J. Japan. Soc. Hort. Sci. 61 (3) : 595 —-601. 1992.

Isozyme Variation in Cucumber (Cucumis sativus L.)

Shiro Isshiki, Hiroshi Okubo and Kunimitsu Fujieda Laboratoryof HorticulturalScience, Faculty of Agriculture,Kyushu University,Fukuoka 812

Summary The Indian wild cucumber (Cucumis sativus L. var. hardwickii Kitamura) and 81 accessions of cucumber (C. sativus) were examined for isozyme variations of six enzymes. Variations were observed in malate dehydrogenase (MDH), 6-phosphogluconic de- hydrogenase (6-PGD), phosphoglucomutase (PGM) and phosphoglucose isomerase (PGI), whereas shikimate dehydrogenase (SKDH) and isocitrate dehydrogenase (IDH) were found to be monomorphic. The variations implied that the Indian wild cucumber is a distant relative of the cultivat- ed cucumber, but the relationships between ecotype differentiations among cucumber culti- vars and isozyme phenotypes were not recognized. The one-seed analysis of MDH isozymes was useful in evaluating the seed purity of F1 cucumber, since the isozyme variations were scattered in commercial cultivars in respective diverse ecotypes.

Staub et al. (1985b) identified variations of seven Introduction isozyme loci in 57 cultivars of cucumber, but they Isozyme variations have been extensively used as did not refer to the phylogenic relationships among genetic markers for characterization of species and cultivars. Knerr et al. (1989) revealed the varia- cultivars (Bringhurst et al., 1981; Dewald et al., tions at 18 allozyme loci in 757 cucumber plant in- 1988; Mundges et al., 1990), establishment of troductions and constructed a cluster analysis phylogenetic relationships among taxa (Crawford, dendrogram by country of origin. Staub and 1983; Hirai et al., 1986; Perl-Treves et al., 1986; Fredrick (1988) described the inheritance of three Staub et al., 1985a) and genetic studies in many enzyme loci in cucumber. plants (Arus and Orton, 1983; Rick, 1983; Staub The purposes of this study were to reveal genet- and Fredrick, 1988; Weeden and Marx, 1984; ic variations of isozymes and to furnish biochemi- Weeden and Lamb, 1987; Wendel and Parks, cal information on the origin and breeding of 1982). The enzyme variations have been also ap- cucumbers. plied in plant breeding successfully as genetic mar- kers for hybrid confirmation and for convenient Materials and Methods screening of seedlings with target characters (Arus Experiment 1 et al., 1985, 1982; Colby and Peirce, 1988; Soost et al., 1980; Tanksley and Jones, 1981; Weeden The Indian wild cucumber, C. sativus L. var. and Provvidenti, 1988; Woods and Thuran, 1976). hardwickii Kitamura, and 81 accessions of C. sati- Compared with other conventional markers, iso- vus collected from Bangladesh, Sri Lanka, zymes have some advantages, such as environmen- Thailand, Indonesia, Philippines, China, Japan and tal stability and codominant expression (Gottlieb, Holland were examined (Table 1). 1981); the latter simplifies genetic analysis. Recog- The plants were grown in plastic pots filled with nizing genetic variations of isozymes is essential vermiculite in a glasshouse in May 1991 and the for advances in systematics, genetics, and breed- young developing leaves were sampled for enzyme ing of crop plants. analysis. Extraction of leaf tissue for electrophoresis and Received for publication 31 January 1992. procedure of the electrophoresis and gel staining

595 596 S. ISSHIKI, H. OKUBO AND K. FUJIEDA

Table 1. Isozyme phenotypes observed for MDH, 6-PGD, PGM and PGI in one Indian wild cucumber (Cucumis sativus L. var. hardwickii) and 81 accessions of C. sativus. J. Japan. Soc. Hort. Sci. 61 (3): 595-601. 1992. 597

Table 1. (Continued). 598 S. ISSHIKI, H. OKUBO AND K. FUJIEDA were done following the protocols of Wendel and isozymes. Cotyledons and true leaves of 'Natsu- Parks (1982) and Wendel (1983). After the elec- fushinari' were also examined for comparison of trophoretic run, gels were sliced horizontally and the enzyme expression. The electrophoretic proce- stained for shikimate dehydrogenase (SKDH; EC dures were the same as those of Experiment 1. 1.1.1.25), isocitrate dehydrogenase (IDH; EC 1.1.1.42), malate dehydrogenase (MDH; EC Results 1.1.1.37), 6-phosphogluconic dehydrogenase Experiment 1 (6-PGD; EC 1.1.1.44), phosphoglucomutase (PGM; EC 2.7.5.1) and phosphoglucose isomerase (PGI; Among six enzymes assayed, two enzyme sys- EC 5.3.1.9). The stained gels were washed with tems, SKDH and IDH, did not yield any detecta- water and dried to preserve them. ble variations. The variations were found in the For the purpose of evaluation, banding patterns other four enzyme systems. of the enzymes were recorded and compared Two regions of activity were observed in MDH among accessions. The inheritance of the isozymes (Fig. 1). Three different electromorphs were used in this study was not investigated. However, present in the most anodal region, MDH-1, based on reports for cucumber (Knerr et al., 1989; whereas the banding patterns at the MDH-2 region Staub and Fredrick, 1988; Staub et al., 1985a, were monomorphic. The best resolution was ob- 1985b), other Cucumis species (Perl-Treves et al., tained in this enzyme. 1986; Staub et al., 1985a) and other organisms The different bands observed in gels stained for (Kephart, 1990; Gottlieb, 1981), it was possible to 6-PGD were grouped into two regions (Fig. 1). assign genetic bases to the observed electro- Both regions, 6-PGD-1 and 6-PGD-2, showed two morphs. The nomenclature in this study is as fol- phenotypes. lows; the isozyme banding regions were numbered Two regions of activity, PGM-1 and PGM-2, and the phenotypes were designated as AA, BB were found in PGM (Fig. 1). Three different band- and CC from most anodal to cathodal. ing patterns appeared at PGM-1, and two at PGM-2. Experiment 2 When gels were stained for PGI isozymes, two Seeds of Japanese cultivars `Natsufushinari', zones of activity were shown (Fig. 1). The resolu- 'Santo' and their hybrid were examined for MDH tion of the PGI-1 region was too poor to identify

Fig. 1. Schematic illustration of isozyme banding patterns (above) and phenotypic designations (below) for MDH, 6-PGD, PGM and PGI in cucumber. J. Japan. Soc. Hort. Sci. 61 (3) : 595-601. 1992. 599

the banding pattern, but three different phenotypes PGI-2 AA; however, CC phenotype was found in '83 were clearly observed at PGI-2. -01' (China) , BB in '90-01', '83-15' (China) and The isozyme phenotypes of the above four en- Knongkai' (Thailand). ' zymes are listed in Table 1, and the phenotype fre- Experiment 2 quencies are shown in Table 2. All the accessions, except the Indian wild one, were divided into nine Hybrid confirmation with MDH isozymes was horticultural groups following ecotypic classification performed since, the enzyme gave the best resolu- by Fujieda (1973). tion in Experiment 1. Most of the 82 accessions showed CC phenotype The seed and cotyledon of 'Natsufushinari' in MDH-1. Phenotype BB was found in six acces- showed the same zymogram as the true leaf ex- sions in the north Chinese type of Chinese culti- cept the bands below 40 mm which were observed vars and south and north Chinese type of Japanese only in the seed and cotyledon (Figs 1 and 2). The cultivars, whereas AA phenotype was observed hybrid seed exhibited both parental bands, distin- only in three accessions in south Chinese (Japan) guishable from those of parents (Fig. 2). Additional and Japanese hybrids. A relatively broad range of intense bands which migrated to an intermediate variant distribution was observed in 6-PGD-1, com- zone between parental bands were observed in pared with other enzyme systems. The frequency MDH-1 region (Fig. 2). This indicated the dimer- of the 6-PGD-1 variants, AA and BB, was 27 and ic structure of the enzymes in MDH-1, as in other 73%, respectively (Table 2). Phenotype 6-PGD-1 plants (Kephart, 1990). AA was recognized in north and south Chinese Discussion (Japan) and south-east Asian cucumbers with high frequency. Only the Indian wild cucumber exhibit- Cucumis sativus var. hardwickii which grows in ed BB in 6-PGD-2, whereas all of the other acces- Nepal Himalaya, has the same chromosome num- sions showed AA. All accessions were ber n=7, as the cultivated cucumbers, and can be monomorphic for the PGM-1 AA, except for the crossed with them. It is considered to be a wild Indian wild cucumber and 'Santo' which were de- cucumber (Imazu and Fujishita, 1956). Four of six termined as PGM-1 BB and CC, respectively. For isozyme phenotypes (Tables 1 and 2) are common PGM-2 the BB was observed only in Sri Lanka na- to the cucumber cultivars and var. hardwickii, in- tive 'S10-1' and two English forcing type acces- dicating the close relationships between them. We sions, whereas the rest exhibited AA. Ninety five analyzed only one accession of var. hardwickii. percent of the accessions examined possessed However, the existence of var. hardwickii specific

Table 2. Phenotype frequencies (%) of isozymes observed for MDH, 6-PGD, PGM and PGI in one Indian wild cucum- ber (Cucumis sativus var. hardwickiil and 81 accessions of C. sativus. 600 S. ISSHIKI, H. OKUBO AND K. FUJIEDA

Hybrid cultivars are common in commercial cu- cumber production. Varietal seed purity is an es- sential quality in commercial seed lots (Arus et al., 1982). Our seed analysis could be useful in evalu- ating the purity of F1 cucumber seeds since the variant MDH phenotypes are scattered in the com- mercial F1 cultivars in diverse ecotypes, e.g., AA of 'Harigaya' and 'Kumamotofushinari' of the south Chinese (Japan) and 'lzumiharu' of the Japanese hybrids; and BB of 'Takaido' in the south Chinese type (Japan), and 'Risshu', 'Mitaniyokusei', 'Santo' and 'Kofune' in the north Chinese type

(Japan) (Table 1).

Literature Cited

Fig. 2. MDH phenotypes of seeds in 'Natsufushinari', 'San- Arus, P., C.R. Shields and T. J. Orton. to' and their F1. 1985. Application of isozyme electrophoresis for purity testing and cultivar identification of F1 hybrids of Brassica oleracea. Euphytica 34 : 651-657. Arus, P., S.D. Tanksley, T.J. Orton and R.A. Jones. phenotypes, i.e., 6-PGD-2 BB and PGM-1 BB im- 1982. Electrophoretic variation as a tool for deter- plies the genetic peculiarity of the variety. Varie- mining seed purity and for breeding hybrid varieties ty hardwickii, therefore, may be one of the of Brassica oleracea. Euphytica 31 : 417-428. ancestral forms of cultivated cucumber. Staub Arus, P. and T.J. Orton. 1983. Inheritance and linkage et al. (1985b) arrived at the same conclusion as we relationships of isozyme loci in Brassica oleracea. J. have. Hered. 74 : 405-412. Cultivated cucumbers in China have been divid- Bringhurst, R.S., S. Arulsekar, J.F. Hancock Jr. and V. ed mainly into two ecotypes, north Chinese and Voth. 1981. Electrophoretic characterization of strawberry cultivars. J. Amer. Soc. Hort. Sci. south Chinese (Kumazawa and Kanazawa, 1948). 106 : 684-687. The south Chinese type of Japanese cultivars was Colby, L.W. and L.C. Peirce. 1988. Using an isozyme introduced from south China centuries ago, marker to identify doubled haploids from anther cul- whereas the north Chinese type of Japanese culti- ture of asparagus. HortScience 23 : 761-763. vars came from north China during the Meiji era Crawford, D.J. 1983. Phylogenetic and systematic in- (Kumazawa and Kanazawa, 1948). The current ferences from electrophoretic studies. p.257-287. Japanese hybrids (spring and summer types) were In : S.D. Tanksley and T.J. Orton (eds.). Isozymes developed from the above two ecotypes (Kumaza- in plant genetics and breeding. Part A. Elsevier, wa and Akiya, 1963). Since the range of isozyme Amsterdam. Dewald, M. G., G. A. Moore and W. B. Sherman. variations in south Chinese (Japan) was broad and 1988. Identification of pineapple cultivars by iso- the variations included that of north Chinese zyme genotypes. J. Amer. Soc. Hort. Sci. (Japan) and Japanese hybrids, the relationships be- 113 : 935-938. tween ecotype differentiations among cucumber Fujieda, K. 1973. The complete book of cultivation of cultivars and isozyme variations were difficult to cucumber. p.42-65. Nogyo Tosho, Tokyo. (In establish. Japanese). South Chinese (China) and south-east Asian ac- Gottlieb, L. D. 1981. Electrophoretic evidence in plant cessions showed similar phenotypes which suggests populations. Prog. Phytochem. 7 : 1-46. Hirai,M., I. Kozaki and I. Kajiura. 1986. Isozyme anal- that they are closely related. ysis and phylogenic relationships of citrus. Japan. J. The PGM-2 BB phenotype, which was not ob- Breed. 36 : 377-389. served in Asian cucumbers except 'S10-1', could Imazu, T. and N. Fujishita. 1956. Cucumbers. p. distinguish European from Asian cucumbers. This 213-228. In : H. Kihara (ed.). Land and crops of phenotype would be one clue to the understanding Nepal Himalaya, Fauna and Flora Res. Soc. Kyoto of cultivar differentiations. Univ., Kyoto, Japan. J. Japan. Soc. Hort. Sci. 61 (3) : 595-601. 1992. 601

Kephart, S.R. 1990. Starch gel electrophoresis of plant Staub, J.E., L. Fredrick and T. L. Marty. 1985a. Elec- isozymes : a comparative analysis of techniques. trophoretic variation in cross-compatible wild diploid Amer. J. Bot. 77 : 693-712. species of Cucumis. Can. J. Bot. 65 : 792-798. Knerr, L. D., J.E. Staub, D. J. Holder and B. P. May. Staub, J.E., R.S. Kupper, D. Schuman, T.C. Wehner and 1989. Genetic diversity in Cucumis sativus L. as- B. May. 1985b. Electrophoretic variation and en- sessed by variation at 18 allozyme coding loci. The- zyme storage stability in cucumber. J. Amer. Soc. or. Appl. Genet. 78 : 119-128. Hort. Sci. 110 : 426-431. Kumazawa, S. and R. Akiya. 1963. Cucumber. p. Tanksley, S.D. and R. A. Jones. 1981.Application of al- 20-42. In : S. Kumazawa (ed.). The discussion of de- cohol dehydrogenase allozymes in testing the puri- tail in vegetable gardening. Yokendo, Tokyo. (In ty of F1 hybrids of tomato. HortScience Japanese). 16: 179-181. Kumazawa, S. and S. Kanazawa. 1948. Introduction of Weeden, N.F. and G.A. Marx. 1984. Chromosomal lo- the north Chinese cucumber and breeding of 'Suyo'. cations of twelve isozyme loci in Pisum sativum. J. J. Hort. Assoc. Japan. 17 : 39-42. (In Japanese). Hered. 75 : 365-370. Mundges, H., W. Kohler and W. Friedt. Weeden, N.F. and R.C. Lamb. 1987. Genetics and 1990. Identification of rape seed cultivars (Brassica linkage analysis of 19 isozyme loci in apple. J. napus) by starch gel electrophoresis of enzymes. Eu- Amer. Soc. Hort. Sci. 112: 865-872. phytica 45 : 179-187. Weeden, N.F. and R. Provvidenti. 1988. A marker lo- Perl-Treves, R., D. Zamir, N. Navot and E. Galun. cus, Adh-1, for resistance to pea enation mosaic vi- 1986. Phylogeny of Cucumis based on isozyme rus in Pisum sativum. J. Hered. 79 : 128-131. variability and its comparison with plastome Wendel, J.F. 1983. Electrophoretic analysis of genet- phylogeny. Theor. Appl. Genet. 71 : 430-436. ic variation in wild and cultivated Camellia japoni- Rick, C.M. 1983. Tomato. p. 147-165. In : S.D. Tanks- ca L. Ph. D. Thesis, University of North Carolina, ley and T. J. Orton (eds.). Isozymes in plant genet- Chapel Hill, North Carolina. ics and breeding. Part B. Elsevier, Amsterdam. Wendel, J.F. and C.R. Parks. 1982. Genetic control of Soost, R. K., T. E. Williams and A. M. Torres. isozyme variation in Camellia japonica L. J. Hered. 1980. Identification of nucellar and zygotic see- 73 : 197-204. dlings of citrus with leaf isozymes. HortScience Woods, S. and D.A. Thuran. 1976. The use of seed 15 : 728-729. acid phosphatases in the determination of the puri- Staub, J.E. and L. Fredrick. 1988. Inheritance of three ty of Fi hybrid Brussels sprout seed. Euphytica enzyme loci in cucumber. HortScience 23 : 757-758. 31 : 707-712.

キ ュ ウ リに お け る ア イ ソザ イム変 異

一 色 司郎 ・大久保 敬 ・藤枝 國光

九州大学農学部 812 福 岡市東区箱崎

摘 要

イ ン ド野 生 キ ュ ウ リ(Cucumis sativus L. var. har- イ ム変 異 か ら， イ ン ド野 生 キ ュ ウ リは栽 培 キ ュ ウ リ と Kitamura)お dwickiiよ び 栽 培 キ ュ ウ リ(C.sativus) は 遠 縁 で あ る こ と が 示 唆 さ れ た.キ ュ ウ リの 生 態 型 と 81系 統 に つ い て，6種 類 の 酵 素 の ア イ ソ ザ イ ム 分 析 を ア イ ソザ イ ム変 異 との 間 に 明 確 な 関 連 は 認 め られ なか っ 行 っ た.シ キ ミ酸 デ ヒ ドロ ゲ ナ ー ゼ(SKDH)と イ ソ た が， こ れ ら の ア イ ソザ イ ム 変 異 は 生 態 型 の 異 な る 品 ク エ ン酸 デ ヒ ドロ ゲ ナ ー ゼ(IDH)で は 変 異 が 認 め ら 種 群 に 分 散 し て い る こ とお よ びMDHで は単 粒 種 子 分 れ ず， リン ゴ酸 デ ヒ ドロゲ ナ ー ゼ(MDH)，6一 ホ ス ホ 析 が 可 能 な こ とが 確 か め られ， 種 子 の 純 度 検 定 や キ ュ グ ル コ ン 酸 デ ヒ ドロ ゲ ナ ー ゼ(6-PGD)， ホ ス ホ グ ル ウ リ育 種 に お け る 遺 伝 マ ー カ ー と し て 利 用 で き る見 通 コ ム ター ゼ(PGM)お よ び ホ ス ホ グ ル コ イ ソ メ ラー ゼ し を得 た。 (PGI)に お い て 変 異 が 認 め ら れ た。 こ れ ら の ア イ ソ ザ