Isolation of an 800 kb contiguous DNA fragment encompassing a 3.5-cM region of chromosome 1 in Arabidopsis using YAC clones

The apetalal mutation of Arabidopsis affects floral meristem identity and the development of sepal and petal primordia of the flower. We mapped the available RFLP markers on chromosome 1 that are in the general vicinity of apetalal on a fine structure map and then chose the closest RFLP as a starting point for contiguous DNA (contig) generation. We report here a contig of about 800 kilobases (kb) that spans a 3.5 cM region of chromosome 1. We used genomic libraries of Arabidopsis prepared in yeast artificial chromosome (YAC) vectors and the detailed characterization of 19 YACs is reported. RFLPs displayed by the end fragments from the walk were mapped to align and correlate the genetic and physical maps for this region of chromosome 1. In this segment of the genome, 1 cM corresponds to a little over 200 kb of physical distance.

Population genetic structure and conservation of Asian elephants (Elephas maximus) across India

This study examines the population genetic structure of Asian elephants (Elephas maximus) across India, which harbours over half the world's population of this endangered species. Mitochondrial DNA control region sequences and allele frequencies at six nuclear DNA microsatellite markers obtained from the dung of free-ranging elephants reveal low mtDNA and typical microsatellite diversity. Both known divergent clades of mtDNA haplotypes in the Asian elephant are present in India, with southern and central India exhibiting exclusively the β clade of Fernando et al. (2000), northern India exhibiting exclusively the α clade and northeastern India exhibiting both, but predominantly the α clade. A nested clade analysis revealed isolation by distance as the principal mechanism responsible for the observed haplotype distributions within the α and β clades. Analyses of molecular variance and pairwise population FST tests based on both mitochondrial and microsatellite DNA suggest that northern-northeastern India, central India, Nilgiris (in southern India) and Anamalai-Periyar (in southern India) are four demographically autonomous population units and should be managed separately. In addition, evidence for female philopatry, male-mediated gene flow and two possible historical biogeographical barriers is described.

Genetic analysis of an Indian family with members affected with Waardenburg syndrome and Duchenne muscular dystrophy

Purpose: Waardenburg syndrome (WS) is characterized by sensorineural hearing loss and pigmentation defects of the eye, skin, and hair. It is caused by mutations in one of the following genes: PAX3 (paired box 3), MITF (microphthalmia-associated transcription factor), EDNRB (endothelin receptor type B), EDN3 (endothelin 3), SNAI2 (snail homolog 2, Drosophila) and SOX10 (SRY-box containing gene 10). Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder caused by mutations in the DMD gene. The purpose of this study was to identify the genetic causes of WS and DMD in an Indian family with two patients: one affected with WS and DMD, and another one affected with only WS. Methods: Blood samples were collected from individuals for genomic DNA isolation. To determine the linkage of this family to the eight known WS loci, microsatellite markers were selected from the candidate regions and used to genotype the family. Exon-specific intronic primers for EDN3 were used to amplify and sequence DNA samples from affected individuals to detect mutations. A mutation in DMD was identified by multiplex PCR and multiplex ligation-dependent probe amplification method using exon-specific probes. Results: Pedigree analysis suggested segregation of WS as an autosomal recessive trait in the family. Haplotype analysis suggested linkage of the family to the WS4B (EDN3) locus. DNA sequencing identified a novel missense mutation p.T98M in EDN3. A deletion mutation was identified in DMD. Conclusions: This study reports a novel missense mutation in EDN3 and a deletion mutation in DMD in the same Indian family. The present study will be helpful in genetic diagnosis of this family and increases the mutation spectrum of EDN3.

Differences in host species relationships and biogeographic influences produce contrasting patterns of prevalence, community composition and genetic structure in two genera of avian malaria parasites in southern Melanesia

1. Host-parasite interactions have the potential to influence broadscale ecological and evolutionary processes, levels of endemism, divergence patterns and distributions in host populations. Understanding the mechanisms involved requires identification of the factors that shape parasite distribution and prevalence. 2. A lack of comparative information on community-level host-parasite associations limits our understanding of the role of parasites in host population divergence processes. Avian malaria (haemosporidian) parasites in bird communities offer a tractable model system to examine the potential for pathogens to influence evolutionary processes in natural host populations. 3. Using cytochrome b variation, we characterized phylogenetic diversity and prevalence of two genera of avian haemosporidian parasites, Plasmodium and Haemoproteus, and analysed biogeographic patterns of lineages across islands and avian hosts, in southern Melanesian bird communities to identify factors that explain patterns of infection. 4. Plasmodium spp. displayed isolation-by-distance effects, a significant amount of genetic variation distributed among islands but insignificant amounts among host species and families, and strong local island effects with respect to prevalence. Haemoproteus spp. did not display isolation-by-distance patterns, showed marked structuring of genetic variation among avian host species and families, and significant host species prevalence patterns. 5. These differences suggest that Plasmodium spp. infection patterns were shaped by geography and the abiotic environment, whereas Haemoproteus spp. infection patterns were shaped predominantly by host associations. Heterogeneity in the complement and prevalence of parasite lineages infecting local bird communities likely exposes host species to a mosaic of spatially divergent disease selection pressures across their naturally fragmented distributions in southern Melanesia. Host associations for Haemoproteus spp. indicate a capacity for the formation of locally co-adapted host-parasite relationships, a feature that may limit intraspecific gene flow or range expansions of closely related host species.

Genetic analysis of Indian tasar silkmoth (Antheraea mylitta) populations

Indian tasar silkmoth, Antheraea mylitta is an economically important wild silkmoth species distributed across India. A number of morphologically and ethologically well-defined ecotypes are known for this species that differ in their primary food plant specificity. Most of these ecotypes do not interbreed in nature, but are able to produce offspring under captive conditions. Microsatellite markers were developed for A. mylitta, and out of these, ten well-behaved microsatellite loci were used to analyze the population structure of different ecoraces. A total of 154 individual moths belonging to eight different ecoraces, were screened at each locus. Hierarchical analysis of population structure using Analysis of MOlecular VAriance (AMOVA) revealed significant structuring (F-ST = 0.154) and considerable inbreeding (F-IS = 0.505). A significant isolation by distance was also observed. The number of possible population clusters was investigated using distance method, Bayesian algorithm and self organization maps (SOM). The first two methods revealed two distinct clusters, whereas the SOM showed the different ecoraces not to be clearly differentiated. These results suggest that although there is a large degree of phenotypic variation among the different ecoraces of A. mylitta, genetically they are not very different, and the phenotypic differences may largely be a result of their respective ecology.

Molecular genetic studies on voltage-gated ion channels

Several different methods have been employed in the study of voltage-gated ion channels. Electrophysiological studies on excitable cells in vertebrates and molluscs have shown that many different voltage-gated potassium (K⁺) channels and sodium channels may coexist in the same organism. Parallel genetic studies in Drosophila have identified mutations in several genes that alter the properties of specific subsets of physiologically identified ion channels. Chapter 2 describes molecular studies that identify two Drosophila homologs of vertebrate sodium-channel genes. Mutations in one of these Drosophila sodium-channel genes are shown to be responsible for the temperature-dependent paralysis of a behavioural mutant para^ts. Evolutionary arguments, based on the partial sequences of the two Drosophila genes, suggest that subfamilies of voltage-gated sodium channels in vertebrates remain to be identified.

In Drosophila, diverse voltage-gated K⁺ channels arise from alternatively spliced mRNAs generated at the Shaker locus. Chapter 3 and the Appendices describe the isolation and characterization of several human K⁺-channel genes, similar in sequence to Shaker. Each of these human genes has a highly conserved homolog in rodents; thus, this K⁺-channel gene family probably diversified prior to the mammalian radiation. Functional K⁺ channels encoded by these genes have been expressed in Xenopus oocytes and their properties have been analyzed by electrophysiological methods. These studies demonstrate that both transient and noninactivating voltage-gated K⁺ channels may be encoded by mammalian genes closely related to Shaker. In addition, results presented in Appendix 3 clearly demonstrate that independent gene products from two K⁺-channel genes may efficiently co-assemble into heterooligomeric K⁺ channels with properties distinct from either homomultimeric channel. This finding suggests yet another molecular mechanism for the generation of K⁺-channel diversity.

Inferring Population Genetic Structure in Widely and Continuously Distributed Carnivores: The Stone Marten (Martes foina) as a Case Study

The stone marten is a widely distributed mustelid in the Palaearctic region that exhibits variable habitat preferences in different parts of its range. The species is a Holocene immigrant from southwest Asia which, according to fossil remains, followed the expansion of the Neolithic farming cultures into Europe and possibly colonized the Iberian Peninsula during the Early Neolithic (ca. 7,000 years BP). However, the population genetic structure and historical biogeography of this generalist carnivore remains essentially unknown. In this study we have combined mitochondrial DNA (mtDNA) sequencing (621 bp) and microsatellite genotyping (23 polymorphic markers) to infer the population genetic structure of the stone marten within the Iberian Peninsula. The mtDNA data revealed low haplotype and nucleotide diversities and a lack of phylogeographic structure, most likely due to a recent colonization of the Iberian Peninsula by a few mtDNA lineages during the Early Neolithic. The microsatellite data set was analysed with a) spatial and non-spatial Bayesian individual-based clustering (IBC) approaches (STRUCTURE, TESS, BAPS and GENELAND), and b) multivariate methods [discriminant analysis of principal components (DAPC) and spatial principal component analysis (sPCA)]. Additionally, because isolation by distance (IBD) is a common spatial genetic pattern in mobile and continuously distributed species and it may represent a challenge to the performance of the above methods, the microsatellite data set was tested for its presence. Overall, the genetic structure of the stone marten in the Iberian Peninsula was characterized by a NE-SW spatial pattern of IBD, and this may explain the observed disagreement between clustering solutions obtained by the different IBC methods. However, there was significant indication for contemporary genetic structuring, albeit weak, into at least three different subpopulations. The detected subdivision could be attributed to the influence of the rivers Ebro, Tagus and Guadiana, suggesting that main watercourses in the Iberian Peninsula may act as semi-permeable barriers to gene flow in stone martens. To our knowledge, this is the first phylogeographic and population genetic study of the species at a broad regional scale. We also wanted to make the case for the importance and benefits of using and comparing multiple different clustering and multivariate methods in spatial genetic analyses of mobile and continuously distributed species.

Patterns of genetic variation in the endangered European mink (Mustela lutreola L., 1761)

Background: The European mink (Mustela lutreola, L. 1761) is a critically endangered mustelid, which inhabits several main river drainages in Europe. Here, we assess the genetic variation of existing populations of this species, including new sampling sites and additional molecular markers (newly developed microsatellite loci specific to European mink) as compared to previous studies. Probabilistic analyses were used to examine genetic structure within and between existing populations, and to infer phylogeographic processes and past demography. Results: According to both mitochondrial and nuclear microsatellite markers, Northeastern (Russia, Estonia and Belarus) and Southeastern (Romania) European populations showed the highest intraspecific diversity. In contrast, Western European (France and Spain) populations were the least polymorphic, featuring a unique mitochondrial DNA haplotype. The high differentiation values detected between Eastern and Western European populations could be the result of genetic drift in the latter due to population isolation and reduction. Genetic differences among populations were further supported by Bayesian clustering and two main groups were confirmed (Eastern vs. Western Europe) along with two contained subgroups at a more local scale (Northeastern vs. Southeastern Europe; France vs. Spain). Conclusions: Genetic data and performed analyses support a historical scenario of stable European mink populations, not affected by Quaternary climate oscillations in the Late Pleistocene, and posterior expansion events following river connections in both North-and Southeastern European populations. This suggests an eastern refuge during glacial maxima (as already proposed for boreal and continental species). In contrast, Western Europe was colonised more recently following either natural expansions or putative human introductions. Low levels of genetic diversity observed within each studied population suggest recent bottleneck events and stress the urgent need for conservation measures to counteract the demographic decline experienced by the European mink.

Genetic evidence of two sibling species within the Contracoecum ogmorhini Johnson & Mawson 1941 complex (Nematoda; Anisakidae) from otariid seals in boreal and austral regions

Genetic variation of Contracaecum ogmorhini (sensu lato) populations from different otariid seals of the northern and southern hemisphere was studied on the basis of 18 enzyme loci as well as preliminary sequence analysis of the mitochondrial cyt b gene (260 bp). Samples were collected from Zalophus californianus in the boreal region and from Arctocephalus pusillus pusillus, A. pusillus doriferus and A. australis from the austral region. Marked genetic heterogeneity was found between C. ogmorhini (sensu lato) samples from the boreal and austral region, respectively. Two loci (Mdh-2 and NADHdh) showed fixed differences and a further three loci (Iddh, Mdh-1 and 6Pgdh) were highly differentiated between boreal and austral samples. Their average genetic distance was DNei = 0.36 at isozyme level. At mitochondrial DNA level, an average proportion of nucleotide substitution of 3.7% was observed. These findings support the existence of two distinct sibling species, for which the names C. ogmorhini (sensu stricto) and C. margolisi n. sp., respectively, for the austral and boreal taxon, are proposed. A description for C. margolisi n. sp. is provided. No diagnostic morphological characters have so far been detected; on the other hand, two enzyme loci, Mdh-2 and NADHdh, fully diagnostic between the two species, can be used for the routine identification of males, females and larval stages. Mirounga leonina was found to host C. ogmorhini (s.s.) inmixed infections with C. osculatum (s.l.) (of which C. ogmorhini (s.l.) was in the past considered to be a synonym) and C. miroungae; no hybrid genotypes were found,confirming the reproductive isolation of these three anisakid species. The hosts and geographical range so far recorded for C. margolisi n. sp. and C. ogmorhini (s.s.) are given.

Exploring factors shaping population genetic structure of the freshwater fish Sinocyclocheilus grahami (Teleostei, Cyprinidae)

Phylogeographical analyses on Sinocyclocheilus grahami samples from seven localities within the Lake Dianchi Basin in China were conducted to explore the main factors shaping population structure within this species. Phylogenetic and network analyses reve

Genetic status of selected fish taxa in relation to conservation of genetic and species diversity in the Victoria and Kyoga Lake basins

Cichlids are known for their explosive radiation especially in the African Great Lakes marked with a high level of lake endemism. These fishes have been characterized mainly along trophic and habitat differences, by variation in morphological structures such as teeth and jaws and by differences in body shape and coloration. Cichlids are important as a microcosm of macroevolution. The explosive radiation, young evolutionary scale, and the isolation of groups characterized with high levels of endemism and presence of living fossils makes the group important for evolutionary and genetic studies. Lake Victoria region cichlids which are isolated and relatively more recent in evolution were the last to be appreciated in their diversity. Recently Ole Seehausen has found scores of rock fishes in Lake Victoria which were up to then thought to be absent from the Lake and only known to occur in Lakes Malawi and Tanganyika. Greenwood put together the species groups of Lake Victoria, and later in the early 1980's revised the classification of haplochromine species to reflect the phyletic origin and interrelationship of the various groups in Lake Victoria region. Melan Stiassny has been interested in early evolution of cichlids while the likes of Paul Fuerst and Lees Kaufman and Axel Meyer have been interested and are working to explain the speciation mechanisms responsible for the explosive radiation and evolution of cichlids. Locally S.B Wandera and his student Getrude Narnulemo are spearheading the biodiversity and taxonomic studies of cichlids in Lake Victoria region

Isolation of Y- and X-linked SCAR markers in yellow catfish and application in the production of all-male populations

P>Sex controls have been performed in some farmed fish species because of significant growth differences between females and males. In yellow catfish (Pelteobagrus fulvidraco), adult males are three times larger than female adults. In this study, six Y- and X-linked amplified fragment length polymorphism fragments were screened by sex-genotype pool bulked segregant analysis and individual screening. Interestingly, sequence analysis identified two pairs of allelic genes, Pf33 and Pf62. Furthermore, the cloned flanking sequences revealed several Y- and X-specific polymorphisms, and four Y-linked or X-linked sequence characterized amplified region (SCAR) primer pairs were designed and converted into Y- and X-linked SCAR markers. Consequently, these markers were successfully used to identify genetic sex and YY super-males, and applied to all-male population production. Thus, we developed a novel and simple technique to help commercial production of YY super-males and all-male populations in the yellow catfish.

Microsatellite marker isolation and cultured strain identification in Carassius auratus gibelio

Microsatellites have become the preferred molecular markers for strain selection and genetic breeding in fish. In this study a total of 105 microsatellites were isolated and identified in gibel carp (Carassius auratus gibelio) by microsatellite sequence searches in GenBank and other databases and by screening and sequencing of positive clones from the genomic library enriched for AG and GATA repeats. Moreover, nineteen microsatellites were randomly selected to design locus-specific primer pairs, and these were successfully used to identify and discriminate different cultured strains of gibel carp including strains A, D, L, and F. Three different types of microsatellite pattern were distinguished by the number and length of fragments amplified from the 19 primer pairs, and some microsatellite primer pairs were found to produce different microsatellite patterns among strains and strain-specific fragments. In addition, some duplicated alleles were also detected in two microsatellite patterns. Therefore, the current study provides direct molecular markers to discriminate among different cultured strains for selective breeding and aquaculture practice of gibel carp.

Isolation and characterization of microsatellites in Chinese soft-shelled turtle, Pelodiscus sinensis

Fifteen polymorphic microsatellite loci were developed for the Chinese soft-shelled turtle (Pelodiscus sinensis) from the (GT)(n) microsatellite-enriched genomic library, using the fast isolation by amplified fragment length polymorphism of sequences containing repeats protocol. The polymorphism of all 15 loci ranged from two to seven alleles with observed heterozygosities ranging from 0.03 to 0.98 (mean 0.43) in one population of 40 individuals. These novel loci will be helpful for understanding the population structure at genetic level and marker-assisted breeding of this vulnerable species.

Isolation and characterization of polymorphic microsatellites in a sex-reversal fish, rice field eel (Monopterus albus)

The rice field eel (Monopterus albus) is a fish of economic importance in China and some Asian countries. From a (GT)(n)-enriched genomic library, 30 microsatellites were developed by employing the fast isolation by AFLP of sequences containing repeats (FIASCO) protocol. Thirteen loci exhibited polymorphism with two to 13 alleles (mean 7.9 alleles/locus) in a test population and observed heterozygosity ranged from 0.3125 to 0.9688 (mean 0.7140). These loci should provide sufficient level of genetic variation to study the fine-scale population structure and reproductive ecology of the species.