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1 Jour. Agri. Sci., Tokyo Univ. of Agric.,.0 (,), ++. +,- (,**+).0, ++. +,-,**+ * ** ** ** +-, : Salverinus malma S. leucomaenis S.l. imbrius, + S. malma malma S.m. miyabei, : S.l.f. leucomaenis : S.l.f. pluvius S.l.f. japonica S.l.f. imbrius. mtodna DNA + Tf Es Hb Cell X Tf D F H L o 3 F *.1+, J *.3+1 Es - Eso A B o Eso F I S o Eso D + D, o Hb, A B A B Cell X A B - Tf Es Hbo Cell X Es, Mu Mu A B A B Tf Es Hbo Mu, mtodna - + Tf Mu : Salmonidae Salvelinus + -. * **

2 115 / Salvelinus malma Salvelinus leucomaenis Salvelinus leucomaenis imbrius, Salvelinus malma malma Salvelinus malma miyabei, : Salvelinus leucomaenis f. leucomaenis : Salvelinus leucomaenis f. pluvius Salvelinus leucomaenis f. japonica Salvelinus leucomaenis f. imbrius. : , 3, +*, ++ DNA mtodna +,, +- DNA +. mtodna DNA + Salvelinus malma +0. Salvelinus leucomaenis 10 :.. : -, Salvelinus pluvius Hb 2,*** rpm +*,* -,*** rpm +* Hb,* 2*, Tf Es Tf Es HPAGE +33* +/,.*,** + mm +, /, mm *.- mm -* ma / Hb Hb YOKOHAMA IEF,3.+, ml *.3 N N, ml ml + *.- ml -* ml ph 0 2 LKB *.1/ ml ph 1 3 LKB,.,/ ml +0* +,* *.. mm Hb 1/ 2 +0* mm : ADVANTEC No. /,0 / ph..* 0.* LKB. NaOH +./ cm - / mm +,,** V. W. Cell X Cell X +/ SDS sodium dodecyl sulfate SDS PAGE / ml 0,* ml +* SDS +** ml, +* ml *.. SDS *./ M Tris ph 0.2,* ml,** ml +** / + BPB / ml +/ ma, *.+ W V R /* V V 1 V V -*,* V V 1 V V Mu Mu Cell X,*

(D, F, H, J and L). Es types were classified into - loci (Es-I, Es-II and Es-III). Number in the brackets is examined specimens in all collections.

3 116 Fig. + Electrophoretic patterns and nomenclature of serum transferrin (Tf) type and serum esterase (Es) type in charr fish, genus Salvelinus (by H-PAGE method) Remarks ; Tf type was composed of five bands (D, F, H, J and L). Es types were classified into - loci (Es-I, Es-II and Es-III). Number in the brackets is examined specimens in all collections. Each set of band patterns was representative of each locus. *., g +** ml + ml +./ ml -,*** rpm +* /* ml 0,* ml +* SDS +** ml, +* ml *.. SDS *./ M Tris ph 0.2,* ml,** ml +** / + BPB +* ml +/ ma, + Tf Es Tf H PAGE Tf Tf D F H J L o +, Tf. / J. D F H L o F *.1+, FH *.+,* + FL HL Fig., Schematic electrophoretic patterns of serum transferrin (Tf) type and serum Esterase (Es) type in charr fish, genus Salvelinus (by H-PAGE method) Remarks ; Tf type was composed of five bands (D, F, H, J and L). Es types were classified into - loci (Es-I, Es-II and Es-III). Each set of band patterns was basic and representative of bands in each locus., J +.*** * *** + Tf Es ao Es - Eso D + D, o Eso A B o Eso F I S o +, Eso o, D + D, D + D, *.0// D, *.001 *./** Eso - D + D,. Eso, A B o o *./.. A *.--0 / o 0 Eso

4 117 Table, Apperance Table + Apperance frequency of transferrin (Tf) types frequency of transferrin (Tf) types among places of catch in Hokkaido (Salvelinus malma)

5 118 Table - Apperance frequency of esterase (Es) types Table. Apperance frequency of esterase-i (Es-I) types among places of catch in Hokkaido (Salvelinus leucomaenis) Table / Apperance frequency of esterase-ii (Es-II) types Table 0 Apperance frequency of esterase-ii (Es-II) types among places of catch in Hokkaido (Salvelinus malma) o / Eso Eso Eso - F I S o IS I *.. *.,1, 1 Eso IS I S F 2 o +.*** * *** 1 + FS, Hb Hb IEF Hbo A B Hbo F S - Hbo,

6 119 Table 2 Apperance Table 1 Apperance frequency of esterase-iii ( Es-III ) types frequency of esterase-iii ( Es-III ) types among places of catch in Hokkaido ( Salvelinus malma)

120 A B AB B *.,3- *.1*1 3 B +* A - A +.*** *.3/, +.*** 3 Hbo F No.,/ : + S F FS *.3/, *.*.2 3 IEF Hbo SGE +2 Hb : +31/ IEF Hbo Hbo SGE - Cell X Cell X SDS PAGE +1,,*+ Da Hb Cell X A B.

- Electrophoretic patterns of Hemoglobin (Hb) type in charr fish, genus Salvelinus (by IEF) Remarks ; Hb-I type was

Mu, A B A A +.*** B B +.*** *.3/, +.*** +.

7 120 A B AB B *.,3- *.1*1 3 B +* A - A +.*** *.3/, +.*** 3 Hbo F No.,/ : + S F FS *.3/, *.*.2 3 IEF Hbo SGE +2 Hb : +31/ IEF Hbo Hbo SGE - Cell X Cell X SDS PAGE +1,,*+ Da Hb Cell X A B. Cell X, A B B B *.2.2 *.23, +.*** * Cell X Tf Es A +, +- Fig. - Electrophoretic patterns of Hemoglobin (Hb) type in charr fish, genus Salvelinus (by IEF) Remarks ; Hb-I type was classified into - types (A, AB and B). Hb-II type was composed of, banes (F and S).. Mu.,,.** kda 00,,01 kda Mu A B. Mu, A B A A +.*** B B +.*** *.3/, +.*** +. Mu Table 3 Apperance frequency of hemoglobin (Hb) types Table +* Apperance frequency of hemoglobin (Hb) types among places of catch in Hokkaido (Salvelinus malma)

muscular protein (Mu) type in charr fish, genus Salvelinus (by +/ SDS-PAGE)

Cell X type was classified into - types (A, AB and B).

protein (Cell X) types Table +, Apperance frequency of blood cell protein (Cell

8 121, AB Tf Es Hbo Mu, mtodna Fig.. Electrophoretic patterns of blood cell membrane protein (Cell X) type and muscular protein (Mu) type in charr fish, genus Salvelinus (by +/ SDS-PAGE) Remarks ; Mu type was classified into - types (A, AB and B). Cell X type was classified into - types (A, AB and B). / + Tf F J FJ Eso FS Hbo B Mu A B AB Table ++ Apperance frequency of blood cell protein (Cell X) types Table +, Apperance frequency of blood cell protein (Cell X) types among places of catch in Hokkaido (Salvelinus malma) Table +- Apperance frequency of blood cell protein (Cell X) types among places of catch in Hokkaido (Salvelinus leucomaenis)

9 122 Table +. Apperance frequency of muscular protein (Mu) types + +30, /2 /3 0/ 12, +311 Salverinus malma,..3 / *.+ - +,+ +-*. +31-,*,./,.1 / +330 /, * +/- +3, ** +-* 2 GOTO, A., TAKAHASHI, M. and YAMAZAKI, F.,+323. Whitespotted and red-spotted morphs as a phenotypic variation of the Japanease Charr Savelinus leucomaenis in the river of southern Hokkaido, Japan. Phisiol. Ecol. Jap., +,.,+.,2. 3 MITSUBOSI, T., GOTO, A.andYAMAZAKI, F.,+33,. Genetic di#erentiation of the Dolly Varden Salvelinus malma in Hokkaido, Japan. Bull. Fac. Fish. Hokkaido Univ.,.-(.), +/ * NAKAJIMA, M.andFUJIO, Y.,+33/. Genetic Di#erentiation among Local Populations of Japanese Char Salvelinus leucomaenis. Fisheries Science, 0+(+), ++ +/ ,, , MCVEIGH, H.P. and DAVIDSON, W.S., A salmonid phylogeny inferred from mitochondrial cytochrome b gene sequences. Journal of Fish Biology, -3, (Supplement A),,,1,2,. +- WILLIAM, J. Berg and FERRIS, S.D., Restriction Endonuclease Analysis of Salmonid Mitochondrial DNA. Can. J. Fish. Aquat. Sci.,.+, +*.+ +* RAPD,/,1-0 +/ +33* /,/ YOKOHAMA, M., An additional classification for equine Hb-a type. Japanese Journal of Electrophoresis, -/ (+), MEDICAL TECHNOLOGY, YOSHIYASU, K.andHUMOTO, Y.,+31,. Starch-gel Electrophoresis of Freshwater Salmonid Fishes in Southwest Japan-I. Genus Salvelinus (Char). Bulletin of the Society of Scientific Fisheries, -2(1), /

10 123 Detection of Protein Polymorphism of Charr Fishes in the Genus Salvelinus. By Kouta ISHII*, Ryusuke IMAEDA**, Kenta WADA** and Michinari YOKOHAMA** (Received February +-,,**+/Accepted June +.,,**+) Summary : The classification of Salvelinus in Japan has been controversial and has not been established. According to one theory, Salvelinus is classified into two species (S. malma and S. leucomaenis) and + subspecies (S. leucomaenis imbrius). According to another theory, S. malma is classified into two subspecies (S. malma malma and S.m. miyabei), and S. leucomaenis, together with Salvelinus l. f. pluvius, S.l. f. japonica, and S.l. f. imbrius. There is no established classification of Salvelinus according to morphological characteristics in Japan. To develop new biochemical markers in addition to isozymes and mt-dna and genomic DNA polymorphism, the detection methods of which have already been established, we examined polymorphic loci of blood proteins and muscular proteins using Salvelinus caught in Hokkaido. +) Examination of blood protein types In Salvelinus, the serum transferrin (Tf) type, serum esterase (Es) type, hemoglobin (Hb) type, and blood cell membrane protein (Cell X) type were examined. Concerning the Tf type [basic bands ; D, F, H, L ; inapparent type (-)], the F type among 3 phenotypes was a characteristic phenotype in S. malma (incidence, *.1+,) while the J type was a characteristic phenotype in S. leucomaenis (incidence, *.3+1). Among the - domains of the Es type, the Es-II domain [basic bands ; A, B ; inapparent type (-)] and the Es-III domain [basic bands ; F, I, S ; inapprent type (-)] were characteristic of S. leucomaenis. Concerning the Hb type, there were two domains (I and II), and polymorphism was observed in the I domain (basic bands ; A and B). The incidence of A was high in S. leucomaenis, and that of B incidence was high in S. malma. The Cell X type (basic bands ; A and B) did not di#er among the species but could be classified into - phenotypes. The appearance frequencies among local populations di#ered in S. malma, Tf type, Es type (II and III domain), Hb-I type and Cell X type, and in S. leucomaenis, Es-I domain.,) Examination of the muscular protein (Mu) type In Salvelinus, concerning the Mu type (basic bands A and B), the incidence of the A type was high in S. malma, and that of the B type was high in S. leucomaenis. Thus, there were di#erences according to the Tf type, Es type (I, II and III domains), Hb-I domain, and the Mu type that allow classification into two groups (S. malma and S. leucomaenis). The same as for isozymes and mt-dna, a locus deteciton method that can clarify inter- and intra-species di#erence was established. -) Confirmation of inter-species hybrids In a S. leucomaenis individual collected in Murii River, Tf and Mu phenotypes that appear to be heterotypes of S. malma and S. leucomaenis were detected. This individual was S. leucomaenis by appearance, showing no characteristics of S. malma, and therefore, may have resulted from crossing between S. malma and S. leucomaenis. Key Words : charr fish, protein polymorphism loci, classification, biochemical markers, local variation * Graduate School of Bioindustry, Tokyo University of Agriculture ** Laboratory of Animal Resources, Department of Bioproduction, Faculty of Bioindustry, Tokyo University of Agriculture

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