Euphytica 88 : 47-53, 1996.

47© 1996 Kluwer Academic Publishers. Printed in the Netherlands.

Genetic differentiation between summer and autumn maturing cuitivars ofsoybean (Glycine mar (L.) Merrill) in Kyushu district of Japan

Toshiyuki Hirata, Megumi Kaneko, Jun Abe & Yoshiya ShimamotoLaboratory of Plant Genetics and Evolution, Faculty of Agriculture, Hokkaido University, Sapporo 060, Japan

Received 12 May 1995 ; accepted 7 August 1995

Key words : soybean, Glycine max, landrace, isozyme, differentiation, maturity

Summary

135 soybean landraces and pure line selections from Kyushu district of Japan were assayed for isozyme and seedprotein loci in order to determine the genetic structure of the groups of summer and autumn maturing cultivars .Out of the 16 tested loci, Dial, Enp, Estl, and 771 exhibited a marked difference in allelic frequency between bothgroups. The summer cultivar group had a high frequency for Dial-b, Enp-b, Estl -a and 7I-b, whereas Dial-a,Enp-a, Estl -b and 77-a were predominant in the autumn group . The analysis of multi-locus genotypes revealed thatboth groups mostly consisted of different multi-locus genotypes . The allelic combination of Dial-b Enp-b Estl-aTi-b was most frequently observed in the summer cultivars, whereas four genotypes, Dial -a Enp-a Estl -a Ti-a,Dial-a Enp-a Estl-b 71-a, Dial-a Enp-b Estl-b 7i-a and Dial-a Enp-a Estl-b 7i-b, occupied most of the autumncultivars. These results indicated that both groups were appreciably differentiated from each other . The summercultivar group also included a few accessions having the multi-locus genotypes observed predominantly in theautumn group or Acol-b characteristic of the landraces native to northern Japan . It seems likely that the summercultivar group was not phyletically derived from a single common ancestor, but partly involves the landraces withearly maturity derived from northern Japan . Dial, Enp, Estl and 7i are useful genetic markers to trace the originand dissemination paths of Japanese soybean landraces .

Introduction

Two cultivar groups of soybean (Glycine max (L.)Merrill) with different maturity, summer and autumnmaturing cultivars, are cultivated consecutively inKyushu, a southern district of Japan, despite the longfrost-free season which makes any cropping systempossible. This discontinuous cultivation is mainly dueto an avoidance of pod feeding by insects in early sum-mer (Fukui & Arai, 1951). The summer maturing cul-tivars in Kyushu possess a distinctive maturing habitwhich is easily discriminated from the landraces withearly maturity adapted to northern Japan; the durationof seed-filling is short relative to the duration fromgermination to anthesis, in contrast to the landraces ofnorthern Japan which usually exhibit a relatively longduration of seed-filling (Nagata, 1959, 1960). From ananalysis of geographical variation for maturing habits

and stem termination, Nagata (1959, 1960) suggest-ed that the summer cultivars had become establishedthrough a different phyletic line from the autumn cul-tivars and the landraces of northern Japan . Hymowitz& Kaizuma (1979) also indicated the different originsof both cultivar groups, based on an analysis of Kunitztrypsin inhibitor (Ti) and amylase isozyme (Spl ) . Bothstudies suggest that the Japanese soybean landracesbecame established through the genetic interminglingof two distinct sources of introductions, i .e . a short-season cropping type and a full-season cropping type,each of which brought the summer and autumn matur-ing cultivars in Kyushu, respectively. It is thus a firstpriority to determine the origin of the summer andautumn cultivars in Kyushu in order to understand thedissemination and differentiation of Japanese soybeanlandraces. The purpose of this study is to determine thegenetic structure of the summer and autumn maturing

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Table 1 . Soybean accessions assayed in this study and classification of cultivar groups

Accession

Name Number Name Number

Summer cultivar groupABURA DAIZU 00031625 KIN DAIZU 00033033AKA BAN 00032979 KISAYA 00032994AKA WASE 00031628 KISAYA (1) 00032992AKA WASE 00032980 KISAYA (2) 00032993AKANIDA 00033145 KISAYA (NATSU) 00033012AMAKUSA 00030734 KUROSAYA 00033013AOCHI 00032858 MATSUBA DIAZU 00031627AOCHI (NATSU) 00033014 MATSUURA 00032977AO DAIZU 00032987 MIKURI DAIZU 00032996CHIKUGO DAIZU 00033027 MISAKI DAIZU 00032981DOYOU MAME 00033003 NAGAHAKEI2-1 00033214EKOU DAIZU 00031617 NATSU KURAKAKE 00033005FUUFU DAIZU 00032997 ONI HADAKA (1) 00033198GENNAI 00031433 SANGOU WASE 00033032GOKU DAIZU 00030735 SHIMABARA 00031614HAJINOMI 00033029 SHIMABARA WASE 00030731HAYAKIN 00033026 SHIN HOUNEN 00033019HAZENOMI DAIZU 00032988 SHIN SIMABARA 00033162HIGO DAIZU 00030654 SHIRASAYA 1 00033118ICHIGOU WASE 00033030 SHIRO HADAKA 1 00032986ISHIHARA DAIZU 00032991 SHIROKUCHI 1 00032995JIN NAI 00032978 SHIROKUCHI DAIZU 00031629KAGOSHIMA NATSU DAIZU 00032318 SHIROME 00032974KAIRYOU GIONBOU 00033022 SHIROME 00031626KAIRYOU SHIROME 00033150 TOPPA 00032999KANAGAWA WASE 00033151 WASE KIN 00033000KANEKO 00033025 WASSE KURO DAIZU 00033148KASUGA ZAIRAI 00033021 WASE NATSU 00033020KIN 00033024 YAMAGA ZAIRAI 83 00033226

Autumn cultivar groupAKANITA 1 00033064 ASO ZAIRAI 83 00033262AKI DAIZU 1 00031225 BAN KURO DAIZU 00033049AMAKUSA NOUICHI 00033144 BANSE1 30 00033107ARAO ZAIRAI 83 00033228 BUNSEI 00033058ASHIKITA ZAIRAI 83 00033230 COLNAGASAKI/1983/ 00033215ASO 1 00033094 YOSHINAGA 1FUSANARI DAIZU 00033055 MISAO DAIZU 00033002GIN DAIZU 00032677 NAGANO ZAIRAI 00033108GINSUI ZAIRAI 00033037 (YAMAGUCHI)GOKU DAIZU 1 00033195 NANKAN ZAIRAI 83 00033232

cultivars native to Kyushu district of Japan by esti-mating allelic frequencies at 16 loci coding for nineenzymes and one seed protein, and hence discuss theorigin of both cultivar groups .

Materials and methods

135 landraces and pure line selections establishedin Kyushu district of Japan were analyzed . Thesewere obtained from the plant germplasm collectionsmaintained at the National Institute of Agrobiologi-

49

cal Resources (Tsukuba, Japan) . The maturity type (Ito V) proposed by Fukui & Arai (1951) was deter-mined for each accession on the basis of observation atan experimental field of Hokkaido University and thecultivar description data issued by Kyushu NationalAgricultural Experiment Station and Tohoku NationalAgricultural Experiment Station . The 58 accessions oftypes I and II were classified into the summer cultivargroup, the 70 accessions of types IV and V into theautumn cultivar group, and the remaining 7 accessionsof type III into the intermediate cultivar group (Table1) .

Table] . Continued

Accession

Name Number Name Number

HACHIRA DAIZU 00033074 OKA DAIZU 00033041HAKUSUI ZAIRAI 83A 00033218 OKAHARA ZAIRAI 83 00033231HAKUSUI ZAIRAI 83B 00033219 OOITA AKI DAIZU 1 00033124HIKAGE DAIZU 00033054 OOITA AKI DAIZU 2 00033044HITOYOSHI 00033142 OOTSUURA 00033193HONDO 00033196 OOURA 00033053HOSHINO ZAIRAI 00033038 OUKUCHI ZAIRAI 83 00033261ICHINOMIYA ZAIRAI 83A 00033220 OUSHOKU AKI DAIZU 00031434ICHINOMIYA ZAIRAI 83B 00033221 RINOU 00033068ITSUKI ZAIRAI 83A 00033247 RYUUSUI 00033143ITSUKI ZAIRAI 83B 00033248 SAGA ZAIRAI 00032768ITSUKI ZAIRAI 83D 00033249 SHICHIJOU ZAIRAI 83A 00033256IZUMI 00033187 SHIMOBABA 00033186IZUMI ZAIRAI 83A 00033237 SHIRO DAIZU 00033039IZUMI ZAIRAI 83B 00033238 SOUTA DAIZU 00033206IZUMI ZAIRAI 83C 00033239 TAKIMIZU 00033048KAOU ZAIRAI 83A 00033234 TAMANA 1 00033065KAOU ZAIRAI 83B 00033235 TOMOCHI ZAIRAI 83A 00033242KAWAHARA 00033052 TOMOCHI ZAIRAI 83B 00033243KIIRO 00033202 TORA MAME 00033006KUMAJI 1 00033061 TOUYOU ZAIRAI 83A 00033244KURO DAIZU 00033051 TOUYOU ZAIRAI 83B 00033245MAGARIKAWA ZAIRAI 00033045 UCHIDA 00033057MENOTAKE 00033059 YABE ZAIRAI 83 00033229MIKAWA ZAIRAI 83 00033233 YONEDAKE 00033146MITAMA 00033191 ZAIRAI KURO DAIZU 00033149

Intermediate cultivar groupAZE DAIZU 00032975 NIBAN DAIZU 00032971IZARI 34 00033115 SHIRO NEKOASHI 00031621KOMUTA 00033004 SHIRONEKO SEN 00033001KUDAO ZAIRAI 00033197

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Table 2 . Allelic frequencies and genetic diversity statistics for 11 polymorphic loci in three cultivargroups

Locus Allele Cultivar groupSummer Autumn Intermediaten=58

n=70

n=7

Acol

a

0.966

1 .000

1 .000b

0.034

0.000

0.000Aco4

a

0.121

0.226

0.190b

0.879

0.774

0.714c

0.000

0.000

0.096Ap

a

0.172

0.043

0.000b

0.828

0.957

1 .000Dial

a

0.224

0.949

0.714b

0.776

0.051

0.286Enp

a

0.183

0.760

0.571b

0.817

0.240

0.429Estl

a

0.661

0.247

0.000b

0.339

0.753

1 .000Idhl

a

0.566

0.655

0.571b

0.434

0.345

0.429ldh2

a

0.856

0.941

0.857b

0.144

0.059

0.143Mpi

a

0.000

0.000

0.143b

0.810

0.590

0.286c

0.190

0.410

0.571Pgml

a

0.052

0.314

0.214b

0.948

0.686

0.786Ti

a

0.241

0.782

0.643b

0.759

0.218

0.357

n: the number of accessions tested .1 calculated between summer and autumn cultivar groups .2 calculated among three cultivar groups .3 x2 value for homogeneity between summer and autumn cultivar groups .* * : significant at 5% and 1% levels, respectively .

16 loci were assayed coding for nine enzymes andone seed protein, aconitase (Acol, Aco2, Aco3, Aco4and Aco5), acid phosphatase (Ap), diaphorase (Dial),endopeptidase (Enp), esterase (Est]), isocitrate dehy-drogenase (Idhl and Idh2), leucine aminopeptidase(Lap]), mannose-6-phosphate isomerase (Mpi), phos-phoglucomutase (Pgml and Pgm2), and Kunitz trypsininhibitor (111) . The analysis of isozyme and Kunitztrypsin inhibitor followed the methods described byAbe et al. (1992) and Hymowitz & Hadley (1972),respectively . At least three seeds were examined indi-vidually for each accession . When heterogeneity wasdetected within an accession, three more seeds wereexamined .

The genetic diversity of each cultivar group and thedegree of genetic differentiation between groups wereevaluated by Nei's (1973) genetic diversity and coeffi-cient of genetic differentiation (Gst) . Gst was calculat-ed as (Ht-Hs)/Ht where Hs and Ht represent the meangenetic diversity within cultivar groups and the geneticdiversity in the total population, respectively .

Results

A total of 29 alleles were observed for the 16 loci exam-ined. All the alleles have been genetically determined(Hymowitz & Hadley, 1972 ; Hildebrand et al ., 1980 ;Gorman et al ., 1983 ; Griffin & Palmer, 1987 ; Bult &

Gstl Gst2 x23 Hr

0.018 0.023 2.49 0.029

0.019 0.025 2.41 0.302

0.044 0.081 5 .88* 0.173

0.542 0.391 71 .5** 0.467

0.334 0.231 42.5** 0.500

0.173 0.352 22.3** 0.484

0.008 0.007 1 .07 0 .475

0.020 0.016 2.63 0 .179

0.058 0 .142 7.24** 0 .448

0.115 0 .075 14.0** 0 .317

0.292 0 .213 37 .5** 0 .496

Table 3. Observed and expected (parenthesis) numbers of accessions which belong toeach of multi-locus genotypes for Dial, Enp, Estl and T

n: the number of accessions tested .

Kiang, 1989; Kiang & Bult, 1991) . Allelic designa-tion followed Griffin & Palmer (1987) for Acol, Aco2,Aco3, Aco4 and Enp, Kiang & Bult (1991) for AcoS,Bult & Kiang (1989) for Est], and Palmer & Kilen(1987) for the other loci .

Allelic frequencies and genetic diversity statisticsfor 11 polymorphic loci are presented in Table 2 . Mostof the accessions tested consisted of a single genotype .No heterozygous seed was detected, as expected sincesoybean is a highly self-fertile species with natural out-crossing rates of less than 0 .5% to about 1% (Carlson& Lersten, 1987) . Of the 16 loci scored, Aco2, Aco3,Aco5, Lap] and Pgm2 were monomorphic in this study,all accessions having Aco2-b, Aco3-a, Aco5-a, Lapl-band Pgm2-a. Aco4 and Mpi had three alleles and theremaining nine loci had two alleles . Genetic diversity inthe total population (Ht) was high for Dial, Enp, Estl,Idhl, Mpi and Ti . Including the five monomorphic loci,the mean genetic diversity in the total population was0.242 with an average of 1 .82 alleles per locus .

A marked difference of allelic frequency betweenthe summer and autumn cultivar groups was recog-nized for Dial, Enp, Est] and Ti, as indicated by highGst values (Table 2). The chi-square test revealed thatthe difference of allelic frequencies was highly signifi-cant for these four loci. The summer cultivar group had

51

a high frequency for Dial-b, Enp-b, Estl-a and Ti-b,whereas Dial-a, Enp-a, Estl-b and Ti-a were predom-inant in the autumn group. The chi-square test alsoindicated that the allelic frequencies differed signifi-cantly between the two groups for Ap, Mpi and Pgml .Ap-a was observed in a low frequency in the summercultivar group but was rare in the autumn group, where-as Pgml-a was rare in the summer group but was foundin a moderate frequency in the autumn group . The fre-quency of Mpi-c was high in the autumn cultivar groupcompared with the summer group . In addition, Acol-b, a rare allele characteristic of the landraces native tonorthern Japan (Hirata et al ., 1993), was observed intwo accessions of the summer cultivar group, 'ABURADAIZU' and `SHIMABARA, but was not observed inthe autumn group . The mean genetic diversity for the16 loci was almost the same in both summer (0 .198)and autumn (0.193) cultivar groups, irrespective oftheir different allelic constitutions .

Allelic frequencies in the intermediate cultivargroup were similar to those in the autumn groupalthough a slight difference was observed for Est] andMpi. The intermediate cultivar group also had Aco4-c('IZARI 34') and Mpi-a (`KOMITFA' ), both of whichwere rarely observed in Japanese soybean landraces(Hirata et al., 1993) .

Allelic combination Cultivar groupDial Enp Estl Ti Summer

n=58Autumnn=67

Intermediaten=6

a a a a I ( 0.4) 13( 9 .4) 0(0.0)a a a b 0( 1 .2) 0 ( 2 .6) 0(0.0)a a b a 1 ( 0.2) 26 (28.5) 2(l .6)a a b b 1 (0.6) 8 ( 8.0) 0(0.9)a b a a 0( 1 .7) 2( 2 .9) 0(0.0)a b a b 3( 5 .3) 1 (0 .8) 0(0.0)a b b a 0( 0 .9) 9( 9 .0) 0(l .2)a b b b 7 ( 2.7) 4( 2 .5) 2(0.7)b a a a 1 (1 .3) 0( 0.5) 0(0.0)b a a b 3(4.1) 0(0.1) 0(0.0)b a b a I ( 0.7) 3( 1 .5) l(0.6)b a b b 2( 2 .1) 1 (0 .4) 0(0.3)b b a a 6 ( 5.9) 0 ( 0.2) 0(0.0)b b a b 25 (18 .4) 0 ( 0 .0) 0(0.0)b b b a 4 ( 3 .0) 0 ( 0 .5) l(0 .5)

b b b b 3(9.5) 0( 0 .1) 0(0 .3)

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Table 3 shows the observed and expected num-bers of accessions which belong to each of multi-locusgenotypes for the four diagnostic loci (Dial, Enp, Estland Ti) . For the polymorphic accessions, the multi-locus genotype was constructed with the most frequent-ly observed genotype at each locus . Four accessions inwhich two homozygous genotypes at any locus wereobserved in an equal frequency were thus excludedfrom the analysis. Of the 16 possible multi-locus geno-types, fifteen were observed in the total population, ofwhich thirteen were observed in the summer cultivargroup, nine in the autumn group and four in the inter-mediate group . The observed numbers of accessionsfor each of multi-locus genotypes fitted the expectedones from random association of allelic frequencies forthe four loci in both summer (X 2 = 7.99, df = 4, 0 .05 <p < 0.10) and autumn (X2 = 1 .80, df = 4, 0 .75 < p <0.90) groups.

The summer and autumn cultivar groups each con-sisted of different allelic combinations (Table 3) . Sev-en allelic combinations were common to both groups,and the six were unique to the summer group, the twoto the autumn group . The accumulative frequency ofthe six genotypes unique to the summer cultivar groupreached 72.4%. In particular, the allelic combinationof Dial-b Enp-b Estl-a Ti-b was observed in 25 ofthe 58 accessions tested. In the autumn cultivar group,on the other hand, four allelic combinations, Dial-aEnp-a Estl-a Ti-a, Dial-a Enp-a Estl-b Ti-a, Dial-aEnp-b Estl -b 7i-a and Dial -a Enp-a Estl -b Ti-b, occu-pied 83.6% of the 67 accessions tested . The summercultivar group also involved three accessions whichpossessed the multi-locus genotypes characteristic ofthe autumn group ; 'DOYOU MAME' (Dial-a Enp-aEstl-a Ti-a), `AKANIDA' (Dial-a Enp-a Estl-b Ti-a)and 'MATSUURA' (Dial-a Enp-a Estl-b T -b) .

Discussion

Hymowitz & Kaizuma (1979) and Kaizuma et al .(1980) reported that the summer maturing cultivarsnative to Kyushu district of Japan mostly had 7i-bwhich was not frequently observed in the autumn cul-tivars. Our result for Ti also confirmed the resultsobtained in the previous investigations . In addition,a marked difference in allelic frequency between thetwo cultivar groups was recognized for Dial, Enp andEstl of the 15 isozyme loci examined . The resultsfor isozyme and Ti loci thus demonstrated the geneticdifferentiation of landraces associated with maturing

habits in Kyushu . Dial, Enp and Estl, together withTi, are useful genetic markers to trace the origin anddissemination paths of Japanese soybean landraces .

Nagata (1959, 1960) pointed out that the sum-mer maturing cultivars in Kyushu had been estab-lished from a short-season cropping type introducedfrom central-south China via Taiwan and Okinawa,and the autumn maturing cultivars from a full-seasoncropping type introduced from northcentral China viaKorea. However, from the finding that Ti-b, charac-teristic of the summer cultivars, was rarely observedin the germplasm from China and Taiwan, Hymowitz& Kaizuma (1979, 1981) indicated that the summermaturing cultivars had evolved not in China but ineither Kyushu or Korea . The predominant occurrenceof T-a in Chinese soybean cultivars was confirmed byWang et al . (1986) who found that of the 1858 acces-sions tested only ten introduced from northern andsoutheastern China and southeastern Tibet had TI-b .In addition, of the three isozyme alleles characteristicof the summer cultivar group, Dial-b and Enp-b wereobserved in common in the germplasm from Japan,Korea, Taiwan and China, whereas Estl-a was rarelyobserved in the latter three (Hirata et al ., 1993) . Thusthe isozyme data suggest that the summer maturingcultivars of Kyushu became established after cultivat-ed soybeans had been disseminated into Japan.

Of the 15 multi-locus genotypes constructed withthe alleles at the four diagnostic loci, six were uniqueto the summer cultivar group, of which Dial-b Enp-bEstl-a Ti-b was most frequently observed . This multi-locus genotype has not been detected in the germplasmfrom China, Taiwan and Korea we have surveyed so far,asexpected from the finding that either of Estl -aand Ti-b or both were rarely observed in these regions (Hirataet al ., 1993). All of the multi-locus genotypes con-structed here, on the other hand, have been observedin the landraces adapted to northern Japan where theseloci were polymorphic as well (Hirata et al ., 1993) . Themulti-locus genotypes frequently observed in northernJapan were those predominant to the autumn cultivargroup in Kyushu (Hirata et al ., in preparation) . Thisis consistent with the hypothesis proposed by Nagata(1959, 1960) and Hymowitz & Kaizuma (1979) thatthe landraces with early maturity adapted to northernJapan became established from a full-season croppingtype which brought the autumn maturing cultivars inKyushu. Furthermore, our results demonstrated thatthe summer cultivar group also involved a few acces-sions which possessed the multi-locus genotypes pre-dominant in the autumn cultivar group or Acol-b char-

acteristic of the landraces of northern Japan . It seemslikely that the summer cultivar group in Kyushu was notphyletically derived from a single common ancestor,and partly involves the derivatives from the landraceswith early maturity adapted to northern Japan .

The hypotheses on the dissemination paths of soy-beans into Japan which have been proposed so far(Hymowitz & Kaizuma, 1979 ; Sugiyama, 1992) most-ly stem from the basic studies of Nagata (1959, 1960)who first suggested a significant role of the summermaturing cultivars native to Kyushu on the phylogenyof Japanese soybean landraces . The present study forisozyme and Ti loci revealed that most of the summercultivars possessed their own multi-locus genotypes forDial, Enp, Estl and Ti. It is thus necessary to deter-mine the origin of the landraces of these multi-locusgenotypes, especially those of Dial-b Enp-b Estl -a Ti-b representative of the summer cultivar group . For thatreason, a comparative study of morphology and oth-er biochemical and molecular markers is to be madebetween the summer cultivar group in Kyushu and thelandraces with early maturity of northern Japan whichshare this distinctive allelic combination . Geograph-ical distributions of the alleles at the four diagnosticloci should also be evaluated in detail not only for cul-tivated soybeans but also for wild soybeans, Glycinesoja Sieb. et Zucc., which is considered as a possiblesource of the alleles .

Acknowledgements

This study was partly supported by a fellowshipof the Japan Society for the Promotion of Sciencefor Japanese Junior Scientists, and by grant-in-aid03454033 from the Ministry of Education, Scienceand Culture, Japan .

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