A Polymerase Chain Reaction Method for Identification of Five Major Meloidogyne Species

A polymerase chain reaction (PCR) method for discriminating Meloidogyne incognita, M. arenaria, M. javanica, M. hapla, and M. chitwoodi was developed. Single juveniles were ruptured in a drop of water and added directly to a PCR reaction mixture in a microcentrifuge tube. Primer annealing sites were located in the 3'' portion of the mitochondrial gene coding for cytochrome oxidase subunit II and in the 16S rRNA gene. Following PCR amplification, fragments of three sizes were detected. The M. incognita and M. javanica reactions produced a 1.7-kb fragment; the M. arenaria reaction, a 1.1-kb fragment; and the M. hapla and M. chitwoodi reactions resulted in a 0.52-kb fragment. Digestion of the amplified product with restriction endonucleases allowed discrimination among species with identically sized amplification products. Dra I digestions of the 0.52-kb amplification product produced a characteristic three-banded pattern in M. chitwoodi, versus a two-banded pattern in M. hapla. Hinf I digestion of the 1.7-kb fragment produced a two-banded pattern in M. javanica, versus a three-banded pattern in M. incognita. Amplification and digestion of DNA from juveniles from single isolates of M. marylandi, M. naasi, and M. nataliei indicated that the diagnostic application of this primer set may extend to less frequently encountered Meloidogyne species.     

The use of an arbitrarily primed PCR product for the specific detection of Brucella

A 1.3 kb Brucella-specific DNA fragment produced through the use of arbitrarily primed polymerase chain reaction (AP-PCR) was tested for its specificity by DNA–DNA hybridization to Brucella and non-Brucella bacteria. The digoxigenin (DIG)-labelled 1.3 kb DNA fragment hybridized with Brucella abortus and Brucella melitensis but did not hybridize with other non-Brucella bacteria tested. The sensitivity of the reaction was determined; as little as 150 fg DNA or 30 Brucella cells could be detected. The specificity and sensitivity of the 1.3 kb DNA fragment combined with the simplicity and speed of the technique suggest the potential of this fragment as a DNA probe for the quick and reliable detection of Brucella organisms.     

Isolation and Confirmation of <Emphasis Type="Italic">Listeria</Emphasis> Species from Seafood off Goa Region by Polymerase Chain Reaction

Several food borne outbreaks have highlighted the importance of Listeria monocytogenes to the public health and have been recognized as an emerging, important food borne pathogen, and a causative agent of listerioses. A number of genes are involved in the manifestation of Listeria virulence, hlyA is one among them. In the present study, 111 marine fish samples including prawns, finfishes and bivalves were screened for the presence of Listeria species. The isolates were characterized biochemically and further L. monocytogenes were confirmed by polymerase chain reaction (PCR) technique using the hlyA gene as a tool to differentiate between L. monocytogenes and other non-pathogenic Listeria species. Out of 111 samples 5 (4.5%) samples were positive for L. monocytogenes. Among the three different types of samples bivalves were found to have maximum percent (12.5) of L. monocytogenes followed by prawns (3.84) and finfishes (2.9). Among all the 111 samples, 15 (13.51%) samples were positive for other Listeria species. It was observed that Listeria occurrence is more in shellfishes than in fin fishes. All the isolates were sensitive towards five different antibiotics in sequence ciprofloxacin > sulphafurazole > norfloxacin > ampicillin and gentamicin.     

Real-time PCR versus conventional PCR for malaria parasite detection in low-grade parasitemia

We have optimized a faster and cheaper real-time PCR and developed a conventional genus specific PCR based on 18S rRNA gene to detect malaria parasites in low-grade parasitemias. Additionally, we compared these PCRs to the OptiMAL-IT test. Since there is no consensus on choice of standard quantitative curve in real-time assays, we decided to investigate the performance of parasite DNA from three different sources: "genome", amplicon and plasmid. The amplicon curve showed the best efficiency in quantifying parasites. Both PCR assays detected 100% of the clinical samples tested; the sensitivity threshold was 0.5 parasite/mul and no PCR positive reaction occurred when malaria parasites were not present. Conversely, if OptiMAL-IT were employed for malaria diagnosis, 30% of false-negative results could be expected. We conclude that PCR assays have potential for detecting malaria parasites in asymptomatic infections, in evaluation of malaria vaccine molecule candidates, for screening blood donors, especially in endemic areas, or even in monitoring malaria therapy.     

Multiplex PCR detection of waterborne intestinal protozoa: microsporidia, Cyclospora, and Cryptosporidium

Recently, emerging waterborne protozoa, such as microsporidia, Cyclospora, and Cryptosporidium, have become a challenge to human health worldwide. Rapid, simple, and economical detection methods for these major waterborne protozoa in environmental and clinical samples are necessary to control infection and improve public health. In the present study, we developed a multiplex PCR test that is able to detect all these 3 major waterborne protozoa at the same time. Detection limits of the multiplex PCR method ranged from 10(1) to 10(2) oocysts or spores. The primers for microsporidia or Cryptosporidium used in this study can detect both Enterocytozoon bieneusi and Encephalitozoon intestinalis, or both Cryptosporidium hominis and Cryptosporidium parvum, respectively. Restriction enzyme digestion of PCR products with BsaBI or BsiEI makes it possible to distinguish the 2 species of microsporidia or Cryptosporidium, respectively. This simple, rapid, and cost-effective multiplex PCR method will be useful for detecting outbreaks or sporadic cases of waterborne protozoa infections.     

Revision of the subfamily Opiinae (Hymenoptera,Braconidae) from Hunan (China), including thirty-six new species and two new genera

The species of the subfamily Opiinae (Hymenoptera: Braconidae) from Hunan (Oriental China) are revised and illustrated. Thirty-six new species are described: Apodesmia bruniclypealis Li & van Achterberg, sp. n., Apodesmia melliclypealis Li & van Achterberg, sp. n., Areotetes albiferus Li & van Achterberg, sp. n., Areotetes carinuliferus Li & van Achterberg, sp. n., Areotetes striatiferus Li & van Achterberg, sp. n., Coleopioides diversinotum Li & van Achterberg, sp. n., Coleopioides postpectalis Li & van Achterberg, sp. n., Fopius dorsopiferus Li, van Achterberg & Tan, sp. n., Indiopius chenae Li & van Achterberg, sp. n., Opiognathus aulaciferus Li & van Achterberg, sp. n., Opiognathus brevibasalis Li & van Achterberg, sp. n., Opius crenuliferus Li & van Achterberg, sp. n., Opius malarator Li, van Achterberg & Tan, sp. n., Opius monilipalpis Li & van Achterberg, sp. n., Opius pachymerus Li & van Achterberg, sp. n., Opius songi Li & van Achterberg, sp. n., Opius youi Li & van Achterberg, sp. n., Opius zengi Li & van Achterberg, sp. n., Phaedrotoma acuticlypeata Li & van Achterberg, sp. n., Phaedrotoma angiclypeata Li & van Achterberg, sp. n., Phaedrotoma antenervalis Li & van Achterberg, sp. n., Phaedrotoma depressiclypealis Li & van Achterberg, sp. n., Phaedrotoma flavisoma Li & van Achterberg, sp. n., Phaedrotoma nigrisoma Li & van Achterberg, sp. n., Phaedrotoma protuberator Li & van Achterberg, sp. n., Phaedrotoma rugulifera Li & van Achterberg, sp. n., Li & van Achterberg,Phaedrotoma striatinota Li & van Achterberg, sp. n., Phaedrotoma vermiculifera Li & van Achterberg, sp. n., Rhogadopsis latipennis Li & van Achterberg, sp. n., Rhogadopsis longicaudifera Li & van Achterberg, sp. n., Rhogadopsis maculosa Li, van Achterberg & Tan, sp. n., Rhogadopsis obliqua Li & van Achterberg, sp. n., Rhogadopsis sculpturator Li & van Achterberg, sp. n., Utetes longicarinatus Li & van Achterberg, sp. n. and Xynobius notauliferus Li & van Achterberg, sp. n. Areotetes van Achterberg & Li, gen. n. (type species: Areotetes carinuliferus sp. n.) and Coleopioides van Achterberg & Li, gen. n. (type species: Coleopioides postpectalis sp. n. are described. All species are illustrated and keyed. In total 30 species of Opiinae are sequenced and the cladograms are presented. Neopius Gahan, 1917, Opiognathus Fischer, 1972, Opiostomus Fischer, 1972, and Rhogadopsis Brèthes, 1913, are treated as a valid genera based on molecular and morphological differences. Opius vittata Chen & Weng, 2005 (not Opius vittatus Ruschka, 1915), Opius ambiguus Weng & Chen, 2005 (not Wesmael, 1835) and Opius mitis Chen & Weng, 2005 (not Fischer, 1963) are primary homonymsandarerenamed into Phaedrotoma depressa Li & van Achterberg, nom. n., Opius cheni Li & van Achterberg, nom. n. andOpius wengi Li & van Achterberg, nom. n., respectively. Phaedrotoma terga (Chen & Weng, 2005) comb. n.,Diachasmimorpha longicaudata (Ashmead, 1905) and Biosteres pavitita Chen & Weng, 2005, are reported new for Hunan, Opiostomus aureliae (Fischer, 1957) comb. n. is new for China and Hunan; Xynobius maculipennis(Enderlein, 1912) comb. n. is new for Hunan and continental China and Rhogadopsis longuria (Chen & Weng, 2005) comb. n. is new for Hunan. The following new combinations are given: Apodesmia puncta (Weng & Chen, 2005) comb. n., Apodesmia tracta (Weng & Chen, 2005) comb. n., Areotetes laevigatus (Weng & Chen, 2005) comb. n., Phaedrotoma dimidia (Chen & Weng, 2005) comb. n., Phaedrotoma improcera (Weng & Chen, 2005) comb. n., Phaedrotoma amputata (Weng & Chen, 2005) comb. n., Phaedrotoma larga (Weng & Chen, 2005) comb. n., Phaedrotoma osculas (Weng & Chen, 2005) comb. n., Phaedrotoma postuma (Chen & Weng, 2005) comb. n., Phaedrotoma rugulosa (Chen & Weng, 2005) comb. n., Phaedrotoma tabularis (Weng & Chen, 2005) comb. n., Rhogadopsis apii (Chen & Weng, 2005) comb. n., Rhogadopsis dimidia (Chen & Weng, 2005) comb. n., Rhogadopsis diutia (Chen & Weng, 2005) comb. n., Rhogadopsis longuria (Chen & Weng, 2005) comb. n., Rhogadopsis pratellae(Weng & Chen, 2005) comb. n., Rhogadopsis pratensis (Weng & Chen, 2005) comb. n., Rhogadopsis sculpta (Chen & Weng, 2005) comb. n., Rhogadopsis sulcifer (Fischer, 1975) comb. n., Rhogadopsis tabidula(Weng & Chen, 2005) comb. n., Xynobius complexus (Weng & Chen, 2005) comb. n., Xynobius indagatrix (Weng & Chen, 2005) comb. n., Xynobius multiarculatus (Chen & Weng, 2005) comb. n.The following (sub)genera are synonymised: Snoflakopius Fischer, 1972, Jucundopius Fischer, 1984, Opiotenes Fischer, 1998, and Oetztalotenes Fischer, 1998, with Opiostomus Fischer, 1971; Xynobiotenes Fischer, 1998, with Xynobius Foerster, 1862; Allotypus Foerster, 1862, Lemnaphilopius Fischer, 1972, Agnopius Fischer, 1982, and Cryptognathopius Fischer, 1984, with Apodesmia Foerster, 1862; Nosopoea Foerster, 1862, Tolbia Cameron, 1907, Brachycentrus Szépligeti, 1907, Baeocentrum Schulz, 1911, Hexaulax Cameron, 1910, Coeloreuteus Roman, 1910, Neodiospilus Szépligeti, 1911, Euopius Fischer, 1967, Gerius Fischer, 1972, Grimnirus Fischer, 1972, Hoenirus Fischer, 1972, Mimirus Fischer, 1972, Gastrosema Fischer, 1972, Merotrachys Fischer, 1972, Phlebosema Fischer, 1972, Neoephedrus Samanta, Tamili, Saha & Raychaudhuri, 1983, Adontopius Fischer, 1984, Kainopaeopius Fischer, 1986, Millenniopius Fischer, 1996, and Neotropopius Fischer, 1999, with Phaedrotoma Foerster, 1862.     

应用RT—PCR检测流产胎儿组织中猪繁殖与呼吸综合征病毒

根据猪繁殖与呼吸综合征病毒（ＰＲＲＳＶ）美洲型膜蛋白和核衣壳蛋白基因序列,设计了一对含有ＥｃｏＲＩ和ＢａｍＨＩ酶切位点的引物,用ＲＴ－ＰＣＲ对四个流产猪场的病料进行了检测,扩增出约９１８ｂｐ的基因片段。通过病毒分离、酶切鉴定和序列分析证实为ＰＲＲＳＶ感染。结果说明应用所设计的引物进行ＲＴ－ＰＣＲ快速检测ＰＲＲＳ是可行的,为我国快速特异诊为ＰＲＲＳ和ＰＲＲＳＶ强毒株的深入研究奠定了基础。     

Evolutionary history of the Afro-Madagascan Ixora species (Rubiaceae): species diversification and distribution of key morphological traits inferred from dated molecular phylogenetic trees

Background and Aims

Previous work on the pantropical genus Ixora has revealed an Afro-Madagascan clade, but as yet no study has focused in detail on the evolutionary history and morphological trends in this group. Here the evolutionary history of Afro-Madagascan Ixora spp. (a clade of approx. 80 taxa) is investigated and the phylogenetic trees compared with several key morphological traits in taxa occurring in Madagascar.

Methods

Phylogenetic relationships of Afro-Madagascan Ixora are assessed using sequence data from four plastid regions (petD, rps16, rpoB-trnC and trnL-trnF) and nuclear ribosomal external transcribed spacer (ETS) and internal transcribed spacer (ITS) regions. The phylogenetic distribution of key morphological characters is assessed. Bayesian inference (implemented in BEAST) is used to estimate the temporal origin of Ixora based on fossil evidence.

Key Results

Two separate lineages of Madagascan taxa are recovered, one of which is nested in a group of East African taxa. Divergence in Ixora is estimated to have commenced during the mid Miocene, with extensive cladogenesis occurring in the Afro-Madagascan clade during the Pliocene onwards.

Conclusions

Both lineages of Madagascan Ixora exhibit morphological innovations that are rare throughout the rest of the genus, including a trend towards pauciflorous inflorescences and a trend towards extreme corolla tube length, suggesting that the same ecological and selective pressures are acting upon taxa from both Madagascan lineages. Novel ecological opportunities resulting from climate-induced habitat fragmentation and corolla tube length diversification are likely to have facilitated species radiation on Madagascar.     

Molecular identification and real-time quantitative PCR (qPCR) for rapid detection of Thelohanellus kitauei, a Myxozoan parasite causing intestinal giant cystic disease in the Israel carp

Intestinal giant-cystic disease (IGCD) of the Israel carp (Cyprinus carpio nudus) has been recognized as one of the most serious diseases afflicting inland farmed fish in the Republic of Korea, and Thelohanellus kitauei has been identified as the causative agent of the disease. Until now, studies concerning IGCD caused by T. kitauei in the Israel carp have been limited to morphological and histopathological examinations. However, these types of diagnostic examinations are relatively time-consuming, and the infection frequently cannot be detected in its early stages. In this study, we cloned the full-length 18S rRNA gene of T. kitauei isolated from diseased Israel carps, and carried out molecular identification by comparing the sequence with those of other myxosporeans. Moreover, conventional PCR and real-time quantitative PCR (qPCR) using oligonucleotide primers for the amplification of 18S rRNA gene fragment were established for further use as methods for rapid diagnosis of IGCD. Our results demonstrated that both the conventional PCR and real-time quantitative PCR systems applied herein are effective for rapid detection of T. kitauei spores in fish tissues and environmental water.     

Anaplasma platys: an improved PCR for its detection in dogs

This study compares two PCR assays for the detection of Anaplasma platys in dog blood using primers based on the A. platys 16S rRNA gene. The first approach utilized a standard PCR protocol composed of a single-step direct amplification using an Ehrlichia genus-specific primer set. The second assay being a nested PCR screen that first involved a universal bacterial primer set that amplified the majority of the 16S rRNA gene, followed by the nested round of PCR using an A. platys-specific primer set. Of the 22 dogs sampled, 10 were found to contain A. platys DNA using both protocols, and an additional two dogs were found positive using the nested technique. An extract of A. platys positive genomic DNA was serially diluted and comparison of sensitivities determined between the nested PCR, and a direct assay using A. platys-specific primers. The nested protocol demonstrated an increased sensitivity by at least 2 orders of magnitude when compared to the direct assay alone. Our results indicated that the nested PCR assay with its increased sensitivity would be useful for experimental research investigations as well as offer the potential for use as a routine test in diagnostic pathology.     

New Neotropical species of Chimarra (Trichoptera, Philopotamidae)

Ten new Neotropical species of Chimarra are described in the subgenera Chimarra, Chimarrita, and Otarrha. New species in the subgenus Chimarra include, in the Chimarra ortiziana group: Chimarra calorisp. n. (southeastern Brazil) and Chimarra onchyrhinasp. n. (Venezuela); in the Chimarra picea group: Chimarra inchoatasp. n. (Venezuela), Chimarra nicehuhsp. n. (Venezuela), and Chimarra sunimasp. n. (Colombia); and in the Chimarra poolei group: Chimarra caucasp. n. (Colombia) and Chimarra desiraesp. n. (Bolivia). New species in the subgenus Chimarrita include, in the Chimarra simpliciforma group: Chimarra curvipenissp. n. (SE Brazil) and Chimarra latiforcepssp. n. (SE Brazil). A single new species in the subgenus Otarrha is also described: Chimarra soroasp. n. (Cuba). Males and females for all of the new species are illustrated, except for Chimarra desirae, for which female specimens were unavailable. Additionally, the female of Chimarra (Chimarrita) camella, which was previously unknown, is illustrated.     

DNA Extraction from Protozoan Oocysts/Cysts in Feces for Diagnostic PCR

PCR detection of intestinal protozoa is often restrained by a poor DNA recovery or by inhibitors present in feces. The need for an extraction protocol that can overcome these obstacles is therefore clear. QIAamp® DNA Stool Mini Kit (Qiagen) was evaluated for its ability to recover DNA from oocysts/cysts directly from feces. Twenty-five Giardia-positive, 15 Cryptosporidium-positive, 15 Entamoeba histolytica-positive, and 45 protozoa-free samples were processed as control by microscopy and immunoassay tests. DNA extracts were amplified using 3 sets of published primers. Following the manufacturer''s protocol, the kit showed sensitivity and specificity of 100% towards Giardia and Entamoeba. However, for Cryptosporidium, the sensitivity and specificity were 60% (9/15) and 100%, respectively. A series of optimization experiments involving various steps of the kit''s protocol were conducted using Cryptosporidium-positive samples. The best DNA recoveries were gained by raising the lysis temperature to the boiling point for 10 min and the incubation time of the InhibitEX tablet to 5 min. Also, using a pre-cooled ethanol for nucleic acid precipitation and small elution volume (50-100 µl) were valuable. The sensitivity of the amended protocol to Cryptosporidium was raised to 100%. Cryptosporidium DNA was successfully amplified by either the first or the second primer set. When applied on parasite-free feces spiked with variable oocysts/cysts counts, ≈ 2 oocysts/cysts were theoretically enough for detection by PCR. To conclude, the Qiagen kit with the amended protocol was proved to be suitable for protozoan DNA extraction directly from feces and support PCR diagnosis.     

A real-time PCR diagnostic method for detection of Naegleria fowleri

Naegleria fowleri is a free-living amoeba that can cause primary amoebic meningoencephalitis (PAM). While, traditional methods for diagnosing PAM still rely on culture, more current laboratory diagnoses exist based on conventional PCR methods; however, only a few real-time PCR processes have been described as yet. Here, we describe a real-time PCR-based diagnostic method using hybridization fluorescent labelled probes, with a LightCycler instrument and accompanying software (Roche), targeting the Naegleria fowleriMp2Cl5 gene sequence.Using this method, no cross reactivity with other tested epidemiologically relevant prokaryotic and eukaryotic organisms was found. The reaction detection limit was 1 copy of the Mp2Cl5 DNA sequence. This assay could become useful in the rapid laboratory diagnostic assessment of the presence or absence of Naegleria fowleri.     

Phylogenetic utility of the nuclear genes AGAMOUS 1 and PHYTOCHROME B in palms (Arecaceae): an example within Bactridinae

Background and Aims

Molecular phylogenetic studies of palms (Arecaceae) have not yet provided a fully resolved phylogeny of the family. There is a need to increase the current set of markers to resolve difficult groups such as the Neotropical subtribe Bactridinae (Arecoideae: Cocoseae). We propose the use of two single-copy nuclear genes as valuable tools for palm phylogenetics.

Methods

New primers were developed for the amplification of the AGAMOUS 1 (AG1) and PHYTOCHROME B (PHYB) genes. For the AGAMOUS gene, the paralogue 1 of Elaeis guineensis (EgAG1) was targeted. The region amplified contained coding sequences between the MIKC K and C MADS-box domains. For the PHYB gene, exon 1 (partial sequence) was first amplified in palm species using published degenerate primers for Poaceae, and then specific palm primers were designed. The two gene portions were sequenced in 22 species of palms representing all genera of Bactridinae, with emphasis on Astrocaryum and Hexopetion, the status of the latter genus still being debated.

Key Results

The new primers designed allow consistent amplification and high-quality sequencing within the palm family. The two loci studied produced more variability than chloroplast loci and equally or less variability than PRK, RPBII and ITS nuclear markers. The phylogenetic structure obtained with AG1 and PHYB genes provides new insights into intergeneric relationships within the Bactridinae and the intrageneric structure of Astrocaryum. The Hexopetion clade was recovered as monophyletic with both markers and was weakly supported as sister to Astrocaryum sensu stricto in the combined analysis. The rare Astrocaryum minus formed a species complex with Astrocaryum gynacanthum. Moreover, both AG1 and PHYB contain a microsatellite that could have further uses in species delimitation and population genetics.

Conclusions

AG1 and PHYB provide additional phylogenetic information within the palm family, and should prove useful in combination with other genes to improve the resolution of palm phylogenies.     

Validation of the official control method based on Polymerase Chain Reaction (PCR) for identification of authorised probiotic yeast in animal feed

Council Directive 70/524/EEC regulates the application of probiotic (microorganisms) additives in feeding stuffs. In the present study a method for the differentiation and strain identification of authorised probiotic Saccharomyces cereviseae strains in feeding stuffs by Polymerase Chain Reaction (PCR) was validated. Four different samples of animal feeding stuffs containing yeast at levels between 10(5) to 10(7) CFU/g were examined. Samples were enumerated on chloramphenicol glucose yeast extract agar and colonies were selected from these plates for DNA extraction and subsequent analysis. The PCR method using delta sequence primers produced an 'amplified sequence polymorphism' characteristic for the test strain. Feeds supplemented with one of four probiotic yeast strains each were analysed by seven of nine invited laboratories. All laboratories returned valid results with the exception of one laboratory that had insufficiently separated bands on the gel. The method had a good reproducibility for probiotic yeast isolates from feed of all four authorised probiotic yeast strains (APYS) CBS 493.94, APYS CNCM 1-1079, APYS CNCM 1-1077, APYS NCYC SC47 and of a commercially available yeast reference strain, NCYC 81. The PCR method is to be considered by CEN and ISO as official control method for identification of authorised probiotic Saccharomyces cerevisiae strains from feeding stuffs.     

Cytotype diversity and genome size variation in eastern Asian polyploid Cardamine (Brassicaceae) species

Background and Aims

Intraspecific ploidy-level variation is an important aspect of a species'' genetic make-up, which may lend insight into its evolutionary history and future potential. The present study explores this phenomenon in a group of eastern Asian Cardamine species.

Methods

Plant material was sampled from 59 localities in Japan and Korea, which were used in karyological (chromosome counting) and flow cytometric analyses. The absolute nuclear DNA content (in pg) was measured using propidium iodide and the relative nuclear DNA content (in arbitrary units) was measured using 4,6-diamidino-2-phenylindole fluorochrome.

Key Results

Substantial cytotype diversity was found, with strikingly different distribution patterns between the species. Two cytotypes were found in C. torrentis sensu lato (4x and 8x, in C. valida and C. torrentis sensu stricto, respectively), which displays a north–south geographical pattern in Japan. Hypotheses regarding their origin and colonization history in the Japanese archipelago are discussed. In Korean C. amaraeiformis, only tetraploids were found, and these populations may in fact belong to C. valida. C. yezoensis was found to harbour as many as six cytotypes in Japan, ranging from hexa- to dodecaploids. Ploidy levels do not show any obvious geographical pattern; populations with mixed ploidy levels, containing two to four cytotypes, are frequently observed throughout the range. C. schinziana, an endemic of Hokkaido, has hexa- and octoploid populations. Previous chromosome records are also revised, showing that they are largely based on misidentified material or misinterpreted names.

Conclusions

Sampling of multiple populations and utilization of the efficient flow cytometric approach allowed the detection of large-scale variation in ploidy levels and genome size variation attributable to aneuploidy. These data will be essential in further phylogenetic and evolutionary studies.     

Development of a real-time PCR assay for detection of Mytilus species specific alleles: Application to a sampling survey in Scotland

Shellfish aquaculture is a growing industry in Scotland, dominated by the production of the mussel Mytilus edulis, the native species. Recently the discovery of Mytilus galloprovincialis and Mytilus trossulus together with M. edulis and all 3 hybrids in cultivation in some Scottish sea lochs led to questions regarding the distribution of mussel species in Scotland. The establishment of an extensive sampling survey, involving the collection of mussels at 34 intertidal sites and 10 marinas around Scotland, motivated the development of a high-throughput method for identification of Mytilus alleles from samples. Three Taqman®-MGB probes and one set of primers were designed, based on the previously described Me 15/16 primers targeting the adhesive protein gene sequence, and samples were screened for the presence of M. edulis, M. galloprovincialis and M. trossulus alleles using real-time PCR. Mytilus edulis alleles were identified in samples from all 44 sites. Mytilus galloprovincialis alleles were found together with M. edulis alleles extensively in northern parts of the west and east coasts. Mytilus trossulus alleles were identified in samples from 6 sites in the west and south-west of Scotland. Because M. trossulus is generally undesirable in cultivation and therefore preventing the geographical spread of this species across Scotland is considered beneficial by the shellfish aquaculture industry, these 6 samples were further analysed for genotype frequencies using conventional PCR. Although distribution of the non-native species M. galloprovincialis and M. trossulus have proven to be more widespread than previously thought, there is no evidence from our study of either M. trossulus or M. galloprovincialis acting as an invasive species in Scotland. The real-time PCR method developed in this study has proven to be a rapid and effective tool for the identification of M. edulis, M. galloprovincialis and M. trossulus alleles from samples and should prove useful in future surveys, ecological or aquaculture management related studies in both unispecific and mixed species areas of these species.