We have started a breeding program to genetically eliminate. the lipoxygenase isozymes (LOX) from soybean seeds. These enzymes are believed to be lhe main cause of off-flavors in soybean products. LOX are present in the seed as three isozymes encoded by Ihree different genes, which are inherited in a simple mendelian fashion. Mutants lacking each one of these isozymes have been identified in the world gennplasm. To introduce Ihese null ali eles into lhe Brazilian variety Cristalina, three mutant progenitors were chosen PI 408.251 (LOX 1 minus), PI 86.023 (LOX2 minus), and Ichigowase (LOX3 minus). The random amplified polymorphic DNA (RAPD) technique was used to characterize these progenitors, as well as lines lacking LOXI (CRI), LOX3 (CR3), LOXI and 3 (CRI,3), and LOX2 and 3 (UFV 91-263, UFV91-401 and UFV 91-717). The results enabled us to establish lhe fmgerprint of each genotype and the genetic distances among them

BARROS, E. G. de

MOREIRA, M. A.

PAIVA, E.

SEDIYAMA, C. S.

VILARINHOS, A. D.

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Rev. Brasil. Genet. 17,3,287-290 (1994)(Brazil. J. Genetlcs)USE OF THE RANDOM AMPLIFIED POLYMORPHIC DNA TECHNIQUE TOCHARACTERIZE SOYBEAN (Glycine max (L.) Merrill) GENOTYPESAlberto Duarte Vilarinhos I, Everaldo Gonçalves de Barros 1, Edilson Paiva 2,Carlos Sígueyuke Sediyama 1 and Maurílio Alves Moreira 1ABSTRACTWe have started a breeding program to genetically eliminate the lipoxygenase isozymes (LOX) from soybean seeds. These enzymes arebelieved to be lhe main cause of off-flavors in soybean products. LOX are present in the seed as three isozymes encoded by Ihree different genes,which are inherited in a simple mendelian fashion. Mutants lacking each one of these isozymes have been identified in the world gennplasm. Tointroduce Ihese null ali eles into lhe Brazilian variety Cristalina, three mutant progenitors were chosen: PI 408.251 (LOX 1minus), PI 86.023 (LOX2minus), and Ichigowase (LOX3 minus). The random amplified polymorphic DNA (RAPD) technique was used to characterize these progenitors,as well as lines lacking LOXI (CRI), LOX3 (CR3), LOXI and 3 (CRI,3), and LOX2 and 3 (UFV 91-263, UFV 91-401 and UFV 91-717). Theresults enabled us to establish lhe fmgerprint of each genotype and the genetic distances among them,INTRODUCTIONIn spite of its high protein content, soybean ismainly used as an oil source in Brazil (Panizzi, 1987). Themain obstacle for soybean protein consumption in Westemcountries is the beany-flavor present in soybean products(Rackis, 1979). One of the major causes of theseoff-flavors is the association of short-chain carboniliccompounds with the protein fraction. These organiccompounds are end products of a series of reactions whichstart with the hydroperoxidation of polyunsaturatedfatty-acids catalyzed by lipoxygenases (Axelrod, 1974).These enzymes are dioxygenases which occur as threeisozymes (LOX 1 to 3) in dry soybean seed. Each isozymeis encoded by a single dominant gene, inherited in amendelian fashion (reviewed in Mack et aI., 1987). It hasbeen suggested that the best way to improve flavor insoybean products is to genetically eliminate these enzymesfrom the seed (Kitamura et aI., 1983). Mutants lacking eachof the isozymes have been identified (Hildebrand andHymowitz, 1982; Kitamura et aI., 1983; Kitamura, 1984);1 Núcleo de Biotecnologia Aplicada à Agropecuária (13I0AORO)/Universidade Federal de Viçosa (UFV), 36570·000 Viçosa, MO, Brasil. Sendcorrespondence to M.A.M.2 Centro Nacional de Pesquisa de Milho e Sorgo (CNPMS)/Empresa Brasileirade Pesquisa Agropecuária (EMBRAPA), 35701-970 Sele Lagoas, MO, Brasil.however, no double or triple nulls have been found innature (Hajika et aI., 1991). One of the reasons for this factis the tight linkage between the genes encoding for LOX 1and LOX2 (Hajika et aI., 1991).We have started at the Federal University of Viço-sa (UFV), MG, Brazil, a breeding program to introduceLOX deficiency into Brazilian adapted soybean cultivars.So far, severallines lacking LOX1, LOX2, LOX3, LOXland LOX3, LOX2 and LOX3 have been obtained (Moreiraet aI., 1993). Initially, only the variety Cristalina was usedas recurrent progenitor, however, several other adaptedcultivars have been included in this programoWe have used the RAPD (Random AmplifiedPolymorphic DNA) technique to characterize theptogenitors used in our breeding programo Thischaracterization should allow us to fingerprint eachgenotype and facilitate the recovering of the characteristicsof the recurrent parent in a breeding program to introgressnull LOX genes into Brazilian soybean varieties.MATERIAL AND METHODSThe genotypes used were as follows: Cristalina(commercial variety, normal for LOX1, LOX2, andLOX3); PI 408.251 (LOX 1 minus); PI 86.023 (LOX2minus); Ichigowase (LOX3 minus), CRI (Cristalina-derived line lacking LOX1); CR3 (Cristalina-derived linelacking LOX3); CRI,3 (Cristalina-derived line lacking288 Vilarinhos et al:LOXl and 3); and tines UFV 91-263, UFV 91-401, andUFV 91-717 (Cristatina-derived tines lacking LOX2 and3). Lines CRI and CR3 originated from crosses betweenCristalina and PI 408.251 and Ichigowase, respectively,followed by three generations of backcrosses. CRI,3originated from a cross between CRI and CR3 at BCIF2folIowed by severa Icycles of selfing. Lines UFV 91-263,UFV 91-401, and UFV 91-717 were derived from crossesbetween CR3 at BC3F2 with PI 86.023, F2 seed selectionfor absence ofboth LOX2 and 3, folIowed by six cycles ofoutcross and selection for pure lines.To confirm the presence or absence of individualLOX isozymes in the genotypes, seeds were analyzed bysodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE) (Kitamura, 1984). Once their phenotypeswere confirmed, seeds were germinated and planted in thegreen house. Young leaves were harvested and kept at-80°C for DNA extraction (Saghai-Maroof et aI., 1984).DNA extracted from leaves of the differentgenotypes was used as a template for amplificationreactions. Each reaction (25 ul) contained: 50 ng of DNA;100 /lM of each deoxyribonucleoside triphosphate; 1.7mM MgCh; 10 mM Tris-HCl, pH 8.3; 50 mM KCl; 0.4/lM of a single decamer primer (Operon Technologies),and one unit ofTaq DNA polymerase (Perkin Elmer-CetusCorp.). DNA amplification was performed in athermocycler model 9600 (Perkin Elmer-Cetus Corp.) setfor 40 cycles, each consisting of a denaturation step at 94°Cfor 15 seconds, an annealing step at 35°C for 30 seconds,and an elongation step at nOc for one minute. After the40th cycle a final elongation step at nOc was performedfor seven minutes. Amplification products were separatedon 1.2% agarose gels containing 10 /lgfml ethidiumbromide in TBE buffer (90 mM Tris-borate, 1mM EDTA,pH 8.0). DNA bands were visualized under UV light andphotographed with Polaroid film type 667.The twenty-six random primer decamers usedwere as follows: OPA04, OPA07, OPA08, OPB08,OPC03, OPC04, OPC07, OPDO1,OPEO1,OPFO1,OPF02,OPF04, OPF07, OPH02, OPH04, OPI01, OPI03, OPI04,OPI01, OPI03, OPV02, OPV03, OPX01, OPY03,OPV102, and OPW03. Only the most prominent DNAbands were scored. Genetic distances were determined bydividing the number of distinct DNA bands between twogiven genotypes and the total number of amplificationproducts. Data were analyzed through a statistical software(SAEG) developed at the Federal University of Viçosa.RESULTS AND DISCUSSIONThe use of DNA-based markers for plantcharacterization is becoming very common in breedingprograms for severa I crops (Hu and Quiros, 1991; Demekeetal., 1992; Thormannand Osbom, 1992). WeusedRAPDmarkers to study the genetic diversity of progenitors andlines involved in a breeding program to geneticallyeliminate the lipoxygenase enzymes from soybean seeds.Twenty-one out of the twenty-three primers testedgenerated amplification products (Figure 1); however,only 13 of them were informa tive, showing polymorphicbands between at least two of the initial progenitors usedin the breeding program (Table I). Two primers generatedno amplification products. Considering only theinformative primers, one to five DNA bands (440 to 1,800bp) were amplified per primer (Table I). These numbersare in the range obtained for soybean DNA by other authors(Tingey et ai., 1992). A total of 61 amplification productswere generated, 30 of them being polymorphic (Table I).Pairwise genetic distances were between six and 36%(Table lI). As expected, the largest distances were betweenthe progenitor Cristalina, a Brazilian adapted cultivar, andthe other three original progenitors involved in theprogram, which originated in Asia (Table lI). The shortestgenetic distances were among lines UFV 91-263, UFV91-401, and UFV 91-717 (Cristalina-derived lines lackingLOX2 and 3) (Table lI). This may be due to the type ofselection made during the cycles of self-pollination whichoriginated these lines. The genetic distances betweencultivar Cristalina and lines CRI and CR3 were well withinthe range expected for lines derived by three backcrosses,having Cristalina as a recurrent progenitor (Table 11). The1,108 bp ==>Genotypes 1 2 3 4 5 6 7 8 9 10 À.Figure 1 - Typical agarose gel showing RAPD amplified soybean DNAbands produced by primer OPW02. Lanes are as follows: I, Cristalina;2, PI 408.251; 3, PI 86.023; 4, Ichigowase; 5, CRI; 6, CR3; 7, CRI,3; 8,UFV 91-263; 9, UFV 91-401; and 10, UFV 91-717. Arrow points to apolymorphic band obtained with primer OPW02. Lane lambdacorresponds to lambda-phage DNA digested with endonucleases EcoRI, Bam HI, and Hind m.RAPOTechnique to Characterize Soybean Genotypes 289Table I - Distribution of polymorphic DNA markers among soybean genotypes. After DNA amplification with different primers, each genotypewas analyzed for the presence (I) or absence (O) of specific bands. Size range of the bands are indicated on the last column of the table.GenotypesPrimers No.of base2 3 4 5 6 7 8 9 10 pairs (bp)OPA04 (I) I O 1 O O 1 O 1 1 1 1,086OPA04 (11) O 1 1 O 1 O O O O O 960OPA07 (I) O O 1 1 O O O 1 1 1 1,338OPA07 (11) 1 O O O 1 1 1 O O O 771OPA07(1m O O 1 O O O O 1 1 1 682OPA07 (IV) 1 1 1 O 1 1 1 1 1 1 446OPC03 (I) 1 O O O 1 1 1 O O O 1,798OPC03 rm 1 1 1 O 1 O 1 1 1 1 1,201OPC03 (111) 1 O 1 1 1 1 1 1 1 1 834OPC03 (IV) 1 1 1 O O O 1 1 O 1 698OPC03 (V) 1 O O 1 1 1 O I O 1 650OPC04 (I) O 1 O O O I I I O 1 857OPC07 (I) 1 1 I O I I 1 O I 1 906OPC07 (m 1 O O O 1 1 O O I 1 594OPEOl (I) 1 O O O O O O O O O 771OPF04 (I) 1 1 O 1 I 1 O O O O 650OPI03 (I) O 1 1 O O O O O O O 1,161OPI03 (11) O 1 1 1 O O O O O O 682OPI03 (111) 1 O O O I I 1 I I I 492OPJOl (I) 1 O O O O I O O 1 I 960OPJOl (11) 1 1 O O I O O I O O 834OPJOl (111) O O I 1 1 O 1 1 O 1 581OPJOl (IV) O O I I O O I 1 O O 446OPH04 (I) O O O I O O O O O O 1,023OPV03 (I) O 1 I O O O O O O O 1,273OPV03 (11) O 1 1 1 O I O O O O 924OPY03 (I) 1 O O O O O O O O O 1,517OPY03 (11) O O O I O O O O O O 1,328OPY03 (11I) 1 1 I O I I I I I I 961OPW02 (I) 1 O O O 1 O O O O O 1,108Table 11- Pairwise genetic 9istances between soybean genotypes. Distances werecalculated by dividing the number of polyrnorphic bands by the total number ofamplification products and expressed as a percentage.Genotypes 2 3 4 5 6 7 8 9 1012 283 33 154 36 27 235 11 23 28 286 13 24 29 26 157 20 21 20 26 15 168 23 24 16 23 21 23 139 18 26 18 27 20 15 18 1510 18 26 18 27 20 15 15 08 06290 Vilarinhos et aLrelatively large genetic distances between cultivarCristalina and lines CRl,3 and UFV 91-263, UFV 91-401,and UFV 91-717, are also understandable because we hadno intention to develop near-isogenic lines to theprogenitor Cristalina by the end of the breeding processoThese genetic distances were used for cluster analyses andallowed the separation of the genotypes into four distinctgroups within 76% of relative genetic distance (Figure 2).GenotypesRelative 1 S 6 7 8 9 10 2 3 4distance%o248- 16- 20- 24- 28- 32- 36- 40- 44 T- 48- S2- S6- 60I- 64 T- 68- 72 ~-- 76 ---> I I- 80- 84I- 88- 92I- 96-100Figure 2 - Dendogram showing relative genetic distances amongsoybean genotypes. Pairwise genetic distances were caJculated bydividing lhe number of polymorphic DNA bands by the total number ofamplification products, and used for cJuster analyses. Arrow indicatesrelative genetic distance (76%) used to define four distinct groups.Numbers corresponding to each genotype are as for Figure J.ACKNOWLEDGMENTSIIIThis work was supported by PADCT/FINEP and NESTLÉgrant No. 64.92.0005.00. Alberto D. Vilarinhos was supported by afellowship from CNPq.RESUMOFoi iniciado um programa de melhoramento para eliminargeneticamente as isozimas lipoxigenases (LOX) das sementes de soja.Acredita-se que essas enzimas constituam a principal causa de saboresindesejáveis nos produtos derivados da soja. LOX estão presentes nassementes como três isozimas codificadas por três diferentes genes quesão herdados através de herança mendeliana simples. Mutantesdeficientes em cada uma dessas isozimas foram identificados nogermoplasma mundial. Para introduzir esses alelos recessivos navariedade brasileira Cristalina, foram escolhidos três progenitoresmutantes: PI 408.251 (sem LOX 1), PI 86.023 (sem LOX2) e Ichigowase(sem LOX3). A técnica de DNA polimórfico amplificado ao acaso(RAPD) foi utilizada para caracterizar esses progenitores, bem comolinhagens sem LOXl (CRI), sem LOX3 (CR3), sem LOX 1 e LOX3(CRI,3) e sem LOX 2 e LOX3 (UFV 91-263, UFV 91-401 e UFV91-717). Esses resultados permitiram estabelecer o "fmgerpring" decada genótipo e determinar a distância genética entre os mesmos.REFERENCESAxelrod, B. (1974). Lipoxygenases. ACS Adv. Chem. Ser. 136:324-348.Demeke, T., Adams, R.P. and Chibbar, R. (1992). Potentialtaxonornic use of random amplified polymorphic DNA (RAPD):a case study in Brassiea. Theor.Appl. Genet. 84: 990-994.Hajika, M., Igita, K. and Kitamura, K. (1991). Aline lacking ali theseed lipoxygenase isozymes in soybean (Glyeine max (L.) Merrill)induced by gamma-ray irradiation. Jap. J. Breed. 41: 507-509.Hildebrand, D.F. and Hymowitz, T. (1982). Inheritance oflipoxygenase-l activity in soybean seeds. Crop. Sei. 22: 851-853.Hu, J. and Quiros, C.F. (1991). Identification of broccoli andcauliflower cultivars with RAPD markers. Plant Cell Rep. 10:505-511.Kitamura, K. (1984). Biochemical characterization of lipoxygenaselacking mutants, L-I less, L-2 less, and L-3 less soybeans. Agrie.Biol. Chem. 48: 2339-2346.Kitamura, K., Davies, C.S., Kaizuma, N. and Nielsen, N.C. (1983).Genetic analysis of nu li-alieI e for lipoxygenase 3 in soybean seeds.Crop. Sei. 23: 924-927.Mack, A.J., Peterman, T.K. and Seidow, J.N. (1987). Lipoxygenaseisozyme in higher plants: Biochemical properties andphysiological role. Isozymes. Curr. Top. Biol. Med. Res. 13:127-154.Moreira, M.A., Rezende, S.T., Ramos, V. and Barros, E.G. (1993).Hexanal production and TBA number are reduced in soybean[Glyeinemax (L.) Merr.] seeds lacking lipoxygenase isozymes 2and 3. J. Agrie. Food Cliem. 41: 103-106.Panizzi, M.C.C. (1987). Proteína para milhões. Cio Hoje 6: 25-31.Rackis, J.J., Sessa, D.J. and Honig, D.H. (1979). Flavor problems ofvegetable food proteins. J. Amer. ou Chem. Soe. 56: 262-271.Sagha'i-Maroof, M.A., Soliman, R.A., Jorgensen, R.A. and Allard,R.W. (1984). Ribossomal DNA spacer-Iength polymorphisms inbarley: Mendelian inheritance, chromosomal location, andpopulation dynarnics. Proe. Natl.Aead. Sei. USA230: 1350-1354.Tingey, S.V., Rafalski, A. and Williams, G.K. (1992). Genetic analysiswith RAPD markers. Proeeedings of the Simposium (on)Applieations of RAPD Teehnology to Plant Breeding.Minneapolis, CSSA, ASHS, AGA, pp. 3-7.Thormann, C.E. and Osborn, T.C. (1992). Use of RAPD and RFLPmarkers for germplasm evaluation. Proeeedings of Simposium (on)Applieations of RAPD Teehnology to Plant Breeding.Minneapolis, CSSA, ASHS, AGA, pp. 9-11.(Received November 10,1993)

Use of the random amplified polymorphic DNA technique to characterize soybean (Glycine max (L.) Merrill) genotypes.

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