Joint Segregation of Biochemical Loci in Salmonidae. 11. Linkage Associations from a Hybridized Salvelinus Genome

The results of more than 300 painvise examinations of biochemical loci for joint segregation in brook trout (Salvelinus fontimlis) and in the hybridized genome of lake trout (S. namaycush) x brook trout are summarized. Nineteen loci have been assigned to the following eight linkage groupings on the basis of nonrandom assortment, including cases of both classical linkage and pseudolinkage: ODH with PMI with PGI-3, PGI-2 with SDH, ADA-I with AGP-2, AAT-(1,2) with AGP-I with MDH-I, MDH-3 with MDH-4, LDH-3 with LDH-4, IDH-3 with ME-2 and GUS with CPK-I. Pseudolinkage (an excess of nonparental progeny types) was observed only for male testcross parents. The results suggest that this phenomenon involves homeologous chromosome arms as evidenced by the de novo association of presumed duplicate loci in each case. Classical linkage has not been found for the five pairs of duplicate loci examined in Salvelinus, suggesting that not all of the eight metacentrics in the haploid complement involve fusions of homeologous chromosomes. Females consistently showed a greater degree of recombination. is accepted by most authors that the Salmonidae (trouts, salmons, chars, ':raylings and whitefishes) are the derivatives of a tetraploid lineage. It is clear from extensive inheritance and population studies of biochemical loci that (1) segregation is strictly disomic (BAILEY et al. 1970; ALLENDORF and UTTER 1973; ROPERS, ENGEL and WOLF 1973; MAY 1975; ALLENDORF 1975; WRIGHT, HECKMAN and ATHERTON 1975; MAY, STONEKING and WRIGHT 1979; MAY, WRIGHT and STONEKING 1979; STONEKING, MAY and WRIGHT 1979), and (2) there has been a substantial loss of duplicate gene activity (ALLENDORF, UTTER and MAY 1975; ALLENDORF 1978; MAY, WRIGHT and STONEKING 1979). The primary mechanism of diploidization in salmonids appears to be Robertsonian fusions of acrocentric chromosomes as evidenced by (1) intraspecific Robertsonian polymorphism? (OHNO et al. 1969; ROBERTS 1970; GOLD and GALL 1975; GOLD, AVISE and GALL 1977; THORGAARD 1976,1977,1978), and (2) variable 2n chromosome numbers with similar arm numbers in related salmonid species (SIMON 1963; * Authorized for publication as Paper Number 5795 in the Journal Series of the Pennsylvania Agricultural Experiment Station, University Park, Pa., in cooperation with the Beamer Spring Fish Research Station, The Pennsylvania Fish Commisson, Bellefonte, Pa. Supported in part by a Biomedical Research Support Grant, Office of the Vice President for Research, The Pennsylvania State University. Genetics: 9 5 : 707-726 July, 1980. 708 B. MAY, M. STONEKING AND J. E. WRIGHT, J R . OHNO el al. 1965; ROBERTS 1970; DAVISSON, WRIGHT and ATHERTON 1972,1973; CHIARELLI and CAPANNA 1973; GOLD and GALL 1975; ZENZES and VOICULESCU 1975). This particular process of diploidization and the coincidental loss of duplicate gene expression raise three interesting and approachable questions. Was the loss of duplicate gene activity a random single-locus event, or did it involve the loss of whole linkage groups; i.e., are single loci linked more often to other single loci or to members of duplicate loci? Did the fusion of acrocentric chromosomes involve homeologous (formerly homologous in the tetraploid set) or nonhomeologous chromosomes; i.e., are duplicated pairs of loci linked? Finally, did specific fusions take place prior or subsequent to the formation of genera or of species; i.e., are the same linkage relationships shared by related species? An unusual genetic phenomenon, termed “pseudolinkage,” has been observed only in salmonids. This phenomenon is characterized by the occurrence of an excess of nonparental progeny types in the backcrosses of males heterozygous for certain loci; similarly heterozygous females exhibit random assortment ( MORRISON 1970; DAVISSON, WRIGHT and ATHERTON 1973; WRIGHT, HECKMAN and ATHERTON 1975; MAY, WRIGHT and STONEKING 1979). To date, pseudolinkage has been observed in heterozygous males composed of apparently divergent genomes, The small size and large 2n numbers of salmonid chromosomes have prevented the visualization of a definitive meiotic mechanism that would produce such results. A fourth question is raised: How widespread is this phenomenon and, furthermore, are all cases of pseudolinkage produced by a single meiotic event? An extensive examination was initiated of joint segregation of biochemical loci in brook trout (Salvelinus fontinalis) and in a hybridized Salvelinus genomlake trout ( S . n a m q a ~ s h ) x brook trout-in order to examine these questions. The results of these inheritance studies are reported here. MATERIALS AND METHODS Single-pair matings involving brook, lake, splake (lake x brook) and Benner Spring Research Synthetic (BSRS) trout were made in the Fall of 1978 (Table 1). The BSRS strain was derived from crosses involving different inbred brook trout lines, splakes and splake backcross progeny. Ripe fish were killed, the gametes stored at 5” for 12 to 24 hr and the tissues typed electrophoretically to determine the appropriate parents for desired crosses. The progeny were reared at the Pennsylvania Fish Commission’s Upper Spring Creek Hatchery and were typed electrophoretically in the Spring of 1979. The horizontal starch gel electrophoretic techniques used were reported in detail in MAY, WRIGHT and STONEKING (1979). The enzymes studied, the abbreviations, loci and alleles, tissue specificity and appropriate huffer systems are listed in Table 2. Staining following the general methods of ALLENDORP et al. (1977), with the following exceptions: AAT (MAY, WRIGHT and STONEKING 1979), ODH (octanol-2 as substrate; BREWER 1970) and MUP (5 mg 4-methylumbelliferyl phosphate in 10 ml RSL buffer and observed under fluorescent light; UTTER, personal communication). Genic nomenclature and statistical treatment of segregation data follow that of MAY, WRIGHT and STONEKING (1979). The terminology XYZ-I ,2 is used when duplicated X Y Z loci share electrophoretically identical alleles and when one cannot identify which locus is variable (e.g., AAT-I,2 or MDH-3,4). XYZ-(I,2,3) is used when referring to more than one X Y Z locus. LINKAGE ASSOCIATIONS IN SALVELINUS 709 TABLE 1 Sources of the two parents of each family Family Source of female parent Source of male parent Y-21 Splake A* F, of ST Of x ST MC$ Y-23 BSRSS ST 3 Y-25 BSRS ST 3 Y-26 BSRS ST 3 Y-40 Unknown STil Splake A Y-41 Unknown ST Splake Y-42 Unknown ST Splake A Y-43 Splake A ST 22 Y-45 ST Albinoq Splake A Y-50 Unknown ST ST (3x28) x Belle Open** Y-61 BSRS ST 15 Y-77 L T t t Splake B$$ Y-79 N.Y S.V.s$ Splake B Y-80 Belle Open Splake B Y-81 LT Splake B Y-94 N.Y.S.V. BSRS Y-98 Belle Open BSRS * F, cross of S. namaycush (female) from L. Cayuga, N.Y. x S. fontina2i.s (male) from + Single numbers reflect inbred lines of S. fontinalis maintained by brother-sister matings for $Wild male from Marsh Creek, Pa. $ A mixed population of fish derived from multiple crosses of lake, splake, and first backcross 11 Either an ST 2 or Belle Open. T Albino color mutant stock with two generations of full-sib inbreeding. hatchery at Edray, WV. 8 to 12 generations. fish with intercrossed fish of S. fontinalis inbred lines (see Table 2). ** An open bred population of S. fontimlis from the Bellefonte, Pa. hatchery population. $$F, cross of S. namaycush (female) from L. Manitou x S. font inah (male) from Maple, An open bred population of S. fontinalis derived from New York Synthetic V, Rome, N.Y. S. namaycush from Lake Manitou, Ontario. Ontario. RESULTS Enzyme systems: The following isozyme systems have not been described previously in Salvelinus. Descriptions of the variation for other isozymes listed in Table 2 can be found in MAY, WRIGHT and STONEKING (1979) and STONEKING, MAY and WRIGHT (1979). AAT: Three loci have previously been reported in salmonid species. The two muscle loci, AAT-(I,2), have been reported to be variable in SaZveZinus (MAY, WRIGHT and STONEKING 1979), Oncorhynchus (MAY 1975; MAY, UTTER and ALLENDORP 1975) and cutthroat trout (SaZmo clarki) (ALLENDORF and UTTER 1976). A single eye-specific locus, AAT-3, has been described in Oncorhynchus spp. (MAY 1975) and brook trout (STONEKING, WAGNER and HILDEBRAND 1980). We now report a liver-specific locus, AAT-4, with three alleles. Heterodimeric bands are observed following electrophoresis of liver extracts that result from combinations of the protein products of AAT-4 and AAT-3, or the 710 B. MAY, M. STONEKING A N D J. E. WRIGHT, J R . TABLE 2 Enzymes*, loci, tissues and buffers used during this invesiigaiion of joint segregation of biochemical loci in Salvelinus Enzyme E.C. no. Aspartate aminotransferase