to a Nylon membrane and hybridized to the random genomic sequence GP1. Fragments common to both sets, present either in 2n lines or 4n varieties, and fragments with uncertain identification are indicated between A and B. Molecular weight markers are indicated on the left hand side of A. A For the identification of 2n lines, see Table 1. B a - Granola, b - Valetta, c - Darwina, d- Roxy, e - Indira, f- Bintje, g - Hansa, h - Taiga, i - Bodenkraft, j- Datura, k - Amigo, l - Quarta, m - Nena, n - Ponto, o - Isola, p - Saturna, q - Assia, r - Aula, s - Sira, t - Ragna Table 2. Fragment distribution in 2n lines and 4n varieties Total Common 2n 4n no. con- to 2n specific specific sidered and 4n GP1/Taql 32 27 2 3 GP1/RsaI 21 20 1 0 CP6/TaqI 18 16 1 1 CP6/RsaI t 4 12 2 0 GP26/TaqI 11 10 0 1 GP26/RsaI 15 9 4 2 Total 111 94 10 7 % 100% 85% 9% 6% The criterion for choosing the set of 38 diploid lines was their heterogeneity in morphological and agro- nomical traits. Hybrids between S tuberosum and wild Solanum species (lines 6 and 40) were included, as well as largely pure tuberosum line (e.g. line 13, see also Geb- hardt et al. 1989). The population of diploids, therefore, represented an apparently wider gene pool than the group of varieties. Nevertheless, the high percentage of common fragments suggested that the two populations were fairly good representatives of the species Solanum tuberosum The number of fragments present in an individual line, compared to the number of all fragments scored in  the population was positively correlated with the relative heterozygosity of that line within the population (Geb- hardtet al. 1989). Relative heterozygosity indices (RHI) were calculated for the 2n and 4n lines, taking into ac- count the fragments of the three probes separately as well as all fragments combined (RHI = number of fragments per line divided by number of fragments scored in the population). The mean RHI values are shown in Table 3. As expected, the tetraploid varieties had higher hetero- zygosity indices than the diploid lines. Number of patterns The individual polymorphic fragments in the six probe/ enzyme combinations were scored for presence or ab- Table 3. Relative heterozygosity index (RHI) in diploids and tetraploids (standard deviation) 2n 4n GP1 0.47 (0.08) 0.62 (0.06) CP6 0.28 (0.08) 0.50 (0.12) GP26 0.15 (0.07) 0.34 (0.07) GP1 CP6 0.36 (0.06) 0.52 (0.05) GP26 19 sence ('Materials and methods') in the 38 2n lines and 20 4n varieties. From these data the number of different patterns was determined, resulting from the six single probe/enzyme combinations, the 15 possible double combinations and 2 of 60 possible triple combinations. In addition, the number of fragments by which any pair of diploid and tetraploid lines respectively differed from each other was calculated (703 pairwise comparisons for the 38 diploids and 190 for the 20 tetraploids). Table 4 summarizes the results of the computer analy- sis. No single probe/enzyme combination was sufficient in distinguishing all 38 diploid lines and all 20 varieties. However, the combination of probe GPI with the en- zyme TaqI (Fig. 1) came very close by revealing 19 out of 20 and 33 out of 38 possible patterns. From the 15 double and 2 triple combinations, all but 3 distinguished the 20 varieties (indicated by * in Table 4), 6 distinguished the 38 diploids and 5 could differentiate all, diploids as well as tetraploids (GP1/Taq+GP26/Taq, GP1/Rsa+CP6/ Taq, GP1/Rsa + GP26/Taq, GP1/Rsa + GP26/Rsa and GP1/Taq + CP6/Taq + GP26/Taq). In the diploid set, lines closely related by pedigree, such as pairs of full sib lines, differed not only in the indicated most effective combinations but also with sin- gle probe/enzyme combinations: sister lines 20 and 30 with GP1 and CP6 (either enzyme, 1-3 fragments) and Table 4. Pattern comparison of 4n varieties and 2n breeding lines Probe/enzyme combination No. of No. of fragments patterns 4n 2n Min-max no. of differing fragments Mean no. of differing fragments (StD) 4n 2n 4n 2n GP1/Taq 38 19 GP1/Rsa 20 18 CP6/Taq 18 15 CP6/Rsa 13 12 GP26/Taq 11 16 GP26/Rsa 15 17 GP1/Taq + GP1/Rsa 58 19 GP1/Taq + CP6/Taq 56 20 * GP1/Taq+ CP6/Rsa 51 20 * GP1/Taq + GP26/Taq 49 20 * GP1/Taq + GP26/Rsa 53 20 * GP1/Rsa + CP6/Taq 38 20 * GP1/Rsa + CP6/Rsa 33 20 * GP 1/Rsa + GP26/Taq 31 20 * GP 1/Rsa + GP26/Rsa 35 20 * CP6/Taq + Cp6/Rsa 31 t 7 CP6/Taq + GP26/Taq 29 20 * CP6/Taq + GP26/Rsa 33 20 * CP6/Rsa + GP26/Taq 24 20 * CP6/Rsa + GP26/Rsa 28 20 * GP26/Taq + GP26/Rsa 26 18 GPt/Taq + CP6/Taq + GP26/Taq 67 20 * GPI/Taq + CP6/Taq + GP26/Rsa 71 20 * 33 0-14 0-21 7.2 (1.0) 34 0-9 0-14 4.4 (0.8) 24 0-14 0-14 5.2 (1.5) 17 0-9 0-10 3.9 (0.9) 16 0-9 0-7 3.4 (0.7) 12 0-7 0-9 3.4 (0.5) 38* 0-21 1-35 11.5 (1.5) 36 3-26 0-30 12.4 (2.1) 34 2-21 0-29 11.1 (1.4) 38* 4--20 1-27 10.6 (1.3) 37 4-18 0-28 10.6 (1.1) 38 * 1-20 1-23 9.6 (1.9) 37 1-18 0-21 8.3 (1.1) 38* 2-15 1-19 7.8 (I.1) 38* 1-14 1-20 7.7 (0.8) 26 0-20 0-21 9.1 (1.9) 35 1-20 0-19 8.7 (1.7) 34 1-18 0-19 8.6 (1.5) 28 1-15 0-16 7.4 (1.3) 25 2-14 0-15 7.3 (1.0) 17 0-16 0-16 6.8 (1.1) 38* 5-33 1-34 15.8 (2.3) 37 4-31 0-35 15.8 (2.0) 9.0 (2.5) 5.6 (1.7) 5.7 (1.1) 3.2 (1.5) 2.9 (0.7) 2.6 (1.0) 14.6 (3.9) 14.7 (3.1) 12.3 (3.4) 12.0 (2.6) 11.6 (2.8) 11.2 2.0) 8.8 2.5) 8.5 (1.8) 8.2 (2.0) 8.9 (2.2) 8.6 1.2) 8.3 (1.5) 6.0 (1.7) 5.7 (1.9) 5.5 (1.7) 17.6 (3.2) 17.3 (3.4) * see text  20 Fig. 2. Southern blot with DNA from 15 tubers each of the three varieties Assia, Datura and Han- sa, restricted with TaqI and hybri- dized to GP1 as in Fig. 1. The fragments distinguishing the 3 va- rieties are indicated with the same numbers as in Fig. 1. The possible exception in the Hansa-series is marked with an X sister lines 17 and 45 with GP1 and GP26 (either enzyme, 2-7 fragments). The higher the number of fragments in which two genotypes differ, the more reliable would be the distinc- tion between them. Therefore, those probe/enzyme com- binations providing more than one fragment as the min- imum difference would better describe genotypic differ- ences. The mean numbers of differing fragments (calcu- lated from all pairwise comparisons) as well as their minimum-maximum range indicated that - whenever a difference between genotypes was noted - in most cases it could be determined safely, based on several restriction fragments (Table 4). Six of the double probe/enzyme combinations offered at least 2 fragments for the min- imum difference between any of the 20 varieties. Despite the higher heterozygosity of the varieties (Table 3), it was also evident that the number of frag- ments differing in the comparisons between two varieties was similar or sometimes even lower than between two diploid lines (Table 4). Controls To test the reproducibility of the RFLP pattern, the same experiments as for the diploid lines and varieties were performed with DNA extracted from 15 tubers each of the three varieties Assia, Datura and Hansa. The tubers were obtained from the breeder of the variety and srci- nated from 15 different plants. The same probe/enzyme combination, GP1/TaqI, as in Fig. 1 is shown in Fig. 2. Within the varieties the patterns were identical, with only one exception in the Hansa series (indicated in Fig. 2), where one faint additional fragment appeared. The possi- bility of an artifact or of a partially digested fragment cannot be excluded. A discrepancy between the patterns obtained with leaf DNA of an independent source and the tuber DNA was not observed. In contrast, the fingerprints of the three varieties were clearly different from each other. Discussion According to Bailey (1983), the basic criteria to be ful- filled by a character used for variety identification are: (1) distinguishable intervarietal variation, (2) minimal intra- varietal variation, (3) environmental stability, and (4) in case of biochemical or molecular characters, experimen- tal reproducibility. The results reported in this paper
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