Characterization of polymorphic microsatellite markers and genetic diversity in wild bronze featherback, Notopterus notopterus (Pallas, 1769)

Six polymorphic microsatellite DNA loci were identified in the primitive fish, bronze featherback, Notopterus notopterus for the first time and demonstrated significant population genetic structure. Out of the six primers, one primer (NN90) was specific to N. notopterus (microsatellite sequence within the RAG1 gene) and five primers were product of successful cross-species amplification. Sixty-four primers available from 3 fish species of order Osteoglossiformes and families Notopteridae and Osteoglossidae were tested to amplify homologous microsatellite loci in N. notopterus. Fifteen primer pairs exhibited successful cross-priming PCR product. However, polymorphism was detected only at five loci. To assess the significance of these six loci (including NN90) in population genetic study, 215 samples of N. notopterus from five rivers, viz Satluj, Gomti, Yamuna, Brahmaputra and Mahanadi were analyzed. The five sample sets displayed different diversity levels and observed heterozygosity ranged from 0.6036 to 0.7373. Significant genotype heterogeneity (P < 0.0001) and high F_ST (0.2205) over all loci indicated that the samples are not drawn from the same genepool. The identified microsatellite loci are promising for use in fine-scale population structure analysis of N. notopterus.     

Evaluation of Genetic Variation in the Clown Knifefish, Chitala chitala, Using Allozymes, RAPD, and Microsatellites

Twenty-seven enzyme systems, six random amplified polymorphic DNA (RAPD) primers, and two microsatellite loci were tested to determine intraspecific divergence in the natural population of the endangered Indian featherback fish, Chitala chitala, for the first time. The 262 samples of C. chitala were collected from six riverine locations in India: the Satluj, Ganga (Ghagra, Bhagirathi, and Brahmaputra), Mahanadi, and Narmada river systems. The analysis revealed population subdivisions, with an F_ST value from 0.1235 (95% confidence 0.0868–0.1621) for RAPD and a combined F_ST of 0.0344 (95% confidence 0.0340–0.0350) for microsatellite loci. An analysis of 38 allozyme loci did not reveal any polymorphism in the samples from any of the riverine localities; a possible explanation for this could be that the ancestors of Chitala could have faced a population reduction in prehistoric periods, as low allozyme variation is also reported for other species of Chitala from south Asia.     

Isolation and characterization of polymorphic microsatellite loci in yellowtail catfish, Pangasius pangasius (Hamilton, 1822)

A total of nine polymorphic microsatellite loci were obtained from a genomic library of Pangasius pangasius (order Siluriformes, family Pangasiidae). Samples from rivers Bhagirathi (n = 22) and Mahanadi (n = 20) were genotyped for each of the nine microsatellite loci to determine genetic variation. The mean number of alleles per locus was 5.22 in Bhagirathi and 5.78 in Mahanadi; and expected heterozygosity ranged from 0.567 (Bhagirathi) to 0.578 (Bhagirathi). Significant deviation (P < 0.003) from Hardy–Weinberg expectations was evident at three loci, Ppa2 (Bhagirathi), Ppa14 (Mahanadi) and Ppa28 (Bhagirathi and Mahanadi). The identified microsatellite loci were found to be promising for population genetics studies of P. pangasius.     

An analysis of allozyme variation in herring <Emphasis Type="Italic">Clupea pallasii</Emphasis> from the White and Barents Seas

An analysis of genetic variation is made according to four allozyme loci of reproductive (spawning) groups of oligovertebrate herring Clupea pallasii collected in various bays of the White Sea and the south-eastern part of the Barents Sea in 1995–2002. The temporal stability of genetic characteristics during several years is shown. The analysis of genetic variation revealed a significant difference between herring from the south-eastern part of the Barents Sea and spring-spawning and summer-spawning herring from inner bays of the White Sea. The analysis of geographic variation of genetic characteristics of spawning aggregations revealed the change in frequencies of alleles of loci LDH-2* and MDH-4* from the north-east to the south-west along the coast.     

Phylogeography of the African catfish,Chrysichthys maurus (Valenciennes, 1839)

Polymorphism at 19 allozyme loci was studied in 227 individuals of the catfish, Chrysichthys maurus (Valenciennes, 1839), collected from ten populations representing nine different river basins in west Africa. Eleven of the loci studied were polymorphic. Analysis showed that populations clustered according to their geographical origin: eastern populations close to the root represented by a population of a closely related species (C. auratus); then populations from the centre of the species' range, followed by the more western populations. Networks observed, regression of genetic distances versus geographical distances, results of the Mantel test as well as the apomorphic status of two alleles suggested that the genetic differentiation corresponds to an isolation by distance model. These results strongly suggest that the populations initially occurred in the lagoons and rivers of Ivory Coast and from here spread to the western part of the species' range by progressively colonising basins from east to west.     

De novo development and characterization of polymorphic microsatellite markers in a schilbid catfish, <Emphasis Type="Italic">Silonia silondia</Emphasis> (Hamilton, 1822) and their validation for population genetic studies

The stock characterization of wild populations of Silonia silondia is important for its scientific management. At present, the information on genetic parameters of S. silondia is very limited. The species-specific microsatellite markers were developed in current study. The validated markers were used to genotype individuals from four distant rivers. To develop de novo microsatellite loci, an enriched genomic library was constructed for S. silondia using affinity–capture approach. The markers were validated for utility in population genetics. A total number of 76 individuals from four natural riverine populations were used to generate data for population analysis. The screening of isolated repeat sequences yielded eleven novel polymorphic microsatellite loci. The microsatellite loci exhibited high level of polymorphism, with 6–24 alleles per locus and the PIC value ranged from 0.604 to 0.927. The observed (Ho) and expected (He) heterozygosities ranged from 0.081 to 0.84 and 0.66 to 0.938, respectively. The AMOVA analysis indicated significant genetic differentiation among riverine populations (overall F_ST = 0.075; P < 0.0001) with maximum variation (92.5 %) within populations. Cross-priming assessment revealed successful amplification (35–38 %) of heterologous loci in four related species viz. Clupisoma garua, C. taakree, Ailia coila and Eutropiichthys vacha. The results demonstrated that these de novo polymorphic microsatellite loci are promising for population genetic variation and diversity studies in S. silondia. Cross-priming results indicated that these primers can help to get polymorphic microsatellite loci in the related catfish species of family Schilbidae.     

Evidence for spontaneous polyploidization in cassava Manihot esculenta Crantz

Since high levels of genetic diversity may ensure long-term survival of a plant species, it is essential to preserve the genetic diversity of the species. Tipularia japonica and Epipactis papillosa are rare terrestrial orchids in southern Korea with fewer than 50 mature individuals in a population and southern Japan and considered to be threatened (endangered or vulunerable). To obtain knowledge of how the genetic variation of these species is partitioned within and among populations in Korea, I used enzyme electrophoresis to examine the genetic diversity of each eight known populations of the two species from South Korea. Twenty-three (E. papillosa) and 24 putative loci (T. japonica) resolved from 15 enzyme systems revealed no variation either within or among populations of each species (0.0% of the percentage of polymorphic loci, %P). Previous studies, in contrast, showed that their more widely distributed disjunct congeners T. discolor and E. helleborine harbored high allozyme-based genetic diversity within populations in eastern United States (%P = 75%) and in Denmark (%P = 73.6%), respectively. In theory, small population size leads to allelic fixation at many loci over generations within a population, resulting in population genetic divergence or differentiation. In this regard, the complete lack of genetic differences between conspecific populations of T. japonica and E. papillosa cannot be explained by genetic drift. Instead, the present allozyme data suggest that recent origin from the same genetically depauperate ancestral or source population could result in this observation. The current status of T. japonica and E. papillosa (rarity and lack of genetic variation) significantly threatens the long-term survival of the species in Korea.     

Intra- and interpopulation variability of southwestern Kamchatka sockeye salmon Oncorhynchus nerka inferred from the data on single nucleotide polymorphism

The variability of 45 single nucleotide polymorphism (SNP) loci was studied in nine samples of the sockeye salmon Oncorhynchus nerka from the rivers of southwestern Kamchatka. The Wahlund effect, gametic disequilibrium at some loci, and a decrease in interpopulation genetic diversity indices observed in samples from the Bolshaya River outlet can be attributed to the samples’ heterogeneity. Partitioning of the mixed samples using some biological characteristics of the individuals led to a noticeable decrease in the frequency of these phenomena. It was demonstrated that the allelic diversity between the populations within the river accounted for the larger part of genetic variation, as compared to the differentiation between the basins. The SNP loci responsible for intra- and interpopulation differentiation of sockeye salmon from the rivers of southwestern Kamchatka were identified. Some recommendations for field population genetic studies of Asian sockeye salmon were formulated.     

Australian lungfish (Neoceratodus forsteri: Dipnoi) have low genetic variation at allozyme and mitochondrial DNA loci: a conservation alert?

Genetic variation at allozyme and mitochondrialDNA loci was investigated in the Australianlungfish, Neoceratodus forsteri Krefft1870. Tissue samples for genetic analysis weretaken non-lethally from 278 individualsrepresenting two spatially distinct endemicpopulations (Mary and Burnett rivers), as wellas one population thought to be derived from ananthropogenic translocation in the 1890's(Brisbane river). Two of 24 allozyme lociresolved from muscle tissue were polymorphic.Mitochondrial DNA nucleotide sequence diversityestimated across 2,235 base pairs in each of 40individuals ranged between 0.000423 and0.001470 per river. Low genetic variation atallozyme and mitochondrial loci could beattributed to population bottlenecks, possiblyinduced by Pleistocene aridity. Limited geneticdifferentiation was detected among rivers usingnuclear and mitochondrial markers suggestingthat admixture may have occurred between theendemic Mary and Burnett populations duringperiods of low sea level when the drainages mayhave converged before reaching the ocean.Genetic data was consistent with theexplanation that lungfish were introduced tothe Brisbane river from the Mary river. Furtherresearch using more variable genetic loci isneeded before the conservation status ofpopulations can be determined, particularly asanthropogenic demands on lungfish habitat areincreasing. In the interim we recommend amanagement strategy aimed at conservingexisting genetic variation within and betweenrivers.     

Low genetic diversity and evidence of population structure among subspecies of Nerodia harteri, a threatened water snake endemic to Texas

Nerodia harteri is a threatened small-bodied water snake that occupies one of the most restricted ranges of any snake within the continental United States. It is found closely associated with rivers and tributaries in the Colorado and Brazos river basins, which flow through north-central Texas. Nerodia harteri has been at the center of debate owing to conflicts between conservation efforts and the construction of dams that change or destroy its preferred habitat. Additionally, its taxonomic status has also been under contention with some authors recognizing two subspecies, the Brazos water snake (N. h. harteri) and the Concho water snake (N. h. paucimaculata), whereas other authors consider each separate species. Despite its relatively recent discovery during the 1940s, N. harteri has been the subject of several ecological studies, yet no population genetic assessment of either subspecies has been performed to date. We first evaluated the phylogenetic placement of both subspecies among other North American Nerodia using partial sequence data from the mitochondrial gene cytochrome-b. We then tested for population subdivision among four rivers encompassing the range of N. harteri and tested for the presence of admixture between river basins using mitochondrial sequence data (920?bp of cyt-b) and five cross-species amplified microsatellite loci. We found low mitochondrial haplotype diversity represented by two unique haplotypes in each river basin, which were separated by no more than four nucleotide changes. Nuclear loci showed low genetic diversity and population structuring within and among river basins. We did not find conclusive evidence of admixture between basins, and we support the presence of two separate evolutionarily significant units and two separate management units corresponding to each major river basin. Given increasing natural and anthropogenic threats, we recommend continued ecological and genetic monitoring of both subspecies.     

Evidence for Two Highly Differentiated Herring Groups at Goose Bank in the Barents Sea and the Genetic Relationship to Pacific Herring, Clupea pallasi

Genetic studies on Atlantic herring, Clupea harengus, have generally revealed a low level of genetic variation over large geographic areas. Genetically distinct herring populations in some of the Norwegian fjords are exceptions, and juvenile herring from the large oceanic herring, Norwegian Spring Spawners (NSS), are often found in mixture with local fjord populations as well as widely distributed in the Barents Sea. Research surveys in the eastern Barents Sea (Goose Bank) in 1993, 1994 and 2001 included collection of herring samples for allozyme analyses. As expected the results identified juveniles from NSS stock, but an additional unique group of herring (low vertebrae number), being almost fixed for alternative alleles at several allozyme loci, was detected. In some cases, the two groups of herring were taken in the same trawl catches as documented by highly significant departure from Hardy—Weinberg expectation with large excess of homozygotes providing evidence for population mixing. Large genetic differences (Nei's genetic distance = 1.53; F_ST = 0.754) were detected in pairwise comparisons based on five allozyme loci. The two herring groups were also compared with reference samples of Pacific herring, Clupea pallasi, including one sample from Japan Sea and three Alaskan samples. UPGMA dendrogram based on five allozyme loci revealed a close genetic relationship between the low vertebrae herring in the Barents Sea and the group of samples of Pacific herring. Although significant different in allele frequencies, one of the herring samples clustered together with the reference sample from Bering Sea with genetic distance of 0.008 and F_ST value of 0.032. The close genetic relationship found in this paper, suggest a re-evaluation of the taxonomic status of the Barents Sea herring populations investigated.     

Genetic Population Structure of Pacific Hake, Merluccius productus, in the Pacific Northwest

This study presents the first protein electrophoretic study of population structure within the Georgia Basin Pacific hake Distinct Population Segment, as defined under the U.S. Endangered Species Act. Forty-one allozyme loci (29 polymorphic) were analyzed in samples from three Pacific hake spawning populations on the west coast of North America: (1) Port Susan, Puget Sound, Washington (three temporal samples); (2) south-central Strait of Georgia, British Columbia, Canada (two temporal samples); and (3) offshore of southern California (two temporal samples) (total n = 664). Mean heterozygosity over all loci was 12–13% for all populations. Within-population temporal samples were not significantly different from one another, but statistically significant differences were detected at 15 of the 29 polymorphic loci (p < 0.05) among the three populations. Differences at eight of these loci were highly significant (p < 0.001): ADA ^*, ALAT ^*, bGALA ^*, GPI-A ^*, sIDHP ^*, LDH-A ^*, MPI ^*, and PEP-B ^*. The two Georgia Basin populations were significantly different at six loci: bGALA ^*, sIDHP ^*, LDH-A ^*, MPI ^*, PGK ^*, and PGM-2 ^* (p < 0.05). Nei's genetic distance (D) was 0.0006 between Port Susan and Strait of Georgia pooled temporal samples, and 0.005 between these populations and offshore Pacific hake. F_ST was 0.02 and 0.0046 among all three populations and among the Georgia Basin populations, respectively. Both F_ST estimates were significantly greater than zero, and the results suggest a high degree of demographic isolation among all three populations.     

Population genetic structure of three major river Populations of rohu,Labeo rohita (Cyprinidae: Cypriniformes) using microsatellite DNA markers

Determining the genetic structure is essential for developing conservation and stock improvement plans. Four dinucleotide microsatellite loci were analysed to reveal population genetic structure of the Indian major carp,Labeo rohita collected from three major rivers namely the Halda, the Jamuna, and the Padma in Bangladesh. The four loci were polymorphic (P ₉₅) in all the populations. The populations varied in the number and frequencies of alleles as well as heterozygosities in the loci analyzed. Population differentiation (F _ST) value between the Halda and the Jamuna population was significant (P<0.05). Relatively high level of gene flow and low level ofF _ST values were found between the Padma and the Jamuna population. The unweighted pair group method with averages (UPGMA) dendrogram based on genetic distance resulted in two clusters: the Halda population was alone in one cluster whereas the Jamuna and the Padma made another cluster. The results revealed a relatively low level of genetic variability in the river populations ofL. rohita in Bangladesh.     

Genetic divergence in wild population of <Emphasis Type="Italic">Labeo rohita</Emphasis> (Hamilton, 1822) from nine Indian rivers,analyzed through MtDNA cytochrome b region

The present study examined partial cytochrome b gene sequence of mitochondrial DNA for polymorphism and its suitability to determine the genetic differentiation in wild Labeo rohita. The 146 samples of L. rohita were collected from nine distant rivers; Satluj, Brahmaputra, Son, Chambal Mahanadi, Rapti, Chauka, Bhagirathi and Tons were analyzed. Sequencing of 307 bp of Cyto b gene revealed 35 haplotypes with haplotype diversity 0.751 and nucleotide diversity (π) 0.005. The within population variation accounted for 84.21% of total variation and 15.79% was found to among population. The total Fst value, 0.158 (P < 0.05) was found to be significant. The results concluded that the partial cyto b is polymorphic and can be a potential marker to determining genetic stock structure of L. rohita wild population.     

Local-scale genetic structure in the Japanese wild boar (<Emphasis Type="Italic">Sus scrofa leucomystax</Emphasis>): insights from autosomal microsatellites

The Japanese wild boar (Sus scrofa leucomystax) is one of the most widely distributed mammals in Japan. However, its population structure and pattern of gene flow at a regional level are poorly understood. In this study, we investigated the local-scale genetic structure of the Japanese wild boar. In total, 172 individuals sampled in Gifu Prefecture, central Japan, were genotyped for 29 autosomal microsatellite loci. Significant genetic differentiations (F _ST = 0.020–0.128) were detected among some geographical areas. In addition, in the overall population (n = 172), all loci deviating from the Hardy–Weinberg equilibrium exhibited a significant deficit of heterozygotes. These results suggest the presence of genetic substructuring in the local population. Moreover, Bayesian cluster analysis revealed the presence of substructuring within the population, despite the relatively small study area (10,621 km²). Spatial Bayesian cluster analysis showed that the boundaries of subpopulations were generally consistent with landscape features (e.g. main rivers, urban areas and road and train networks). Our study implies that these landscape features play a significant role as a barrier to dispersal and gene flow in the local population of the Japanese wild boar.     

Allozyme variability in populations of trout (<Emphasis Type="Italic">Salmo trutta</Emphasis>) from the rivers of Iran

For the first time, an analysis was carried out of allozyme variability in trout (Salmo trutta) from three rivers of Iran. We studied 23 gene loci coding enzymes: glycerol-3-phosphate dehydrogenase (G3PDH), aspartate aminotransferase (AAT), malate dehydrogenase (MDH), lactate dehydrogenase (LDH), creatine kinase (CK), malic enzyme [NADP-dependent MDH] (MEP), superoxide dismutase (SOD), esterase (EST), and esterase D (EST-D). The obtained data demonstrate the similarity between the trout samples from different rivers of Iran according to genetic characteristics. Taking into account the differences by allozyme markers of allele frequencies and allele composition of some loci, we should expect that Iranian trout diverges significantly in genetics from the other trout populations of the Caspian Sea.     

Genetic differentiation among sea trout, Salmo trutta L., populations from western Norway

Samples of sea trout, Salmo trutta L., were collected by electrofishing in eight rivers of western Norway. The samples consisted mainly of presmolt, but also contained some spawners. Muscle, eye and liver tissue were sampled and analysed for allozyme polymorphism by standard starch gel electrophoresis. Nine polymorphic loci were found. The genotype as well as the allele distributions was significantly different in all but six pair‐wise comparisons, showing the high degree of population differentiation in this anadromous species. Even some nearby rivers displayed very different allele frequencies. The correlation between genetic and geographic distances was nearly significant.     

A triose-phosphate isomerase polymorphism in the Atlantic salmonSalmo salar L.

Atlantic salmon,Salmo salar L., from four European locations show allelic variation at one of three triose-phosphate isomerase (TPI) loci (TPI-3*) when separated on horizontal starch gel electrophoresis, using either eye or liver extracts. Two common alleles (*100 and*103) and one rare allele (*97) segregate atTPI-3* with unambiguous typing being possible by observing the interlocus heterodimers. Family studies demonstrate thatTPI-3* 100 and*103 are of autosomal location and are inherited in a Mendelian fashion.TPI-3* variation can also be typed in adipose fin tissue, allowing nondestructive tissue sampling. Three loci are also active in brown trout,Salmo trutta, with two individuals being homozygous forTPI-3*, as are a small number ofS. salar from eastern Canada. The presence of this additional variable allozyme locus inS. salar is important, since genetic studies in that species have been limited by the low level of allozyme variability detectable.     

Evaluation of cytochrome b mtDNA sequences in genetic diversity studies of <Emphasis Type="Italic">Channa marulius</Emphasis> (Channidae: Perciformes)

Channa marulius (Hamilton, 1822) is a commercially important freshwater fish and a potential candidate species for aquaculture. The present study evaluated partial Cytochrome b gene sequence of mtDNA for determining the genetic variation in wild populations of C. marulius. Genomic DNA extracted from C. marulius samples (n = 23) belonging to 3 distant rivers; Mahanadi, Teesta and Yamuna was analyzed. Sequencing of 307 bp Cytochrome b mtDNA fragment revealed the presence of 5 haplotypes with haplotype diversity value of 0.763 and nucleotide diversity value of 0.0128. Single population specific haplotype was observed in Mahanadi and Yamuna samples and 3 haplotypes in Teesta samples. The analysis of data demonstrated the suitability of partial Cytochrome b sequence in determining the genetic diversity in C. marulius population.     

Bottleneck in the endangered kalibaus, Labeo calbasu (cyprinidae: cypriniformes) populations in Bangladesh revealed by microsatellite DNA marker analysis

Microsatellite DNA marker analysis was carried out to assess the population genetic structure of an endangered carp, Labeo calbasu, collected from three different stocks; the Jamuna River, the Halda River and a Hatchery. Four heterologous microsatellite loci (Lr12, Lr14b, Lr21 and Lr24) identified from rohu (Labeo rohita) were analyzed to test the genetic variability of the target kalibaus stocks. The maximum number of alleles observed in loci Lr12, Lr14b, Lr21 and Lr24 were 10, 7, 8 and 6, respectively. The loci were found to be polymorphic (<P ₉₅) in all the populations. The average number of allele was highest in the Jamuna population (6.75) followed by that of the Halda (5.50) and the Hatchery population (4.25). The observed average heterozygosity (Ho) value was almost similar in all three populations. Except locus Lr12 in the Halda population, significant deviations from the Hardy-Weinberg Equilibrium were detected in all cases due to excess heterozygosity. The population differentiation values (F _ST ) between all the population pairs were significant. The highest genetic distance value (D = 0.295) was measured between the Halda and the Hatchery populations. A recent bottleneck was observed in the Halda and the Hatchery population.