PERMANENT GENETIC RESOURCES: Isolation and characterization of nine microsatellite loci from the chub mackerel, Scomber japonicus (Perciformes, Scombridae)

The stock abundance of the chub mackerel Scomber japonicus - a very important species for fisheries, particularly in Japan - in the Pacific Ocean off Japan has remained at a low level. For studying the population genetics of the chub mackerel, we isolated nine polymorphic microsatellite loci (12-31 alleles/locus; expected heterozygosity, 0.762-0.983) from this species. Cross-species amplification indicated that eight of the nine microsatellite loci in the blue mackerel S. australasicus were polymorphic and functional.     

Differential population structuring of two closely related fish species, the mackerel (Scomber scombrus) and the chub mackerel (Scomber japonicus), in the Mediterranean Sea

Population genetic structures of the mackerel (Scomber scombrus) and chub mackerel (Scomber japonicus) were studied in the Mediterranean Sea. Fragments of 272 bp (S. scomber) and 387 bp (S. japonicus) of the 5'-end of the mitochondrial control region were sequenced from spawning individuals collected off the coasts of Greece, Italy, Spain, and Portugal. High levels of mitochondrial control region haplotypic diversity (> 0.98) were found for both Scomber species. Nucleotide diversity was higher in the mackerel (0.022) than in the chub mackerel (0.017). Global F(ST) values were also higher and significant in the mackerel (0.024, P < 0.0001) as opposed to the chub mackerel (0.003, P > 0.05). Molecular variance analyses showed differential genetic structuring for these two closely related species. There is extensive gene flow between Mediterranean Sea and Atlantic Ocean populations of chub mackerel, which are organized into a larger panmictic unit. In contrast, Mediterranean Sea populations of mackerel show some degree of genetic differentiation and are structured along an east-west axis. The analysed eastern Mediterranean Sea mackerel populations (Greece, Italy) are clearly separated from that of the western Mediterranean Sea (Barcelona), which forms a panmictic unit with eastern Atlantic Ocean populations. The genetic structures of both species showed asymmetric migration patterns and indicated population expansion.     

EST and Mitochondrial DNA Sequences Support a Distinct Pacific Form of Salmon Louse, <Emphasis Type="Italic">Lepeophtheirus salmonis</Emphasis>

Nuclear deoxyribonucleic acid sequences from approximately 15,000 salmon louse expressed sequence tags (ESTs), the complete mitochondrial genome (16,148bp) of salmon louse, and 16S ribosomal ribonucleic acid (rRNA) and cytochrome oxidase subunit I (COI) genes from 68 salmon lice collected from Japan, Alaska, and western Canada support a Pacific lineage of Lepeophtheirus salmonis that is distinct from that occurring in the Atlantic Ocean. On average, nuclear genes are 3.2% different, the complete mitochondrial genome is 7.1% different, and 16S rRNA and COI genes are 4.2% and 6.1% different, respectively. Reduced genetic diversity within the Pacific form of L. salmonis is consistent with an introduction into the Pacific from the Atlantic Ocean. The level of divergence is consistent with the hypothesis that the Pacific form of L. salmonis coevolved with Pacific salmon (Onchorhynchus spp.) and the Atlantic form coevolved with Atlantic salmonids (Salmo spp.) independently for the last 2.5–11 million years. The level of genetic divergence coincides with the opportunity for migration of fish between the Atlantic and Pacific Ocean basins via the Arctic Ocean with the opening of the Bering Strait, approximately 5 million years ago. The genetic differences may help explain apparent differences in pathogenicity and environmental sensitivity documented for the Atlantic and Pacific forms of L. salmonis. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Revision of the genus <Emphasis Type="Italic">Melamphaes</Emphasis> (Melamphaidae): Part 3. Multirakered species: <Emphasis Type="Italic">M. suborbitalis,M. parini</Emphasis>, and <Emphasis Type="Italic">M. acanthomus</Emphasis>

In the third part of the revision of the genus Melamphaes Melamphaidae (Melamphaidae), we examine multirakered species (20 and more rakers at the first gill arch) with seven soft rays in the ventral fin that have a posttemporal (temporal) spine directed anteriorly-upwards, with 14–15 rays in the pectoral fin, and 11 (rarely 12) trunk vertebrae. M. suborbitalis inhabits the Atlantic Ocean (in the north up to 57°N, in the south, up to 40°S), the Indian Ocean (is known in its southwestern part), and the western part of the Pacific Ocean. There is no significant evidence on catches of this species in the eastern part of the Pacific Ocean. Apparently, M. suborbitalis is absent in the tropical waters of the oceans. Until recently, M. parini was known from the holotype caught in the Sea of Okhotsk. Two specimens of this rare species: from the central (the area of the Hawaiian Islands) and the northeastern part of the Pacific Ocean are reported. M. acanthomus is an endemic of the eastern part of the Pacific Ocean where it is known along the coasts of America from California to the northern coast of Chile (approximately between 33°N and 21°S).     

Microsatellite DNA markers for population-genetic studies of blue mackerel (Scomber australasicus) and cross-specific amplification in S. japonicus

Blue mackerel (Scomber australasicus) is targeted by large-scale purse-seiners in the western North Pacific, and its stock structure is still contentious. Herein, we described 10 polymorphic microsatellite loci for blue mackerel. The number of alleles among 32 individuals surveyed ranged from five to 27 (average of 16.2 alleles per locus). Departures from Hardy-Weinberg expectation were observed at two loci. Cross-specific amplification in the congener, S. japonicus, was successful, except for one locus, revealed to be diagnostic for these congeners. These microsatellite loci will be useful tools to address queries in population genetic structure, fishery management unit and taxonomic species status in the genus Scomber.     

New species in the genus <Emphasis Type="Italic">Francisella</Emphasis> (Gammaproteobacteria; Francisellaceae); <Emphasis Type="Italic">Francisella piscicida</Emphasis> sp. nov. isolated from cod (<Emphasis Type="Italic">Gadus morhua</Emphasis>)

A Francisella strain, GM2212, previously isolated from moribund farmed Atlantic cod (Gadus morhua) in Norway, is closely related to Francisella philomiragia among Francisella spp. according to its complete 16S rDNA, 16S-23S intergenic spacer, 23S rDNA, 23S–5S intergenic spacer, 5S rDNA, FopA, lipoprotein TUL4 (LpnA), malate dehydrogenase and hypothetical lipoprotein (LpnB) sequences. A comparison between GM2212 and the type strain of Francisella philomiragia were performed by DNA–DNA hybridization and fatty acid analysis. The DNA–DNA hybridization showed a 70% similarity. The fatty acid analysis showed only minor differences between the Francisella isolates. Due to the inconclusive result from the DNA–DNA hybridisation, major emphasis concerning the status of this isolate is made on previously published molecular, phenotypic and biochemical characters. All characteristics taken together support the establishment of GM2212 as a novel species, for which the name Francisella piscicida sp. nov. is proposed (=CNCM I-3511^T = DSM 18777^T = LMG registration number not yet available).     

The genome of <Emphasis Type="Italic">Streptomyces rimosus</Emphasis> subsp. <Emphasis Type="Italic">rimosus</Emphasis> shows a novel structure compared to other <Emphasis Type="Italic">Streptomyces</Emphasis> using DNA/DNA microarray analysis

DNA/DNA genome microarray analysis together with genome sequencing suggests that the genome of members of the genus Streptomyces would seem to have a common structure including a linear genomic structure, a core of common syntenous Actinomycete genes, the presence of species specific terminal regions and two intermediate group of syntenous genes that seem to be genus specific. We analyzed Streptomyces species using DNA/DNA microarray comparative genome analysis. Only Streptomyces rimosus failed to give a congruent genome pattern for the genes found in Streptomyces coelicolor. We expanded the analysis to include a number of strains related to the type strain of S. rimosus and obtained a similar divergence from the main body of Streptomyces species. These strains showed very close identity to the original strain with no gene deletion or duplication detected. The 16S rRNA sequences of these S. rimosus strains were confirmed as very similar to the S. rimosus sequences available from the Ribosomal Database Project. When the SSU ribosomal RNA phylogeny of S. rimosus is analyzed, the species is positioned at the edge of the Streptomyces clade. We conclude that S. rimosus represents a distinct evolutionary lineage making the species a worthy possibility for genome sequencing.     

Revision of the genus <Emphasis Type="Italic">Melamphaes</Emphasis> (Melamphaidae). 5. Oligo-raker species: <Emphasis Type="Italic">M. indicus</Emphasis>, <Emphasis Type="Italic">M. eurous</Emphasis>, and <Emphasis Type="Italic">M. typhlops</Emphasis>

Three oligo-raker species (?19 rakers on the first gill arch) of the genus Melamphaes out of the “M. typhlops” group are considered. The validity of M. indicus Ebeling is restored. This species inhabits equatorial and tropical waters of the Indian Ocean and the western part of the Pacific Ocean. M. eurous sp. n., which is related to M. indicus, is described from equatorial waters of the eastern part of the Pacific Ocean. M. typhlops (Lowe) inhabiting the northern part of the Atlantic Ocean, from the equatorial zone about to 45° N, is redescribed.     

Introgressive hybridization between the Atlantic and Pacific herring (<Emphasis Type="Italic">Clupea harengus</Emphasis> and <Emphasis Type="Italic">Clupea pallasii</Emphasis>) in the White Sea,Barents and Kara Seas evidenced by microsatellites

In the North of Europe two sister species of herring—the Atlantic Clupea harengus and Pacific Clupea pallasii have post-glacially come into secondary contact. Although the breeding areas of the two species are thought to be separate, previous genetic studies based on allozyme and mitochondrial DNA data have supported the existence of introgression from the Atlantic herring to the Pacific herring. In this study, we employed microsatellite markers to explore the extent of introgressive hybridization between these fishes. Bayesian methods for assigning individuals to populations and identifying admixture proportions were applied to the genetic data based on ten microsatellites. Results indicated that 18 out of 464 Pacific herring (3.88%) were likely admixed. Some of the hybrid individuals contained genotypes suggesting that introgressive hybridization is an ongoing process. Despite some admixture, the Atlantic and Pacific herring gene pools remain sharply distinct (the average proportions of membership to “Atlantic” and “Pacific” clusters were Q_A = 0.998 and Q_P = 0.974, respectively), suggesting that hybridization was not frequent. The discovery of introgression among herring species opens new questions about the mechanisms underlying the observed pattern of genetic population differentiation in the North European Pacific herring.     

Phylogenetic Studies of <Emphasis Type="Italic">Gordonia</Emphasis> Species Based on <Emphasis Type="Italic">gyrB</Emphasis> and <Emphasis Type="Italic">secA1</Emphasis> Gene Analyses

Phylogenetic relations within the genus Gordonia were analyzed using partial gyrB and secA1 gene sequences of 23 type species in comparison with those of 16S rRNA gene. The gyrB and secA1 phylogenies showed agreement with that constructed using 16S rRNA gene sequences. The degrees of divergence of the gyrB and secA1 genes were approximately 3.4 and 1.7 times greater, respectively, than that of 16S rRNA gene. The gyrB gene showed more discriminatory power than either the secA1 or 16S rRNA gene, facilitating clear differentiation of any two Gordonia species using gyrB gene analysis. Our data indicate that gyrB and secA1 gene sequences are useful as markers for phylogenetic study and identification at the species level of the genus Gordonia.     

Detection of <Emphasis Type="Italic">Candida</Emphasis><Emphasis Type="Italic">dubliniensis</Emphasis> in Venezuela

Over the past decades there has been a significant increase in fungal infections caused by Candida species, and continues to be common in immunocompromised individuals infected with the human immunodeficiency virus (HIV). Although Candida albicans remains the fungal species most frequently isolated as an opportunistic oral pathogen, other non-albicans are often identified in this cohort of patients, including C. dubliniensis. This yeast is closely related to and shares many phenotypic characteristics with C. albicans. Colonies of these two species appear morphologically identical when not grown on special media. The shared phenotypic characteristics of C. dubliniensis and C. albicans suggest that many C. dubliniensis isolates may have been misidentified as C. albicans in the past. The present studies aim is to recover and identify C. dubliniensis, and presumptive clinical C. albicans, from the oral cavities of HIV-seropositive individuals, comparing conventional media to obtain a simple, low-cost and reliable identification system for C. dubliniensis. A total of 16 isolates (3,98%) had been obtained from 402 HIV infected individuals with recurrent oropharyngitis and were identified as C. dubliniensis. Out of these C. dubliniensis isolates 19% were resistant, with MICs above 64 μg/ml to fluconazole. This constitutes, to the authors knowledge the first recovery of this organism in Venezuela.     

<Emphasis Type="Italic">Drosophila</Emphasis><Emphasis Type="Italic">P</Emphasis> transposons of the urochordata <Emphasis Type="Italic">Ciona intestinalis</Emphasis>

P transposons belong to the eukaryotic DNA transposons, which are transposed by a cut and paste mechanism using a P-element-coded transposase. They have been detected in Drosophila, and reside as single copies and stable homologous sequences in many vertebrate species. We present the P elements Pcin1, Pcin2 and Pcin3 from Ciona intestinalis, a species of the most primitive chordates, and compare them with those from Ciona savignyi. They showed typical DNA transposon structures, namely terminal inverted repeats and target site duplications. The coding region of Pcin1 consisted of 13 small exons that could be translated into a P-transposon-homologous protein. C. intestinalis and C. savignyi displayed nearly the same phenotype. However, their P elements were highly divergent and the assumed P transposase from C. intestinalis was more closely related to the transposase from Drosophila melanogaster than to the transposase of C. savignyi. The present study showed that P elements with typical features of transposable DNA elements may be found already at the base of the chordate lineage. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Comparison of the <Emphasis Type="Italic">Coffea canephora</Emphasis> and <Emphasis Type="Italic">C. arabica</Emphasis> karyotype based on chromosomal DNA content

Nuclear genome size has been measured in various plants, seeing that knowledge of the DNA content is useful for taxonomic and evolutive studies, plant breeding programs and genome sequencing projects. Besides the nuclear DNA content, tools and protocols to quantify the chromosomal DNA content have been also applied, expanding the data about genomic structure. This study was conducted in order to calculate the Coffea canephora and Coffea arabica chromosomal DNA content, associating cytogenetic methodologies with flow cytometry (FCM) and image cytometry (ICM) tools. FCM analysis showed that the mean nuclear DNA content of C. canephora and C. arabica is 2C = 1.41 and 2.62 pg, respectively. The cytogenetic methodology provided prometaphase and metaphase cells exhibiting adequate chromosomes for the ICM measurements and karyogram assembly. Based on cytogenetic, FCM and ICM results; it was possible to calculate the chromosomal DNA content of the two species. The 1C chromosomal DNA content of C. canephora ranged from 0.09 (chromosome 1) to 0.05 pg (chromosome 11) and C. arabica from 0.09 (chromosome 1) to 0.03 pg (chromosome 22). The methodology presented in this study was suitable for DNA content measuring of each chromosome of C. canephora and C. arabica. The cytogenetic characterization and chromosomal DNA content analyses evidenced that C. arabica is a true allotetraploid originated from a cross between Coffea diploid species. Besides, the same analyses also reinforce that C. canephora is a possible progenitor of C. arabica.     

Genome size variation in some representatives of the genus <Emphasis Type="Italic">Tripleurospermum</Emphasis>

Genome size has been estimated by flow cytometry in 14 populations belonging to eight taxa (seven species, one of them with two varieties) of the genus Tripleurospermum. 2C nuclear DNA amounts range from 4.87 to 9.22 pg, and nuclear DNA amounts per basic chromosome set from 1.99 to 2.75 pg. Statistically significant differences depending on ploidy level, life cycle or environmental factors such as altitude have been found. Also, genome size is positively correlated with total karyotype length. The presence of rhizome is related to nuclear DNA content in these species.This work was supported by project BOS2001-3041-C02-01 of the Spanish government, and one of the authors (S.G.) received a predoctoral grant from the Spanish government.     

Molecular phylogenetic perspective on speciation in the genus <Emphasis Type="Italic">Sebastes</Emphasis> (Scorpaenidae) from the Northwest Pacific and the position of <Emphasis Type="Italic">Sebastes</Emphasis> within the subfamily Sebastinae

In the Northeast and the Northwest Pacific, 26 and 68 species of Sebastes occur, respectively, the similar appearance of some pairs of shallow coastal water species in the two regions having been interpreted as evidence either for migration of ancestral species or of morphological convergence. To distinguish between these competing hypotheses, a portion of the mitochondrial DNA (mtDNA) cytochrome b gene was sequenced from 23 species of Sebastes from the Northwest Pacific and phylogenetic trees constructed so as to include the existing data from Northeastern species of Sebastes. The results clearly indicated separate origins for external morphology in Northeast and Northwest Pacific species. The shallow coastal water species in the Northwest Pacific are thought to have diverged from about 9.8MYA. Furthermore, the phylogenetic position of Sebastes was inferred among the subfamily Sebastinae, Helicolenus, Hozukius, Sebastes, and Sebastiscus, the results suggesting that Helicolenus and Hozukius are more closely related to Sebastes than Sebastiscus.     

Isolation and characterization of polymorphic microsatellite markers in <Emphasis Type="Italic">Cupressus </Emphasis><Emphasis Type="Italic">chenggiana</Emphasis> S. Y. Hu (Cupressaceae)

Cupressus chenggiana S. Y. Hu (Cupressaceae) is an endemic and endangered conifer species in southwest China. In order to study the population genetics and design the effective conservation methods, we aimed to develop microsatellite primers for this species in the present study. We developed eight new microsatellite loci for this species through biotin capture method. Polymorphism of each locus was further assessed in 18 individuals from three geographically distant populations. The number of alleles per locus ranged from 6 to 11 with an average of 8.13. The observed and expected heterozygosities ranged from 0.219 to 0.296 and from 0.374 to 0.470, with averages of 0.254 and 0.417, respectively. We further found that three of nine microsatellite loci developed previously for another congeneric species showed polymorphic banding patters. We performed primer-crossing tests of these loci in the other two congeneric species which are closely related to C. chenggiana (C. gigantea and C. duclouxiana). These microsatellite markers would be effective for analyzing genetic diversity and population genetic structure of this species and its morphological differentiation with the close relatives.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-Mediated Transformation of Common Bermudagrass (<Emphasis Type="Italic">Cynodon dactylon</Emphasis>)

Common bermudagrass, Cynodon dactylon, is a widely used warm-season turf and forage species in the temperate and tropical regions of the world. We have been able to transform the species using Agrobacterium-mediated approach. In seven experiments reported here, a total of 67 plates of calluses and suspensions were infected with Agrobacterium tumefaciens strains, and nine hygromycin B resistant calluses were obtained after selection. Among them two green independent transgenic plants were recovered. The plants growing in pots looked relatively compact at the beginning, but the ploidy level of the plants, as determined by nuclear DNA content, was not altered.