Molecular Genetic Identification Tools for Three Commercially Cultured Oysters (<Emphasis Type="Italic">Crassostrea belcheri</Emphasis>, <Emphasis Type="Italic">Crassostrea iredalei</Emphasis>, and <Emphasis Type="Italic">Saccostrea cucullata</Emphasis>) in Thailand

Abstract Molecular genetic keys for identification of 3 commercially cultured oysters (Crassostrea belcheri, Crassostrea iredalei, and Saccostrea cucullata) in Thailand were developed based on restriction analysis of 18S ribosomal DNA and cytochrome oxidase subunit I (COI). Digestion of the amplified 18S rDNA with Hinf I unambiguously differentiated Crassostrea oysters from Saccostrea oysters and Striostrea (Parastriostrea) mytiloides. In addition, species-specific restriction fragment length polymorphism patterns of C. belcheri, C. iredalei, and S. cucullata were consistently observed when the gel-eluted COI was digested with Mbo I and Dde I. Thirty composite haplotypes were observed across all individuals. Species-specific composite haplotypes were found in C. belcheri (AAAA and AAAB), C. iredalei (AABC and AABU), and S. cucullata (BBCD and BBCE), respectively. The most common composite haplotype of COI in C. belcheri (AAAA), C. iredalei (AABC), and S. cucullata (BBCD) was amplified, cloned, and sequenced. Detection of C. belcheri and C. iredalei based on polymerase chain reaction was further developed using more specific primers (HCO2198 and R372) followed by digestion of a 372-bp product with Mbo I.     

DNA barcoding,phylogenetic relationships and speciation of snappers (genus <Emphasis Type="Italic">Lutjanus</Emphasis>)

The phylogenetic relationships of 13 snapper species from the South China Sea have been established using the combined DNA sequences of three full-length mitochondrial genes (COI, COII and CYTB) and two partial nuclear genes (RAG1, RAG2). The 13 species (genus Lutjanus) were selected after DNA barcoding 72 individuals, representing 20 species. Our study suggests that although DNA barcoding aims to develop species identification systems, it may also be useful in the construction of phylogenies by aiding the selection of taxa. Combined mitochondrial and nuclear gene data has an advantage over an individual dataset because of its higher resolving power.     

Genetic differentiation among natural populations of the lizard complex <Emphasis Type="Italic">Darevskia raddei</Emphasis> as inferred from genome microsatellite marking

The article presents the genetic parameters of the populations of lizards of the Darevskia raddei complex (D. raddei nairensis and D. raddei raddei) and the populations of D. valentini calculated on the basis of the analysis of variability of 50 allelic variants of the three nuclear genome microsatellite-containing loci of 83 individuals. It was demonstrated that the F_st genetic distances between the populations of D. raddei nairensis and D. raddei raddei were not statistically significantly different from the F_st genetic distances between the populations of different species, D. raddei and D. valentini. At the same time, these distances were statistically significantly higher than the F_st distances between the populations belonging to one species within the genus Darevskia. These data suggest deep divergence between the populations of D. raddei raddei and D. raddei nairensis of the D. raddei complex and there arises the question on considering them as separate species.     

Genetic structure of Schrenck newt <Emphasis Type="Italic">Salamandrella schrenckii</Emphasis> populations by mitochondrial cytochrome <Emphasis Type="Italic">b</Emphasis> variation

The nucleotide sequence variation of the mitochondrial cytochrome b gene was studied in Schrenck newt Salamandrella schrenckii (Strauch, 1870) from populations of Primorye and the Khabarovsk region. Phylogenetic analysis revealed two haplotype clusters, southern cluster 1 and northern cluster 2, with a divergence of 3%. Analysis of the mtDNA and cytochrome b amino acid sequence variations made it possible to assume that the modern range of Schrenck newt was colonized from south Primorye northwards. In contrast to the southern cluster, the northern one demonstrated all the signs of demographic expansion (a unimodal distribution of pairwise nucleotide differences, specific results of tests for selective neutrality of mtDNA variation, and a good correspondence of genetic parameters to those expected from demographic expansion models).     

Microsatellite markers developed for a Swedish population of sand lizard (<Emphasis Type="Italic">Lacerta agilis</Emphasis>)

Populations of sand lizards (Lacerta agilis) are declining throughout its north-western range. Here we characterize fifteen new microsatellite markers developed specifically for parentage analysis in a small Swedish population of sand lizards. These loci were screened in the Asketunnan population and a much larger and genetically diverse Hungarian population, with heterozygosities ranging from (0.217–0.875) and (0.400–0.974), respectively. All loci were in Hardy-Weinberg Equilibrium in the Swedish population but eight loci had significant heterozygote deficiencies in the Hungarian population. Two loci were significantly linked in both populations. These microsatellite loci are likely to be applicable in research on other sand lizard populations throughout Europe. An erratum to this article can be found at     

Molecular-genetic diversification of lizard complex D. raddei (Sauria: Lacertidae): early stages of speciation

A number of populations of genus Darevskia lizards were studied using inter-SINE-PCR (IS-PCR). The number and size of PCR-amplified spacers of genomic DNA flanked by SINE-type repeats were compared in 17 populations of the D. raddei species complex along with several other species from the same genus. Nei and Li's (D(NL)) genetic distances between populations were unequivocal. Individual differences among D. r. nairensis sample specimens and between the samples fall into a range of less than 0.1. Individual variability intra each of the D. r. raddei samples--into a range of 0.1-0.2, but the inter samples differences in this subspecies depends on its geographic localizations. Thus the differences between 10 samples from Armenia and Karabach fall into a range of 0.2-0.3, but between all these and two samples from Talysh (Azerbajdzhan)--of 0.3-0.4. At the same time, the variability between both known subspecies (D. r. raddei and D. r. nairensis) reaches the same values. The differences between "good" species D. raddei and D. rudis were about 0.60-0.7. It may be supposed that D. r. raddei is currently undergoing the speciation process. The population of lizards from Turkey which is considered to be close to D. raddei by some researchers and to D. rudis by others is, according to our molecular data, more closely related to the latter species. The difference of homogeneity between other populations was revealed by comparison of the DNL values. Possible phylogeographic pathways of Darevskia species distribution based on the molecular data were proposed.     

Nucleotide sequences of the microsatellite locus <Emphasis Type="Italic">Du215 (arm)</Emphasis> allelic variants in the parthenospecies <Emphasis Type="Italic">Darevskia armeniaca</Emphasis> (Lacertidae)

Using monolocus PCR analysis with the pairs of primers designed for the Du215 locus of Darevskia unisexualis, allelic polymorphism at the orthologous locus in the populations of the related parthenospecies D. armeniaca was investigated. It was demonstrated that Du215 (arm) locus was polymorphic and in the populations of parthenospecies D. armeniaca (n = 127) represented by at least three allelic variants, differing from each other by the size and composition of microsatellite cluster, and by single nucleotide substitutions in flanking DNA. Unlike the Du215 locus, Du215 (arm) was shown contain not only GATA, but also (GACA) repeats, which were absent in D. unisexualis. Thus, in this study, the data on the molecular nature of allelic polymorphism at one of the microsatellite loci of the parthenospecies D. armeniaca were reported.     

Molecular phylogeny and karyotype differentiation in <Emphasis Type="Italic">Paratelmatobius</Emphasis> and <Emphasis Type="Italic">Scythrophrys</Emphasis> (Anura,Leptodactylidae)

Paratelmatobius and Scythrophrys are leptodactylid frogs endemic to the Brazilian Atlantic forest and their close phylogenetic relationship was recently inferred in an analysis that included Paratelmatobius sp. and S. sawayae. To investigate the interspecific relationships among Paratelmatobius and Scythrophrys species, we analyzed a mitochondrial region (approximately 2.4 kb) that included the ribosomal genes 12S and 16S and the tRNAval in representatives of all known localities of these genera and in 54 other species. Maximum parsimony inferences were done using PAUP* and support for the clades was evaluated by bootstrapping. A cytogenetic analysis using Giemsa staining, C-banding and silver staining was also done for those populations of Paratelmatobius not included in previous cytogenetic studies of this genus in order to assess their karyotype differentiation. Our results suggested Paratelmatobius and Scythrophrys formed a clade strongly supported by bootstrapping, which corroborated their very close phylogenetic relationship. Among the Paratelmatobius species, two clades were identified and corroborated the groups P. mantiqueira and P. cardosoi previously proposed based on morphological characters. The karyotypes of Paratelmatobius sp. 2 and Paratelmatobius sp. 3 described here had diploid chromosome number 2n = 24 and showed many similarities with karyotypes of other Paratelmatobius representatives. The cytogenetic data and the phylogenetic analysis allowed the proposal/corroboration of several hypotheses for the karyotype differentiation within Paratelmatobius and Scythrophrys. Namely the telocentric pair No. 4 represented a synapomorphy of P. cardosoi and Paratelmatobius sp. 2, while chromosome pair No. 5 with interstitial C-bands could be interpreted as a synapomorphy of the P. cardosoi group. The NOR-bearing chromosome No. 10 in the karyotype of P. poecilogaster was considered homeologous to chromosome No. 10 in the karyotype of Scythrophrys sp., chromosome No. 9 in the karyotype of Paratelmatobius sp. 1, chromosome No. 8 in the karyotypes of Paratelmatobius sp. 2 and of Paratelmatobius sp. 3, and chromosome No. 7 in the karyotype of P. cardosoi. A hypothesis for the evolutionary divergence of these NOR-bearing chromosomes, which probably involved events like gain in heteochromatin, was proposed.     

Phylogeography and speciation in the <Emphasis Type="Italic">Pseudocrenilabrus philander</Emphasis> species complex in Zambian Rivers

Haplochromine cichlids form the most species-rich lineage of cichlid fishes that both colonized almost all river systems in Africa and radiated to species flocks in several East African lakes. The enormous diversity of lakes is contrasted by a relatively poor albeit biogeographically clearly structured species diversity in rivers. The present study analyzed the genetic structure and phylogeographic history of species and populations of the genus Pseudocrenilabrus in Zambian rivers that span two major African drainage systems, the Congo- and the Zambezi-system. The mtDNA phylogeny identifies four major lineages, three of which occur in the Congo-system and one in the Zambezi system. Two of the Congo-clades (Lake Mweru and Lunzua River) comprise distinct albeit yet undescribed species, while the fish of the third Congo-drainage clade (Chambeshi River and Bangweulu swamps), together with the fish of the Zambezi clade (Zambezi and Kafue River) are assigned to Pseudocrenilabrus philander. Concerning the intraspecific genetic diversity observed in the sampled rivers, most populations are highly uniform in comparison to lacustrine haplochromines, suggesting severe founder effects and/or bottlenecking during their history. Two bursts of diversification are reflected in the structure of the linearized tree. The first locates at about 3.9% mean sequence divergence and points to an almost simultaneous colonization of the sampled river systems. Subsequent regional diversification (with about 1% mean sequence divergence) occurred contemporaneously within the Kafue River and the Zambezi River. The clear-cut genetic biogeographic structure points to the dominance of geographic speciation in this lineage of riverine cichlid fishes, contrasting the importance of in situ diversification observed in lake cichlids.     

Mitochondrial <Emphasis Type="Italic">cox</Emphasis>1 and plastid <Emphasis Type="Italic">rbc</Emphasis>L genes of <Emphasis Type="Italic">Gracilaria vermiculophylla</Emphasis> (Gracilariaceae,Rhodophyta)

The mitochondrial cytochrome c oxidase subunit I gene sequence was recently developed for DNA barcoding of red algal species. We determined the 1245 base pairs of the gene from 27 taxa of an agar-producing species, Gracilaria vermiculophylla, and putative relatives and compared the results with rbcL data from the same species. A total of 392 positions (31.5%) were variable, 282 positions (22.6%) were parsimoniously informative, and average sequence divergence was 13% in an ingroup. Within G. vermiculophylla, pairwise divergence of the gene was variable up to 11 bp (0.9%). Seven recognized haplotypes of cox1 tended to be geographically related. In the aligned 1386 bp of rbcL, three haplotypes were recognized. These results suggest that cox1 is a valuable molecular marker within species and will be very useful in haplotype analyses.     

Molecular characterization,relatedness of <Emphasis Type="Italic">Haematobia irritans</Emphasis> (horn fly) populations,by RAPD-PCR

Haematobia irritans is a hematophagous parasite of cattle that causes significant economic losses in many parts of the world, including Brazil. In the present work, one American, four Brazilian populations of this species were studied by Random Amplified Polymorpht DNA (RAPD) to assess basically genetic variability within and between populations. Ten different decamer random primers were employed in the genomic DNA amplification, yielding 117 fragments in the five H. irritans populations. In Drosophila prosaltans, used as an outgroup, 81 fragments were produced. Forty-three of these fragments were shared by both species. Among the H. irritans samples, that from Rio Branco (Acre State, Brazil) produced the smallest numbers of fragments, polymorphic bands. This high genetic homogenity may be ascribed to its geographic origin (in the Northwest of Brazil), which causes high isolation, low gene flow, unlike the other Brazilian populations, from the South Central region, in which cattle trade is very intensive. Marker fragments (exclusive bands) detected in every sample enabled the population origin to be characterized, but they are also potentially useful for further approaches such as the putative origin of Brazilian populations from North America. Similarity indices [Nei & Li, 1979, Proc. Natl. Acad. Sci. USA 76: 5269–5273], phylogenetic trees, rooted by using the outgroup, produced by the Phylogenetic Analysis using Parsimony (PAUP 4.0-Swofford, 2001) program showed the closest relationships between flies from São José do Rio Preto, Turiúba (both from São Paulo State, Brazil) while flies from the geographically distant Rio Branco showed the greatest differentiation relative to the others.     

Molecular evolution of paclitaxel biosynthetic genes <Emphasis Type="Italic">TS</Emphasis> and <Emphasis Type="Italic">DBAT</Emphasis> of <Emphasis Type="Italic">Taxus</Emphasis> species

Evolutionary patterns of sequence divergence were analyzed in genes from the conifer genus Taxus (yew), encoding paclitaxel biosynthetic enzymes taxadiene synthase (TS) and 10-deacetylbaccatin III-10β-O-acetyltransferase (DBAT). N-terminal fragments of TS, full-length DBAT and internal transcribed spacer (ITS) were amplified from 15 closely related Taxus species and sequenced. Premature stop codons were not found in TS and DBAT sequences. Codon usage bias was not found, suggesting that synonymous mutations are selectively neutral. TS and DBAT gene trees are not consistent with the ITS tree, where species formed monophyletic clades. In fact, for both genes, alleles were sometimes shared across species and parallel amino acid substitutions were identified. While both TS and DBAT are, overall, under purifying selection, we identified a number of amino acids of TS under positive selection based on inference using maximum likelihood models. Positively selected amino acids in the N-terminal region of TS suggest that this region might be more important for enzyme function than previously thought. Moreover, we identify lineages with significantly elevated rates of amino acid substitution using a genetic algorithm. These findings demonstrate that the pattern of adaptive paclitaxel biosynthetic enzyme evolution can be documented between closely related Taxus species, where species-specific taxane metabolism has evolved recently.     

Instability of (GATA)<Subscript> n</Subscript> microsatellite loci in the parthenogenetic Caucasian rock lizard<Emphasis Type="Italic"> Darevskia unisexualis</Emphasis> (Lacertidae)

Mini- and microsatellites, comprising tandemly repeated short nucleotide sequences, are abundant dispersed repetitive elements that are ubiquitous in eukaryotic genomes. In humans and other bisexual species hypervariable mini- and microsatellite loci provide highly informative systems for monitoring of germline and somatic instability. However, little is known about the mechanisms by which these loci mutate in species that lack effective genetic recombination. Here, multilocus DNA fingerprinting was used to study M13 minisatellite and (GATA) _n microsatellite instability in the parthenogenetic Caucasian rock lizard Darevskia unisexualis (Lacertidae). DNA fingerprinting of 25 parthenogenetic families, from six isolated populations in Armenia (comprising a total of 84 siblings), using the oligonucleotide (GATA)₄ as a hybridization probe, revealed mutant fingerprinting phenotypes in 13 siblings that differed from their mothers in several restriction DNA fragments. In three families (8 siblings), the mutations were present in the germline. Moreover, the mutant fingerprint phenotypes detected in siblings were also present in population DNA samples. No intrafamily variations in DNA fingerprint patterns were observed with the M13 minisatellite probe. Estimates of the mutation rate for (GATA) _n microsatellite loci in D. unisexualis showed that it was as high as that seen in some bisexual species, reaching 15% per sibling or 0.95% per microsatellite band. Furthermore, in one case, a somatic (GATA) _n microsatellite mutation was observed in an adult lizard. These findings directly demonstrate that mutations in (GATA) _n microsatellite loci comprise an important source of genetic variation in parthenogenetic populations of D. unisexualis.Communicated by G. P. Georgiev     

Assessment of genetic stability of <Emphasis Type="Italic">in vitro</Emphasis> grown <Emphasis Type="Italic">Dictyospermum ovalifolium</Emphasis>

In the present study, a polymerase chain reaction (PCR)-based method namely inter simple sequence repeat (ISSR) was employed to assess genetic stability in tissue culture-derived Dictyospermum ovalifolium plantlets. To study genomic stability of micropropagated plants, 14 individuals were randomly tagged among a population of 2500 regenerants and were compared with single donor mother plant. A total of 51 clear and reproducible bands ranging from 200 bp to 2.1 kb were scored corresponding to an average of 3.64 bands per primer. Two of the 51 bands were polymorphic (3.92 %) among 14 individuals, thus indicating the occurrence of low level genomic variation in the micropropagated plants. Cluster analysis indicates that genetic similarity values were 0.978 which allows classification of the plants to distinct groups. Further an attempt was made to reintroduce the micropropagated plants into their natural habitat. Over one thousand six hundred fifty plants were successfully established.     

<Emphasis Type="Italic">Sporidiobolus johnsonii</Emphasis> and <Emphasis Type="Italic">Sporidiobolus salmonicolor</Emphasis> revisited

The relationship between Sporidiobolus johnsonii and S. salmonicolor was investigated using rDNA sequence data. Two statistically well-supported clades were obtained. One clade included the type strain of S. johnsonii and the other included the type strain of S. salmonicolor. However, some mating strains of S. salmonicolor were found in the S. johnsonii group. These strains belonged to mating type A2 and were sexually compatible with mating type A1 strains from the S. salmonicolor group. DNA–DNA reassociation values were high within each clade and moderate between the two clades. In the re-investigation of teliospore germination, we observed that the basidia of S. salmonicolor were two-celled. In S. johnsonii, basidia were not formed and teliospore germination resulted in direct formation of yeast cells. We hypothesize that the S. johnsonii clade is becoming genetically isolated from the S. salmonicolor group and that a speciation process is presently going on. We suspect that the observed sexual compatibility between strains of the S. johnsonii and S. salmonicolor groups and the possible genetic flow between the two species has little biological relevance because distinct phenotypes have been fixed in the two taxa and intermediate (hybrid) sequences for LSU and ITS rDNAs have not been detected. An erratum to this article can be found at     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

Molecular genetics of the<Emphasis Type="Italic"> Alhambra</Emphasis> (<Emphasis Type="Italic"> Drosophila AF10</Emphasis>) complex locus of<Emphasis Type="Italic"> Drosophila</Emphasis>

The Alhambra ( Alh) gene is the Drosophila homologue of the human AF10 gene. AF10 has been identified as a fusion partner of MLL, a human homologue of the fly gene trithorax, in infant leukemias. The endogenous function of human AF10 is not known, but may be vital to its role in acute leukemia. This prompted us to analyse Alh function. We describe here the genetic organisation of the Alh locus in D. melanogaster. We show that an independent lethal complementation group encoding a muscle protein ( Mlp84B) is located within an Alh intron. We have already shown that the leucine zipper (LZ) domain of ALH activates several Polycomb group-responsive elements. We further demonstrate that the LZ domain on its own bears the Alh vital function, since it is necessary and sufficient for rescue of Alh mutant lethality. Finally, we demonstrate that, in contrast to a previous report, Alh does not affect position-effect variegation.Communicated by G. Reuter     

Molecular genetic characteristic of dinucleotide microsatellite loci in parthenogenetic lizards <Emphasis Type="Italic">Darevskia unisexualis</Emphasis>

In the present study, the first molecular genetic investigation of dinucleotide (GT) _n microsatellite loci in parthenogenetic lizards Darevskia unisexualis was performed. New polymorphic locus, Du214, (GenBank Ac. No. EU252542) was identified and characterized in detail. It was demonstrated that allele of this locus differed in the size and structure of microsatellite locus, as well as in point mutations, the combinations of which enabled the isolation of stabile fixed double nucleotide substitutions A-A (alleles 2 and 4) and G-T (alleles 1, 3, 5, and 6). Double nucleotide substitutions described were also identified in the orthlogous loci of the parental species genomes, D. raddei (G-T) and D. valentine (A-A). Based on the analysis of allele distribution pattern at this locus in all populations of parthenospecies D. unisexualis, mathematic model was elaborated and realized. Using this model, frequencies of allelic variants for all populations of the species of interest were calculated and population genetic structure of D. unisexualis was characterized. Genetic contribution of each population to the species gene pool was determined. The data obtained demonstrated that microsatellite variation was one of the factors of clonal and genetic diversity of a parthenospecies.