J. AMER. SOC. HORT. SCI. 117(6):996-999.

1992.

Genetic Analysis of Isoenzymes in Raspberry

Johanne C. Cousineau and Danielle J. Donnelly
Department of Plant Science, Faculty of Agricultural and Environmental Sciences, McGill University, Macdonald Campus, 21111 Lakeshore Road, Ste-Anne-de-Bellevue, Que. H9X 3V9, Canada
Additional index words. Rubus, linkage, starch gel electrophoresis Abstract. The inheritance of five isoenzymes was studied in red and purple raspberry F1 progenies (Rubus idaeus L. and Rubus neglectus Peck). Isocitrate dehydrogenase (IDH; EC 1.1.1.42) was a dimeric enzyme present in the cytosol and coded for by one locus (Idh-1). Three of the four crosses analyzed at this locus had deviations from expected ratios, possibly caused by its linkage to a recessive lethal gene. Malate dehydrogenase (MDH; EC 1.1.1.37), phosphoglucoisomerase (PGI; EC 5.3.1.9), and triose phosphate isomerase (TPI; EC 5.3.1.1) were dimeric enzymes with two loci. Each of these three enzymes was present in an organelle and in the cytosol for locus 1 and 2, respectively. Phosphoglucomutase (PGM; EC 2.7.5.1) was monomeric with two loci, Pgm-1 and Pgm-2, located in an organelle and the cytosol, respectively. Each allele at Pgm-1 resulted in the formation of two bands. Although most progenies analyzed supported Mendelian inheritance of polymorphic loci (except for Idh-1), there was a higher than expected number of aberrant segregation ratios observed (18.4%). Analysis of 85 pairs of jointly segregating loci revealed a possible linkage group consisting of Mdh-2, Tpi-2, and Pgm-1.

There are numerous single trait genes described in raspberry (Rubus subgenus Idaeobatus) including some that are linked to one another (Jennings, 1988; Ourecky, 1975). Since many of these gene markers can be difficult to score, it would be beneficial to have easily scored markers like isoenzymes available for use by raspberry breeders. Applications of isoenzyme technology include plant identification (Cousineau and Donnelly, 1992), increased efficiency of gene introgression in interspecific crosses (Tanksley and Rick, 1980), tagging of important economic traits (Rick and Fobes, 1974), and estimating genetic variability in populations (Hamrick, 1989). Most of these applications require that the inheritance of the isoenzymes be understood. Our objectives were to determine the genetic basis and linkage relationships among five polymorphic enzymes in raspberry. Materials and methods Progenies were generated by crossing red and purple raspberry cultivars and selections (Table 1). All seedlings were maintained in a greenhouse at ambient temperatures under natural light. The putative alleles coding for isoenzymes at each locus were determined by sampling leaves from 78 raspberry cultivars (R. idaeus, R. neglectus, and R. occidentalis) for the purpose of cultivar identification (Cousineau and Donnelly, 1992). A band at Rf 0.7, obtained for PGM for the cultivar Prestige, was omitted from this study since it was not possible to assign it with certainty to either of the PGM loci. The inheritance of the following enzymes was studied: IDH, MDH, PGI, PGM, and TPI. Enzyme extraction from leaves, horizontal starch gel electrophoretic procedures, and isoenzyme staining techniques were that of Cousineau and Donnelly (1992), except that to visualize both PGI loci, samples were run on a lithium borate system (electrode buffer, 0.06 M LiOH, 0.3 M boric acid, pH 8.1, and gel buffer: 90% 0.03 M Tris, 0.05 M citric acid, pH 8.5; 10% electrode buffer)(Ridgway et al., 1970).
Received for publication 24 Feb. 1992. Accepted for publication 6 July 1992. Partial financial support from the Natural Sciences and Engineering Research Council of Canada (grant A2236) to D.J.D. and scholarship funds provided by Fonds FCAR, J.W. McConnell Foundation, and Canadian Pacific to J.C.C. are gratefully acknowledged. We thank Harry J. Swartz for donating five of the crosses used in this study. The cost of publishing this paper was defrayed in part by the payment of page charges. Under postal regulations, this paper therefore must be hereby marked advertisement solely to indicate this fact.

Pollen enzymes were analyzed to determine the subcellular localization of the isoenzyme loci (Weeden and Gottlieb, 1980). They were extracted by soaking 30 mg of pollen in 100 l buffer consisting of 100 mM K2HPO4 (pH 7.0) containing 50 l glycerol and 50 mg of polyvinyl pyrrolidone (PVP-40) per milliliter (Raelson and Grant, 1989). After a 6-h incubation at 4C, the supernatant containing the enzymes was absorbed onto filter paper wicks and separated as for leaf samples. The computer program LINKAGE-l was used to perform single locus segregation and linkage analyses (Suiter et al., 1983). For each enzyme, the loci and the alleles were numbered sequentially starting at 1 for the most anodal locus and a for the most anodal allele. The method of Hedrick and Muona (1990) was used to determine the maximum likelihood estimate of the recombination fraction between Idh-1 and a putative lethal gene in a selfed progeny. Results The alleles coding for isoenzymes present in raspberry at each locus are summarized in Fig. 1. For all loci studied, progeny from crosses in which both parents had the same allele were identical to the parents. In crosses where both parents were homozygous for a different allele, all progeny were heterozygous for those two alleles. IDH. One zone of activity (Idh-1) was present in all plants tested, and banding patterns consisted of either one or three bands, which suggested a dimeric structure for this enzyme. Idh-1 was present in soaked pollen, although the heterodimeric band was missing in samples from plants with a three-banded pattern in leaves. This result confirmed the dimeric nature of the enzyme and suggested its presence in the cytosol. Three alleles were found at Idh-1 but three of the four crosses tested deviated from expected ratios (Table 1). MDH. There were two areas of staining visible for MDH. Patterns in both areas were either one or three-banded, confirming the dimeric nature of this enzyme. Analysis of soaked pollen revealed Mdh-2 but not Mdh-1, suggesting the presence of these loci in the cytosol and an organelle, respectively. There appeared to
Abbreviations: IDH, isocitrate dehydrogenase; MDH, malate dehydrogenase; PGI, phosphoglucoisomerase; PGM, phosphoglucomutase; TPI, triose phosphate isomerase.

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J. Amer. Soc. Hort. Sci. 117(6):996-999.

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Table 1. Single-locus segregation and x2 analysis at seven polymorphic loci.

The following red raspberry (R. idaeus L.) and, where indicated, purple raspberry (R. neglectus Peck) cultivars and selections were used to generate progeny (number of seedlings shown in parentheses): 1) Comet Titan and the reciprocal cross (106), 2) Comet Killarney (47), 3) Comet GU63 (58), 4) Comet selfed (47), 5) Festival Titan and the reciprocal cross (72), 6) Festival GU63 (67), 7) Festival GU74 (50), 8) Citadel Glen Prosen (46), 9) Royalty (purple) Glen Prosen (26), 10) Scepter Autumn Bliss (57), 11) Southland spine-free Willamette (28), 12) Brandywine (purple) Glen Garry (48), 13) and Heritage selfed (56). GU63 and GU74 are selections from the Univ. of Guelph raspberry breeding program that were selected from Creston Viking and Hilton Latham crosses, respectively. Data from reciprocal crosses were pooled since there were no differences with respect to the direction of the crosses. y Degrees of freedom were 1, 2, and 3 for 1:1, 1:2:1, and 1:1:1:1 ratios, respectively.

J. Amer. Soc. Hort. Sci. 117(6):996-999.

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Table 2. Number of crosses analyzed by contingency x2 tests for independent assortment of jointly segregating isoenzyme loci (above the diagonal) and number of crosses deviating from independent assortment (below the diagonal).

significant (P < 0.01) association between Pgm-1 and Tpi-2 in five of seven crosses (Table 3). In addition, significant linkage was observed in two of 10 crosses between Mdh-2 and Pgm-1 (P < 0.01 and P 0.05, respectively) and in one cross each with P 0.05 between the following groups of two markers: Mdh-2 and Tpi-1, Mdh-2 and Tpi-2, Pgi-2 and Idh-1, and Tpi1 and Idh-1. Discussion Subunit structure, number of loci, and subcellular localization of isoenzymes are well conserved in plant species, especially for those enzymes catalyzing steps in primary metabolism (Gottlieb, 1982). Results obtained for raspberry isoenzymes correspond to those reported in other plant species for IDH, PGI, PGM, and TPI (Weeden and Wendel, 1989). There were two MDH loci detected in raspberry, a result similar to that observed in Prunus (Byrne, 1989; Mowrey et al., 1990a) and balsam fir [Abies balsamea (L.) Mill.] (Neale and Adams, 1981). Each allele at Pgm-1 resulted in the formation of two bands. This type of inheritance pattern for PGM has been reported for balsam fir (Neale and Adams, 1981), Camellia japonica L. (Wendel and Parks, 1982), maize (Zea mays L.) (Goodman et al., 1980), and in many species of Prunus (Byrne, 1989; Byrne and Littleton, 1988; Mowrey et al., 1990b). The two-banded alleles may be the result of posttranslational modifications or artifacts generated during the extraction procedure. Three of the four crosses tested for Idh-1 inheritance deviated significantly from the expected ratio. This deviation could be caused by the linkage of this locus to a recessive lethal gene. The maximum likelihood estimate of the recombination fraction between the postulated lethal gene and Idh-1 in cross 13 (Heritage selfed) was 0.023 assuming that the gene was fully lethal (lod score = 5.217). A minimum lod score of three or more is needed to demonstrate linkage between loci (Conneally et al., 1985). The majority of the crosses studied did not deviate from expected ratios for Mdh-2, Pgi-2, Pgm-1, Tpi-1, and Tpi-2. However, there were more skewed ratios (average 18.4%) than would be expected. Segregation distortions are not uncommon and many processes, including preferential segregation, pollen tube competition, pollen lethal factors, preferential fertilization, and selective elimination of zygotes have been proposed to explain such distortions (Grant, 1975; Zamir and Tadmor, 1986). Five of seven progeny supported the linkage of Pgm-1 and Tpi-2 in raspberry. Recombination fractions ranging from 0.09 to 0.20 were obtained for these progenies. Comet was a parent in four of the crosses showing linkage but not in the two crosses with independent assortment. The inability to detect linkage in all progenies may reflect differences in genetic background as suggested by the consistent linkage when Comet was used as a parent.

Fig. 1. Interpretative drawing of loci (numbers) and alleles (letters) observed in five polymorphic isoenzymes of raspberry. be two alleles present at Mdh-1, but this finding will require verification since none of the crosses tested segregated at this locus. Seven out of 10 crosses segregating at Mdh-2 fit the expected Mendelian ratios and a total of six alleles were detected. PGI. There were two loci coding for PGI isoenzymes. Pgi-1 was monomorphic in all plants tested and was present in an organelle according to pollen analysis. Three alleles were found at Pgi-2; they segregated normally in 10 of 13 crosses. Based on pollen analysis, this locus coded for a dimeric enzyme that was located in the cytosol. PGM. There were two overlapping zones of activity for PGM. Pgm-1 was present in an organelle and was functional as a monomer, although each of the three alleles detected resulted in the formation of two bands, some of which overlapped one another. Chi-square analysis at this locus showed no deviations from expected ratios in 12 of 13 crosses. Pgm-2 was characterized by the presence of either one or two bands, suggesting a monomeric structure. It was active in soaked pollen, indicating its presence in the cytosol. Two alleles were detected, each one coding for a single band. The only cross segregating at this locus showed a deviation from the expected ratio. More crosses will be needed to confirm the inheritance at this locus. TPI. This enzyme was coded for by two loci that were both dimeric, with two alleles each. Analysis of soaked pollen suggested that Tpi-1 and Tpi-2 were present in an organelle and the cytosol, respectively. All crosses tested, except one at each locus, segregated normally, and the deviations from expected ratios in the distorted crosses were not highly significant (P = 0.04 and 0.03, respectively for Tpi-1 and Tpi-2). Linkage analyses. Eighty-five pairs of jointly segregating loci were tested for independent assortment (Table 2). Only those pairs where at least one or both loci segregated normally were included in the analysis (Bailey, 1961). There was a highly

J. Amer. Soc. Hort. Sci. 117(6):996-999.

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Table 3. Deviations observed from independent assortment in joint segregation analysis of polymorphic loci.

Genotypic classes are given in the same sequence as those in Table 1. A change in the genotype of the first locus is indicated by a slash (/) and a change in the genotype of the second locus is indicated by a comma (,). There was a weak association between Mdh-2 and Pgm-1 in two of ten crosses where the recombination fractions were 0.32 and 0.45, respectively. Since there was also linkage between Pgm-1 and Tpi-2, the three markers may be present in the same linkage group, although additional evidence will be required for confirmation. Linkage between these same markers has been reported in apple (Malus domestica Borkh.)(Weeden and Lamb, 1987), another member of the Rosaceae family. The possibility of linkage conservation in this family will require further study. We have insufficient data to confirm the existence of the following linkage groups: Mdh-2/Tpi-1, Pgi-2/Idh-1, and Pgm1/Tpi-1. Deviations from independent assortment were observed in one of five, four, and six jointly segregating progeny, respectively, with a P value that was not highly significant ( 0.05). Since there was only one progeny co-segregating for Tpi-1/Idh1, the analysis of more crosses would be desirable to confirm the observed deviation from independent assortment. This study describes the mode of inheritance of five polymorphic isoenzymes in raspberry and reveals a possible linkage group consisting of Mdh-2/Tpi-2/Pgm1. The usefulness of isoenzymes as genetic markers has been established in many plant species. Some of the possible applications for raspberry breeders include the acceleration of gene introgression from related species, the evaluation of wild populations for genetic variability, the tagging of traits, and the development of a linkage map. These applications may assist breeders in selecting parents and screening seedlings more efficiently. Literature Cited
Bailey, N.T.J. 1961. Introduction to the mathematical theory of genetic linkage. Oxford Univ. Press, London. Byrne, D.H. 1989. Inheritance of five isozyme loci in apricot. HortScience 24:1015-1016. Byrne, D.H. and T.G. Littleton. 1988. Electrophoretic characterization of diploid plums of the southeastern United States. J. Amer. Soc. Hort. Sci. 113:918-924. Conneally, J.H., J.H. Edwards, K.K. Kidd, J.-M. Lalouel, N.E. Morton, J. Ott, and R. White. 1985. Report of the committee on methods of linkage analysis rind reporting. Cytogenetics Cell Biol. 40:356-359. Cousineau, J.C. and D.J. Donnelly. 1992. Use of isoenzyme analysis to characterize raspberry cultivars and detect cultivar mislabeling. HortScience. 27:1023-1025. Goodman, M.M., C.W. Stuber, K. Newton, and H.H. Weissinger. 1980. Linkage relationships of 19 enzyme loci in maize. Genetics 96:697-710. Gottlieb, L.D. 1982. Conservation and duplication of isozymes in plants. Science 216:373-380. Grant, V. 1975. Genetics of flowering plants. Columbia Univ. Press, New York. Hamrick, J.L. 1989. Isozymes and the analysis of genetic structure in plant populations, p. 87-105. In D.E. Soltis and P.S. Soltis (eds.). Isozymes in plant biology. Dioscorides Press, Portland, Ore.
Hedrick, P.W. and O. Muona. 1990. Linkage of viability genes to marker

loci in selfing organisms. Heredity 64:67-72. Jennings, D.L. 1988. Raspberries and blackberries: Their breeding, diseases and growth. Academic Press, London. Mowrey, B.D., D.J. Werner, and D.H. Byrne. 1990a. Inheritance of isocitrate dehydrogenase, malate dehydrogenase, and shikimate dehydrogenase in peach and peach x almond hybrids. J. Amer. Soc. Hort Sci. 115:312-319. Mowrey, B.D., D.J. Werner, and D.H. Byrne. 1990b. Isozyme survey of various species of Prunus in the subgenus Amygdalus. Scientia Hort. 44:251-260. Neale, D.B. and W.T. Adams. 1981. Inheritance of isozyme variants in seed tissues of balsam fir (Abies balsamea). Can.. J. Bot. 59:1285-1291. Ourecky, D.K. 1975. Brambles, p. 98-129. In: J. Janick and J.N. Moore (eds.). Advances in fruit breeding. Purdue Univ. Press, West Lafayette, Ind. Raelson, J.V. and W.F. Grant. 1989. An isoenzyme study in the genus Lotus (Fabaceae). Experimental protocols and genetic basis of electrophoretic phenotype. Theor. Applied Genet. 77:595-607. Rick, C.M. and J.F. Fobes. 1974. Association of an allozyme with nematode resistance. Rpt. Tomato Genet. Coop. 24:25. Ridgway, G.J., .S.W. Sherburne, and R.D. Lewis. 1970. Polymorphisms in the esterases of atlantic herring. Trans. Amer. Fisheries Soc. 99:147151. Suiter, K.A., J.F. Wendel, and J.S. Case. 1983. LINKAGE-1: A PASCAL computer program for the detection and analysis of genetic linkage. J. Hered. 74:203-204. Tanksley, S.D. and C.M. Rick. 1980. Isozymic gene linkage map of the tomato: Applications in genetics and breeding. Theor. Applied Genet. 57:161-170. Weeden, N.F. and L.D. Gottlieb. 1980. Isolation of cytoplasmic enzymes from pollen. Plant Physiol. 66:400-403. Weeden, N.F. and R.C. Lamb. 1987. Genetics and linkage analysis of 19 isozyme loci in apple. J. Amer. Soc. Hort. Sci. 112:865-872. Weeden, N.F. and J.F. Wendel. 1989. Genetics of plant isozymes, p. 4672. In: D.E. Soltis and P.S. Soltis (eds.). Isozymes in plant biology. Dioscorides Press, Portland, Ore. Wendel, J.F. and C.R. Parks. 1982. Genetic control of isozyme variation in Camellia japonica L. J. Hered. 73:197-204. Zamir, D. and Y. Tadmor. 1986. Unequal segregation of nuclear genes in plants. Bot. Gaz. 147:355-358.

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