Detection of an NAD+-dependent malic enzyme locus in the atlantic salmonSalmo salar and other salmonid fish

Electrophoretic studies of malate oxidoreductases routinely assess variation in two enzymes, malate dehydrogenase (EC 1.1.1.37) and malic enzyme (NADP⁺) (EC 1.1.1.40). By modification of the standard isozyme staining conditions for these enzymes, we have resolved a new NAD⁺-preferring, MgCl₂-requiring malic enzyme which is indicated to be EC 1.1.1.39. The enzyme was detected in 10 salmonid fish species of the generaSalmo, Salvelinus, andOnchoryhncus. Phenotypic variation indicates that the novel enzyme is tetrameric and coded by a single locus. Inheritance inS. salar follows a single-locus model and the phenotypes are unlinked to polymorphisms for_s MDH-3,4* and_m MEP-2*, two malate oxidoreductase loci previously shown to be variable in this species.This work was supported by a contract to E. V. from Fisheries and Oceans Canada, St. John's, Newfoundland, and a postgraduate award to W. C. J. from the Department of Education for Northern Ireland.     

Evolutionary relationship ofHLA-DRB genes inferred from intron sequences

The major histocompatibility complex (Mhc) consists of class I and class II genes. In the humanMhc (HLA) class II genes, nineDRB loci have been identified. To elucidate the origin of these duplicated loci and allelic divergences at the most polymorphicDRBI locus, introns 4 and 5 as well as the 3′ untranslated region (altogether approximately 1,000 base pairs) of sevenHLA-DRB loci, threeHLA-DRBI alleles, and nine nonhuman primateDRB genes were examined. It is shown that there were two major diversification events inHLA-DRB genes, each involving gene duplications and allelic divergences. Approximately 50 million years (my) ago,DRBI ^*04 and an ancestor of theDRB1 ^*03 cluster (DRBI ^*03, DRBI^*15, andDRB3) diverged from each other andDRB5, DRB7, DRB8, and an ancestor of theDRB2 cluster (DRB2, DRB4, andDRB6) arose by gene duplication. Later, about 25 my ago,DRBI ^*15 diverged fromDRBI*03, andDRB3 was duplicated fromDRBI ^*03. Then, some 20 my ago, the lineage leading to theDRB2 cluster produced two new loci,DRB4 andDRB6. TheDRBI ^*03 andDRBI ^*04 allelic lineages are extraordinarily old and have persisted longer than some duplicated genes. The orthologous relationships ofDRB genes between human and Old World monkeys are apparent, but those between Catarrhini and New World monkeys are equivocal because of a rather rapid expansion and contraction of primateDRB genes by duplication and deletion. Correspondence to: Y. Satta     

Sex linkage in salmonids: Evidence from a hybridized genome of brook trout and arctic charr

In second-generation sparctics (Salvelinus fontinalis × Salvelinus alpinus) backcrossed toS. fontinalis, we have identified tight classical linkage of phenotypic sex withLdh-1, Aat-5, andGpi-3. We designate this locusSex-1 and suggest that it may be the primary sex-determining locus in salmonids. Cumulative salmonid gene-to-centromere map distances for the three biochemical loci put the order as centromere—Ldh-1—(Aat-5 andGpi-3), with the latter two loci being tightly linked. An absence of association of phenotypic sex (presumably Sex-1) with these same three loci and other loci known to be linked to these loci is shown in splakes (S. fontinalis × Salvelinus namaycush) and cutbows (Salmo gairdneri × Salmo clarki). These data imply that the linkage ofSex-1 with these loci is found only inS. alpinus and support the view thatSex-1 lies across the centromere from these three loci inS. alpinus, representing a Robertsonian fusion not found in any of the other four species. A similar specific Robertsonian fusion is argued forS. gairdneri, whereSex-1 may be linked across a centromere to another biochemical locus (Ha). These linkage results and chromosomal observations of other investigators suggest thatSex-1 lies on an information-depauperate arm.     

Phylogenetic origin of <Emphasis Type="Italic">Salmo trutta</Emphasis> L 1758 from Sicily,based on mitochondrial and nuclear DNA analyses

The phylogenetic status of brown trout Salmo trutta L 1758 in Sicily is uncertain as some reports describe these trout as S. macrostigma or S. cettii on one hand while other, contradictory reports imply a hatchery origin on the other. In order to clarify this situation, we performed sequence analysis of the mtDNA control region and restriction fragment analysis of the nuclear lactate dehydrogenase (LDH-C1*) gene. A single mitochondrial haplotype (At-s6) found previously in brown trout in Morocco, and two alleles at LDH-C1* (the ancestral*100, at a high frequency, and *90) were revealed. Our results suggest that Sicilian brown trout are native and that they probably colonized Sicily from west to east in an expansion, from the Atlantic Ocean basin, along the North-West African coast. Handling editor: C. Sturmbauer     

Genetic variability inCebus appella paraguayanus: Biochemical analysis of seven loci and variation in Glyoxalase I (E.C.4.4.1.5)

An analysis of seven loci inCebus apella paraguayanus showed that Glyoxalase I was polymorphic due the appearance of two alleles (GLO*2 andGLO*3) with frequencies of 0.955 and 0.045, respectively. Of the two alleles,GLO*2 was electrophoretically similar to the most common allele found in the human andAotus. These results confirmed our previous findings in the same population sample showing that this subspecies has a very low genetic variation among New World primates.     

Inheritance of allozyme loci inBombina: One linkage group established

Two hybridizing European species of fire-bellied toads,Bombina bombina andB. variegata, have alternate electromorphs fixed at a number of allozyme loci. Segregation of alleles at seven allozyme loci (Ldh-1, Mdh-1, Ak, Ck, Gpi, Np, andEst-) was studied in a backross progeny of an F₁ interspecific hybrid male and aB. bombina female. Mendelian inheritance of allozyme forms at all seven loci was ascertained. Except for two loci,Gpi andEst-, which were found to be tightly linked (1 cM apart), other loci showed independent segregation.     

Interspecific Relationships among Charrs Based on Phylogenetic Analysis of Nuclear Growth Hormone Intron Sequences

DNA samples were obtained from six charr species: Salvelinus alpinus, S. malma, S. confluentus, S. leucomaenis, S. namaycush and S. fontinalis and two outgroups: Salmo salar and S. trutta. The C and D introns from the duplicate growth hormone loci (GH1 and GH2) were amplified using the polymerase chain reaction and sequenced from each of the species. Phylogenetic analyses with and without gaps were performed using the beta version of PAUP. The GH introns are evolving relatively slowly, so the data were combined with sequences from the ITS1 and ITS2 for a phylogenetic analysis of the charr species. The GH intron data and the combined data supported two groups of sister species among charrs: S. alpinus and S. malma, and S. confluentus and S. leucomaenis.     

Contribution of genetic distances studies to the taxonomy ofAteles,particularlyAteles paniscus paniscus andAteles paniscus chamek

We studied 20 electrophoretic loci in two populations ofAteles (Ateles paniscus paniscus andAteles paniscus chamek). We observed intrapopulational variation at the following loci: esterase D, glyoxalase 1, adenosine deaminase (A. p. chamek) and carbonic anhydrase 2 (A. p. paniscus). The two populations share the most frequent alleles at 17 loci, but we noted great differences in glyoxalase 1, adenosine deaminase and phosphoglucomutase 1.A. p. paniscus is monomorphic for theGLO1 ^*1 allele, which has a frequency of 6% inA. p.chamek. They did not share alleles in relation to the ADA and PGM1 loci. We found a CA2 allele, named hereCA2 ^*1, which has not been described previously in other neotropical primates (Sampaio et al., 1991a), inA. p. paniscus. The present results suggest that the geographical isolation represented by the Rio Amazonas has lasted long enough to support this level of divergence. These observations taken together with chromosomal findings, led us to endorse the proposal of two distinct species:Ateles paniscus andAteles chamek.     

Genetic variation and hybridization in SwedishSchoenus (Cyperaceae)

Schoenus ferrugineus andS. nigricans have restricted distributions in Sweden and are almost exclusively confined to calcareous fen habitats. AtS. nigricans sites,S. ferrugineus is usually also present, and hybrids are frequently found. In this report, I used allozymes to estimate the amount of gene flow between the two species, and to compare the partitioning of genetic diversity in each of them. Thirteen loci were analysed at eight different enzyme systems. Seven loci were variable between or within the species. The two species had completely different alleles at two of the seven variable loci, whereas there was overlap at five loci. In all, 22 different alleles were found. Six of these alleles were confined toS. nigricans, and five alleles were confined toS. ferrugineus. Nei's genetic identity was 0.55.—InS. ferrugineus, three loci (23%) were polymorphic, and the average number of alleles per polymorphic locus was 2.0 (each polymorphic locus had two alleles). InS. nigricans, three loci (23%) were polymorphic, and the average number of alleles per polymorphic locus was 2.3.—The proportion of genetic diversity due to variation among sites (G _ST) was fairly similar in the two species, mean over loci = 0.12 inS. ferrugineus and 0.15 inS. nigricans. However, the proportion of genetic diversity due to variation among individuals within sites (G _IS) differed markedly between the two species, mean over loci = 0.54 inS. ferrugineus and 0.17 inS. nigricans. Accordingly, there was a much higher individual heterozygosity inS. nigricans than inS. ferrugineus. — Most hybrids were interpreted as F₁ hybrids. However, a small proportion, 0.5–1.6 %, were F_n hybrids or back-crosses.—On the Swedish mainland, all former occurrences ofS. nigricans are extinct, but viable hybrids are still present at a few sites in southernmost Sweden.     

Allozyme polymorphisms within and among open-pollinated and adapted exotic populations of maize

Summary Twelve U.S. Corn Belt open-pollinated and five adapted exotic populations of maize (Zea mays L.) were assayed for allozyme (allele) variation at 13 enzyme marker loci. Extensive allozyme variability was observed in all populations studied. No locus was monomorphic over all populations. Each of the lociIdh2, Got1, Mdh2, Pgd1, andPgd2 expressed two allozymes over all populations,Adh1, Acp1, Prx1, andEst1 each had three allozymes present,Est4, Glu1, andEnp1 had five allozymes, andAcp4 had six allozymes present. Significant deviations of genotypic frequencies were detected from Hardy-Weinberg equilibrium frequencies and 94% of average Fixation Index values indicated heterozygote deficiencies, which suggested that nonrandom mating and/or natural selection favoring homozygotes were possible factors affecting the maintenance or loss of genetic variability marked by these enzyme loci. Genetic distance and cluster analyses indicated that the observed genetic variability at the 13 enzyme loci was closely related to Dent and Flint types of maize.     

Role of nonclassical class I genes of the chicken major histocompatibility complex Rfp-Y locus in transplantation immunity

The chicken major histocompatibility complex (MHC) genes are organized into two genetically independent clusters which both possess class I and class II genes: the classical B complex and the Restriction fragment pattern-Y (Rfp-Y) complex. In this study, we have examined the role of Rfp-Y genes in transplantation immunity. For this we used three sublines, B19H1, B19H2 and B19H3, derived from a line fixed for B19. Southern blots, PCR-SSCP assays using primers specific for Rfp-Y genes, and Rfp-Y class I allele-specific sequencing show that the polymorphisms observed in B19H1, B19H2 and B19H3 are due to the presence of three different Rfp-Y haplotypes. The Rfp-Y class I (YF) alleles in these three haplotypes are highly polymorphic, and RT-PCR shows that at least two YF loci are expressed in each subline. The three sublines show Rfp-Y-directed alloreactivity in that Rfp-Y-incompatible skin grafts are rejected within 15 days, a rate intermediate between that seen in B-incompatible rejection (7 days) and that observed for grafts within the sublines (20 days). We conclude that Rfp-Y has an intermediate role in allograft rejection, likely to be attributable to polymorphism at the class I loci within this region.The sequence data reported are available in the GenBank database under the accession numbers AY257165 (YFVw*15), AY257166 (YFVw*16), AY257167 (YFVIw*15), AY257168 (YFVIw*17), AY257169 (YFw*16), and AY257170 (YFw*17)     

Allozyme divergence among species ofWolffia (Lemnaceae)

The genusWolffia was surveyed electrophoretically at 14 allozyme loci. A total of 133 clones representing 10 of the 11 recognized species was examined. Genetic identities among most pairs of species are zero, with non-zero values ranging from 0.14 to 0.40.Wolffia angusta and the newly describedW. neglecta show the highest similarity, and the former species has an identity of 0.14 withW. australiana. The next highest similarity (0.34) occurs betweenW. globosa of Southeast Asia andW. cylindracea of southern Africa, which until recently, had generally been viewed as members of the same species. Other species showing some common alleles are members of a complex involvingW. arrhiza, W. columbiana, W. cylindracea, andW. globosa. WithinW. arrhiza, plants from South Africa and Europe are easily distinguished electrophoretically because each contains unique alleles at two loci. Strains from other parts of Africa vary at these loci and are not totally distinct from either the plants from South Africa or from Europe. Species ofWolffia are much more divergent at allozyme loci than the majority of congeners of flowering plants. This suggests that the species are quite old and that the difficulties in distinguishing taxa morphologically are the result of reduction rather than lack of divergence due to recent speciation. Because of the lack of shared alleles between the majority of species pairs inWolffia, enzyme electrophoresis provides limited resolution of species relationships in the genus.     

Analysis of F ST outliers at allozyme loci in Pacific salmon: implications for natural selection

Natural selection has been invoked to explain the observed geographic distribution of allozyme allele frequencies for a number of teleost species. The effects of selection on allozyme loci in three species of Pacific salmon were tested. A simulation-based approach to estimate the null distribution of population differentiation (F _ST) and test for F _ST outliers was used. This approach showed that a majority of allozyme loci conform to neutral expectations predicted by the simulation model, with relatively few F _ST outliers found. No consistent F _ST outlier loci were found across species. Analysis of population sub-groups based on geography and genetic identity reduced the number of outlier loci for some species, indicating that large geographic groups may include genetically divergent populations and/or that there is geographic heterogeneity in selection pressure upon allozyme loci. Two outlier allozyme loci found in this analysis, lactate dehydrogenase-B and malic enzyme, have been shown to be influenced by selection in other teleost species. This approach is also useful in identifying allozyme loci (or other genetic markers) that meet assumptions for population genetic study.     

Comparison of allozyme,RFLP, and RAPD markers for revealing genetic variation within and between trembling aspen and bigtooth aspen

We examined genetic variation in allozyme loci, nuclear DNA restriction fragment length polymorphisms (RFLPs), and random amplified polymorphic DNAs (RAPDs) in 130 trembling aspen (Populus tremuloides) and 105 bigtooth aspen (P. grandidentata) trees. In trembling aspen 10 out of 13 allozyme loci assayed (77%) were polymorphic (P), with 2.8 alleles per locus (A) and an expected heterozygosity (H_e) of 0.25. In contrast, bigtooth aspen had a much lower allozyme genetic variability (P=29%; A=1.4; H_e=0.08). The two species could be distinguished by mutually exclusive alleles at Idh-1, and bigtooth aspen has what appears to be a duplicate 6PG locus not present in trembling aspen. We used 138 random aspen genomic probes to reveal RFLPs in HindIII digests of aspen DNA. The majority of the probes were from sequences of low copy number. RFLP results were consistent with those of the allozyme analyses, with trembling aspen displaying higher genetic variation than bigtooth aspen (P=71%, A=2.7, and H_e=0.25 for trembling aspen; P=65%, A=1.8, and H_e=0.13 for bigtooth aspen). The two species could be distinguished by RFLPs revealed by 21 probes (15% of total probes assayed). RAPD patterns in both species were studied using four arbitrary decamer primers that revealed a total of 61 different amplified DNA fragments in trembling aspen and 56 in bigtooth aspen. Assuming a Hardy-Weinberg equilibrium, estimates of P=100%, A=2, and H_e=0.30 in trembling aspen and P=88%, A=1.9, and H_e=0.31 in bigtooth aspen were obtained from the RAPD data. Five amplified DNA fragments were species diagnostic. All individuals within both species, except for 2 that likely belong to the same clone, could be distinguished by comparing their RAPD patterns. These results indicate that (1) RFLPs and allozymes reveal comparable patterns of genetic variation in the two species, (2) trembling aspen is more genetically variable than bigtooth aspen at both the allozyme and DNA levels, (3) one can generate more polymorphic and species-specific loci with DNA markers than with allozymes in aspen, and (4) RAPDs provide a very powerful tool for fingerprinting aspen individuals.     

Allozyme Variation in a Threatened Freshwater Fish, Spotted Murrel (Channa punctatus) in a South Indian River System

Samples of the spotted murrel (Channa punctatus) were collected from three rivers of Tamil Nadu and Kerala. The allozyme variation of C. punctatus was investigated by polyacrylamide gel electrophoresis. Eighteen enzymes were detected, but only 10 (EST, PGM, G3PDH, G6PDH, SOD, GPI, ODH, GDH, XDH, and CK) showed consistent phenotypic variations. Allele frequencies were estimated at the 18 polymorphic loci representing 10 enzymes. Two rare alleles, EST-4*C and G6PDH-2*C, were noted in the Tamirabarani and Kallada populations but were absent in the Siruvani population. The allele frequencies of the Tamirabarani and Kallada populations were similar, except for a few loci. Among the three populations, the maximum genetic distance (0.026) and FST (0.203) were found between the geographically distant Siruvani and Kallada populations. Overall the study showed that among the three populations, the Tamirabarani and Kallada have similar genetic structures.     

Studies on theStellaria longipes complex (Caryophyllaceae): Isozyme variability and the relationship betweenStellaria longipes andS. longifolia

Genetic variation within and the relationship betweenStellaria longipes Goldie andS. longifolia Muhl. were studied. Ten enzyme systems were assessed in eight natural populations ofS. longipes (25 loci) and three ofS. longifolia (20 loci) using starch and polyacrylamide gel electrophoresis. Patterns of population differentiation corresponded to geographic distance. There was no evidence that polyploidS. longipes had greater electrophoretic variability than diploidS. longipes. The isozyme data confirmed extensive population differentiation in these species and, within that context, a relatively close relationship betweenS. longipes andS. longifolia. It was postulated that diploids of these two species might be the progenitors of tetraploidS. longipes.     

Giemsa karyotypes and their evolutionary significance inScilla bifolia,S. drunensis,andS. vindobonensis (Liliaceae)

Quantitatively evaluated C-banded and conventional karyograms are presented for the related speciesScilla bifolia subsp.danubialis Speta,S. drunensis (Speta)Speta, andS. vindobonensis Speta. On the basis of banding patterns and karyotype structureS. bifolia subsp.danubialis (2n = 18, 2×) andS. drunensis (2n = 36, 4×) are quite similar, whileS. vindobonensis (2n = 18, 2×) is entirely different. There is a moderate degree of karyotypic variation withinS. bifolia subsp.danubialis andS. drunensis. However, inS. vindobonensis karyotypes and banding patterns are almost completely stable over a geographical range of about 500 km. The present results confirm the recent taxonomic separation ofS. vindobonensis fromS. bifolia, and suggest a considerable phylogenetic distance between these two diploid species. The results are discussed with reference to the morphological characters of the species and their geographical distribution.Evolution ofScilla and Related Genera, II.Dedicated to Univ.-Prof. Dr.Elisabeth Tschermak-Woess on the occasion of her 60th birthday.     

Isoenzyme variation in the wild relatives ofVicia faba (Fabaceae)

Electrophoretic analysis of five enzyme systems, LAP, PGI, SKDH, SOD and 6-PGDH, among 102Vicia accessions representingV. bithynica and seven species of theV. narbonensis complex, namelyV. eristalioides, V. kalakhensis, V. johannis, V. galilaea, V. serratifolia, V. narbonensis andV. hyaeniscyamus, has been performed. The recorded variation was tentatively assigned to 41 allelic genes at eight loci; intraspecific variation was observed in all species except forV. eristalioides. The results obtained were compared with the corresponding data reported earlier forV. faba. Hierarchical grouping of the investigated taxa, includingV. faba, was based onNei's genetic identities calculated from the allozyme frequency data.Vicia faba andV. bithynica were shown to be most distantly related to one another and to the remaining species investigated.Vicia serratifolia appeared to be a peripheral member of theV. narbonensis complex. The results are discussed with reference to genetic diversity and taxonomic relationships of the species under study.     

Isozyme evidence of relationships withinSphagnum sect.Acutifolia (Sphagnaceae,Bryophyta)

Ten enzyme systems coding for 13 putative loci were studied in 12 European species ofSphagnum sect.Acutifolia (peat mosses). On average seven plants from each of 73 collections made throughout N Europe were analysed. Despite complex morphological variation, all species expressed unique allele combinations. Principal components and cluster analyses based on allele frequencies showed that sect.Acutifolia can be divided into two major groups; one comprisingSphagnum fimbriatum, S. girgensohnii andS. molle; the second comprisingS. capillifolium, S. angermanicum, S. fuscum, S. quinquefarium, S. rubellum, S. subfulvum, S. subnitens andS. warnstorfii. These groups are linked byS. russowii, which is shown to be an allopolyploid, with fixed heterozygosity at 6 to 8 of the loci. The presumed progenitors ofS. russowii areS. girgensohnii andS. rubellum. Allelic data also indicate thatS. teres from sect.Squarrosa is related toS. girgensohnii andS. fimbriatum. The bisexual species tend to be less variable than the unisexual species.     

Phylogenetic relationships inSecale (Poaceae): An isozymatic study

The genetic frequencies of 9 isozyme loci have been estimated in 23 samples of 4 species ofSecale by means of starch gel electrophoresis. The populations ofS. silvestre andS. vavilovii were monomorphic and uniform within each species, those ofS. montanum andS. cereale were polymorphic for most of the isozyme loci. On the basis of isozyme patterns as well as allelic and genotypic frequencies of isozyme loci,S. silvestre can be distinguished fromS. vavilovii, and both fromS. cereale andS. montanum; but there is no clear differentiation between the two latter species. Clusters constructed from genetic distances separateS. silvestre andS. vavilovii, whereasS. cereale andS. montanum were grouped together. The isozymatic data presented here, along with cytogenetic and life habit data, agree with the generally admitted existence of 4 species inSecale, and support the relationships suggested byKhush & Stebbins (1961).