Key words: Glycine max, DNA fingerprinting, cultivar identification, simple repetitive sequences, oligonucleotide probes, soybean. Summary. Soybean DNA fingerprints were analyzed by digoxigenin-labeled ..... a laboratory manual. 2nd edn.
Euphytica 80: 129-136, 1994. O 1994KluwerAcademicPublishers. Printedin the Netherlands.
129
DNA fingerprinting in soybean (Glycine max (L.) Merrill) with oligonucleotide probes for simple repetitive sequences T. Y a n a g i s a w a 1'3'4, M . H a y a s h i 2, A. H i r a i 1 & K. H a r a d a 3,5
i Faculty of Agriculture, The University of Tokyo, Tokyo 113, Japan; 2 Faculty ofAgriculture, Meiji University, Kawasaki 214, Japan; 3 Department of Molecular Biology, National Institute of Agrobiological Resources, Tsukuba 305, Japan; 4 present address: Department of Crop Breeding, National Agriculture Research Center, Tsukuba 305, Japan; 5 present address: Faculty of Horticulture, Chiba Univ., Matsudo 271, Japan Received28 February 1994;accepted 1 September1994
Key words: Glycine max, DNA fingerprinting, cultivar identification, simple repetitive sequences, oligonucleotide probes, soybean
Summary Soybean DNA fingerprints were analyzed by digoxigenin-labeled oligonucleotide probes complementary to simple repetitive sequences. The clearest and most polymorphic patterns were obtained with (AAT)6 as a probe, with which all 47 soybean cultivars tested could be distinguished. However, DNA fingerprints of individuals within cultivars showed the same pattern. Using (CT)8, (GAA)5 or (AAGG)4 as probes, clear polymorphic patterns among cultivars and accessions in the subgenus Soja (Glycine max and Glycine soja) were not observed, while quite different patterns were found in accessions in the subgenus Glycine. The results suggest that G. max and G. soja are closer in their genome structure. DNA fingerprints of reciprocal crosses between cultivars and accessions in the subgenus Soja were similar, and contained bands of both parents. In an F2 population from these crosses, such bands segregated in a Mendelian fashion.
Introduction Soybean (Glycine max (L.) Merrill (2n = 40)) is an important crop especially as a source of proteins and oils. The genus Glycine subgenus Soja consists of two species, the cultivated soybean, G. max, and the wild soybean (G. soja Sieb. & Zucc. (2n = 40)). Several groups have reported that soybean varieties and accessions in the subgenus Soja lack diversity at the DNA sequence level. Doyle & Beachy (1985) found no length heterogeneity or restriction site polymorphisms for six enzymes in the 18S and 25S rDNA tandem repeats. Doyle (1988) surveyed 33 G. max and G. soja accessions for variation in the 5S rRNA genes and found only a single variant. Keim et al. (1989) indicated that soybean shows low levels of diversity at the DNA sequence level. A large part of the polymorphism identified in G. max may be due to genomic rearrangement (Apuya et al., 1988). In these earlier studies, most
of the probes used were randomly selected, single copy genomic and cDNA clones. Several classes of repetitive sequences, such as 'minisatellites' (Jeffreys et al., 1985) and 'microsatellites' (Litt & Luty, 1989; Tautz, 1989) are found to be hypervariable. The variation in such repetitive sequences is mainly the result of changes in copy number of the repeat units, which are a major source of genomic polymorphism (Tautz et al., 1986). Several polymorphic loci can be simultaneously detected if an appropriate probe is used (Jeffreys et al., 1985; Schafer et al., 1988). The occurrence of microsatellites, which are simple repetitive sequences consisting of 2-4 bp core sequences (Hamada et al., 1982; Tautz & Renz, 1984), have been investigated in many organisms (Weising et al., 1991). As Southern hybridization analysis with synthetic oligonucleotide as probes can be used without the need of cloning, this approach has recently been employed for DNA fingerprinting.
130 Previously, we reported the abundance and organization o f 14 simple repetitive sequences in the soybean genome (Hayashi et al., 1993). In this paper we describe the D N A fingerprinting o f soybean (G. max) and its wild relatives using digoxigenin-labeledprobes, which allow discrimination among various accessions, and also describe the mode o f inheritance o f some o f the bands identified in the D N A fingerprints.
Materials and methods
Plant material and DNA isolation Forty-seven different cultivars o f G. max, 14 accessions o f G. soja and 4 accessions of wild perennial relatives were used in this study (listed in Table 1). These cultivars and accessions were chosen to represent geographically diverse origins. Total D N A from leaves was isolated according to a modified CTAB (cetyl trimethyl a m m o n i u m bromide) method (Murray & Thompson, 1980).
Electrophoresis and hybridization D N A was digested with various 4 bp cutter restriction enzymes (AluI, Hinfl, RsaI and Sau3AI) following the supplier's instructions (Toyobo), followed by separation on 0.8% agarose gels in TBE buffer for 24 hr within circulating running buffer. After electrophoresis, the gel was denatured in 0.5 M NaOH/1.5 M NaC1, neutralized in 0.5 M Tris-HC1 (pH 7.4)/1.5 M NaCI and blotted onto a nylon membrane (Hybond N +, Amersham). The membrane was rinsed in 2 x SSC for 5 min, dried at room temperature and then baked for 2 hr at 800 C. Hybridization was performed at Tm-10 ° C for 3 hr in hybridization buffer (5 x SSC, 1% Skim milk (Difco), 0.1% N-lauroylsarcosine, 0.02% SDS). The Tm of each probe was calculated according to Sambrook et al. (1989), oligonucleotides were 3~-end labeled with terminal transferase using DIG- 11-ddUTP (Boehringer Mannheim). The membrane was washed twice in 2 x SSC, 0.1% SDS for 5 min at room temperature, and then twice in 2 x SSC, 0.1% SDS for 15 min at the relevant hybridization temperature. Detection was performed according to the D I G luminescent detection system protocol (Boehringer Mannheim). Signals were detected by exposure of the membrane to X-ray film (New RX; Fuji).
Table 1. Forty-sevencultivars of G. max, 14 accessions G. soja and 4 species of the subgenus Glycine currently used in this study species
cultivars accessions name
origin
G. max (L.) Merrill
Yoshiokadairyuu Ooyachi 2 Okuhara 1 Tokachihadaka Wasemidori Shirotsurunoko Yagi 1 Ani Nemashirazu Yamashirotama Hakuhou (6) Miyagishirome Kimusume lbaraki 1 Bonminori Norin 2 Oojiro Tochigikimusume 1 Shirohanasai 1 Bukoumame Karihatakiya Oohamadaizu Ginjiro Sakagami 2 Oguradaizu Fuji 4 Syuzenjizairai Natsudaizu 1 Udadaizu Yukikorogashi Tamanishiki Shirodaizu Akiyoshikurakake Chuusei 11 Hanashirazu Aochi Kanagawawase Matsuura Kairyoushirome Aso 1 Oushokuakidaizu Ooitaakidaizu 1 Kindaizu Shirokuchi 1 Kouandaa Aohiguu Manseiouhakusyu Chugokunanbu H4
Hokkaido "
Aomori Akita Iwate Fukushima Miyagi Ibaraki
Gunma Tochigi " Saitama Niigata Ishikawa Nagano
Shizuoka Mie Nara Hyogo Shimane Yamaguchi Kagawa Tokushima Ehime Fukuoka Saga Nagasaki Kumamoto Ooita Miyazaki Kagoshima Okinawa Taiwan China China
131
Table 1. Continued. species
cultivars accessions name
origin
G. soja Sieb. & Zucc.
Sarugawa Kikonaigawa Tsurumame Tsurumame Tsurumame Madaraoohatsurumame COL/IBARAKI/1983/WATANABE-3 COLtAICHI/1981/TANEDA Tsurumame COL/OKAYAMA/1983/ISHIDA COL/KAGISHIMA/1983/SHINCHI
Hokkaido
G. soja Sieb. & Zucc. 148 6 G. soja Sieb. & Zucc. 147 5 G. soja Sieb. & Zucc. 145 3 G. soja Sieb. & Zucc. G. soja Sieb. & Zucc. 40003 G. soja Sieb. & Zucc. 50005 G. soja Sieb. & Zucc. 146 4 G. soja Sieb. & Zucc. 80002 G. soja Sieb. & Zucc. 90019 G. soja Sieb. & Zucc. 110001 G. soja Sieb. & Zucc. 110003 G. soja Sieb. & Zucc. 110009 G. clandestina Wendl. G. falcata Benth. G. tomentella Hayata G. tabacina (LaGill.) Benth.
unknown Lindeman Miyakojimatsurumame
Akita Tokyo Saitama Tochigi Ibaraki Aichi Shimane Okayama Kagoshima China China China Australia " Okinawa
Fig. 1. Cultivar and accession-specificDNA fingerprints of 14 G. max (lanes a-n) and six G. soja (lanes o-t) using (AAT)6 as a probe. DNA was digested with Rsal. (a) Shirotsurunoko, (b) Manseiouhakusyu, (c) Miyagishirome, (d) Bonminori, (e) Shuzenjizairai, (f) Norin 2, (g) Sakagami 2, (h) Yukikorogashi, (i) Oguradaizu, (j) Shirodaizu, (k) Kouandaa, (1) Chugokunanbu H4, (m) Aochi, (n) Matsuura, (o) 147, (p) 110009, (q) 145, (r) 146, (s) 148, (t) 50005. Molecular sizes are indicated (kb).
132
Fig. 2. DNA fingerprints of five individuals of 'Bonminori' with (AAT)6 as a probe. DNA was digested with Hinfl (lanes a-e) or Sau3AI (lanes f-j). Molecular sizes are indicated (kb).
Results
Fig. 3. DNAfingerprintsofsevencultivarsofG. maxwith(CT)8 asa probe. DNA was digested with RsaI, lanes a-g. (a) Miyagishirome,
DNA fingerprints for cultivar identification
(b) Sakagami 2, (c) Shuzenjizairai, (d) Aochi, (e) Matsuura, (f) Kouandaa, (g) Chugokunanbu H4. Molecular sizes are indicated (kb).
The clearest and most polymorphic DNA fingerprints were obtained with the sequence (AAT)6. DNA fingerprints of 47 cultivars of G. max and 14 accessions of G. soja (listed in Table 1) showed different and highly reproducible patterns. Representative results from RsaI-digested DNA fingerprints of 14 cultivars of G. max and 6 accessions of G. soja are shown in Fig. 1. Similar clear bands were also obtained from Hinfl, AluI and Sau3AIdigested DNA (data not shown), by which all the cultivars could also be distinguished from each other. Therefore, clear polymorphism could be detected irrespective of the restriction enzyme used. DNA fingerprints of related cultivars, such as 'Bonminori' (Fig. 1; lane d) and 'Norin 2' (Fig. 1; lane f) showed several common bands, indicating that the DNA fingerprints may reflect the extent of the genetic relationship. DNA fingerprints of five individuals of 'Bonminori' were
almost identical (Fig. 2; Hinfl- and Sau3AI-digested, lanes a-e), suggesting that these DNA fingerprints may be cultivar-specific. Using (CT)8 (Fig. 3), (GATA)4, (GGAT)4 and (GACA)4 (data not shown) as probes, the polymorphism among cultivars was not so clear as that using (AAT)6 (Fig. 1). With (GAA)5 as a probe, the patterns were relatively homogeneous in the subgenus Soja (G. max (lanes a-c) and G. soja (lanes d, e)), but quite different in the subgenus Glycine (G. clandestina (lane f), G. falcata (lane g), G. tomentella (lane h) and G. tabacina (lane i); listed in Table 1) (Fig. 4). Using (AAGG)4 as a probe, relatively faint patterns were found in the subgenus Soja, whereas those in the subgenus Glycine had distinct bands (Fig. 4). The use of (TG)10, (TCC)5 or (GTG)5 as probes produced strong, smeared signals without any distinct patterns (data not
133
Fig. 4. DNA fingerprintsof six speciesusing(GAA)5or (AAGG)4as probes.DNA was digestedwithRsaI. (lanes a-c; G. max)(a) Bonminori, (b) Norin 2, (c) Matsuura, (lanes d, e; G. soja) (d) 145, (e) 148, (lane f) G. clandestina, (lane g) G.falcata, (lane h) G. tomentella, (lane i) G. tabacina.
shown), suggesting that these motifs may be very abundant and highly dispersed in the genome. Different patterns were observed with each probe, therefore the patterns were considered to be probe-specific. Inheritance
of (AAT)6D N A
fingerprints
DNA fingerprints of F1 plants of reciprocal parental crosses were very similar and contained the bands of both parents (Parental accessions; Madaraoohathurumame (Fig. 5; lane P1) and 147 (Fig. 5; lane P2); G. soja)). DNA fingerprints of 30 individual plants of an F2 population were analyzed. The DNA fingerprints of 14 plants in the F2 population, in which three bands segregated independently are shown in Fig. 5. The segregation ratios did not deviate significantly from 3:1 at the 5% level, indicating a Mendelian inheritance of the bands.
Discussion
The analysis of DNA fingerprints using radioisotope labeled oligonucleotide probes complementary to simple repetitive sequences have been reported in chickpea (Weising et al., 1992), banana (Kaemmer et al., 1992), sugarbeet (Schmidt et al., 1993), oilseed rape (Poulsen et al., 1993), tomato (Vosman et al., 1993) and grapevine (Thomas et al., 1993). DNA fingerprints with (GATA)4 as a probe produced polymorphic patterns in 13 different accessions of chickpea, and in 15 different cultivars of tomato. In addition, DNA fingerprints with (GATA)4 could distinguish particular individuals within accessions in chickpea, oilseed rape and sugarbeet. The intensity of signals is found to vary when (GTG)5, (GGAT)4, (CT)s, (GATA)4, (GACA)4 or (TCC)5 are used as probes in plants (Weising et al., 1991). These results indicate that the organization and abundance of a specific microsatellite motif vary
134
Fig. 5. DNA fingerprints of parental accessions(Pj ; Madaraoohathurumameand P2; 147 G soja), F1 (1:'2 x Pl), and the 14 F2 populations with (AAT)6as a probe. DNA was digested with RsaI. Molecularsizes are indicated (kb). among plant species, and that the identification of suitable motifs is essential for DNA fingerprinting. We had previously shown that (AAT) motifs were abundant in the soybean genome (Hayashi et al., 1993). In this paper, we demonstrate that DNA fingerprints with (AAT)6 as oligonucleotide probes were the clearest among the various probes used, and that all cultivars tested could be clearly distinguished using (AAT)6.In contrast, the banding patterns of individuals within a cultivar were almost the same, making the (AAT)6 probes ideally suited for cultivar identification in soybean. The DNA fingerprint patterns, using (CT)8, (GAA)5 or (AAGG)4, were relatively homogeneous among accessions of the subgenus Soja, but were polymorphic among species of the subgenus Glycine. These observations suggest that G. max and G. soja are very close in their genome structure, especially since G. soja is generally accepted as the wild ancestor of cultivated soybean. Indeed, these two species can be easily crossed, have similar genomes, and have
the same assigned genome symbol of GG (Hymowitz et al., 1991). Very low levels of diversity has been found between these two species using various molecular markers, such as seed storage proteins (Staswick et al., 1983), isozymes (Fuchsman & Palmer, 1985) and cytoplasmic DNA (Sisson et al., 1978; Shoemaker et al., 1986). We have also observed that with some motifs the DNA fingerprints showed low levels of diversity in the subgenus Soja. Conservative DNA fingerprints in each species will be informative for analyzing the relationship between the Glycine species. We have found that (AAT),~ motifs are hypervariable in the soybean genome, possibly because replication slippage is potentially dependent on the ATcontent of the sequences involved (Schl6tterer & Tautz, 1992), so (AAT) repeats may be apt to make errors in replication and have high mutation rates, compared to other motifs. Distinct bands, in our fingerprints, that were larger than 2 kb suggest that some (AAT),~ motifs are clustered in these DNA fragments.
135 DNA fingerprints of the F1 progeny, using (AAT)6 as a probe, showed bands of both parents, suggesting that this probe allows the identification of natural and artificial hybrids between cultivars or species. The (AT),~ and (ATT),~ motifs from published sequence data were previously identified in soybean (Akkaya et al., 1992; Morgante & Olivieri, 1993), and our data show that these repeats are abundant in the whole genome. Morgante & Olivieri (1993) estimated that (TAT) microsatellites occur in the soybean genome at a frequency of approximately once every 150 kb and one microsatellite (di- and trinucleotides) once every 50 kb. The isolation of microsatellite markers from DNA libraries has recently been initiated in plants (Zhao & Kochert, 1992, 1993). Hypervariable DNA markers, such as microsatellites, will contribute to developing the genetic map of a particular species (Beckmann & Soller, 1990). Our result also demonstrates that nonradioactive techniques will be important in the applied fields, and can be routinely used for fingerprinting purposes.
Acknowledgements We thank Professors A. Tatara, N. Tsutsumi, (The Univ. of Tokyo) and T. Akihama (Meiji Univ.), and Dr. K. Kadowaki (Natl. Inst. Agrobiol. Resour.) for their valuable comments and suggestions.
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