Multi-locus microsatellite analysis supports the hypothesis of an autochthonous focus of Echinococcus multilocularis in northern Italy

Echinococcus multilocularis is characterised by a wide geographical distribution, encompassing three continents (North America, Asia and Europe) yet very low genetic variability is documented. Recently, this parasite has been detected in red foxes (Vulpes vulpes) circulating in an Alpine region of Italy, close to Austria. This finding raised the question as to whether an autochthonous cycle exists in Italy or whether the infected foxes originated from the neighbouring regions of Austria. Studies have shown that multi-locus microsatellite analysis can identify genomic regions carrying mutations that result in a local adaptation. We used a tandem repeated multi-locus microsatellite (EmsB) to evaluate the genetic differences amongst adult worms of E. multilocularis collected in Italy, worms from neighbouring Austria and from other European and extra-European countries. Fluorescent PCR was performed on a panel of E. multilocularis samples to assess intra-specific polymorphism. The analysis revealed four closed genotypes for Italian samples of E. multilocularis which were unique compared with the other 25 genotypes from Europe and the five genotypes from Alaska. An analysis in the Alpine watershed, comparing Italian adult worms with those from neighbouring areas in Austria, showed a unique cluster for Italian samples. This result supports the hypothesis of the presence of an autochthonous cycle of E. multilocularis in Italy. EmsB can be useful for 'tracking' the source of infection of this zoonotic parasite and developing appropriate measures for preventing or reducing the risk of human alveolar echinococcosis.

Population structure of spotted sunfish (Lepomis punctatus) in the Florida Everglades as revealed by DNA microsatellite analysis

Genetic diversity can be used to describe patterns of gene flow within and between local and regional populations. The Florida Everglades experiences seasonal fluctuations in water level that can influence local population extinction and recolonization dynamics. In addition, this expansive wetland has been divided into water management regions by canals and levees. These combined factors can affect genetic diversity and population structure of aquatic organisms in the Everglades. We analyzed allelic variation at six DNA microsatellite loci to examine the population structure of spotted sunfish (Lepomis punctatus) from the Everglades. We tested the hypothesis that recurrent local extinction and recent regional divisions have had an effect on patterns of genetic diversity. No marked differences were observed in comparisons of the heterozygosity values of sites within and among water management units. No evidence of isolation by distance was detected in a gene flow and distance correlation between subpopulations. Confidence intervals for the estimated F-statistic values crossed zero, indicating that there was no significant genetic difference between subpopulations within a region or between regions. Notably, the genetic variation among subpopulations in a water conservation area was greater than variation among regions (Fsp>FPT). These data indicate that the spatial scale of recolonization following local extinction appears to be most important within water management units.

Microsatellite analysis of pacu broodstocks used in the stocking program of Paranapanema River, Brazil

O monitoramento da diversidade genética é fundamental em um programa de repovoamento. Avaliouse a diversidade genética de pacu Piaractus mesopotamicus (Holmberg, 1887) em duas estações de piscicultura em Andirá -Paraná, Brasil, utilizadas no programa de repovoamento do Rio Paranapanema. Foram amplificados seis loci microssatélite para avaliar 60 amostras de nadadeira. O estoque de reprodutores B apresentou maior número de alelos e heterozigose (alelos: 22 e H O: 0,628) que o estoque de reprodutores A (alelos: 21 e H O: 0,600). Alelos com baixos níveis de frequência foram observados nos dois estoques. Os coeficientes positivos de endogamia no locus Pme2 (estoque A: F IS = 0,30 e estoque B: F IS = 0,20), Pme5 (estoque B: F IS = 0,15), Pme14 (estoque A: F IS = 0,07) e Pme28 (estoque A: F IS = 0,24 e estoque B: F IS = 0,20), indicaram deficiência de heterozigotos. Foi detectada a presença de um alelo nulo no lócus Pme2. As estimativas negativas nos loci Pme4 (estoque A: F IS = -0,43 e estoque B: F IS= -0,37), Pme5 (estoque A: F IS = - 0,11), Pme14 (estoque B: F IS = - 0,15) e Pme32 (estoque A: F IS = - 0,93 e estoque B: F IS = - 0,60) foram indicativas de excesso de heterozigotos. Foi evidenciado desequilíbrio de ligação e riqueza alélica baixa só no estoque A. A diversidade genética de Nei foi alta nos dois estoques. A distância (0,085) e identidade (0,918) genética mostraram similaridade entre os estoques, o qual reflete uma possível origem comum. 6,05% da variância genética total foi devida a diferenças entre os estoques. Foi observado um recente efeito gargalo nos dois estoques. Os resultados indicaram uma alta diversidade genética nos estoques de reprodutores e baixa diferenciação genética entre eles, o que foi causado pelo manejo reprodutivo das pisciculturas, redução do tamanho populacional e intercâmbio genético entre as pisciculturas.

Genetic analysis reveals two stocks of skipjack tuna (Katsuwonus pelamis) in the north western Indian Ocean

Skipjack (SJT) (Katsuwonus pelamis) is a medium sized, pelagic, highly dispersive tuna species that occurs widely across tropical and subtropical waters. SJT constitute the largest tuna fishery in the Indian Ocean, and are currently managed as a single stock. Patterns of genetic variation in a mtDNA gene and 6 microsatellite loci were examined to test for stock structure in the northwestern Indian Ocean. 324 individuals were sampled from five major fishing grounds around Sri Lanka, and single sites in the Maldive Islands and the Laccadive Islands. Phylogenetic reconstruction of mtDNA revealed two coexisting divergent clades in the region. AMOVA (Analysis of Molecular Variance) of mtDNA data revealed significant genetic differentiation among sites (ΦST = 0.2029, P < 0.0001), also supported by SAMOVA results. AMOVA of microsatellite data also showed significant differentiation among most sampled sites (FST = 0.0256, P<0.001) consistent with the mtDNA pattern. STRUCTURE analysis of the microsatellite data revealed two differentiated stocks. While the both two marker types examined identified two genetic groups, microsatellite analysis indicates that the sampled SJT are likely to represent individuals sourced from discrete breeding grounds that are mixed in feeding grounds in Sri Lankan waters.

Piecing together an integrative taxonomic puzzle: microsatellite, wing shape and aedeagus length analyses of Bactrocera dorsalis s.l. (Diptera: Tephritidae) find no evidence of multiple lineages in a proposed contact zone along the Thai/Malay Peninsula

Bactrocera dorsalis (Hendel) and B. papayae Drew & Hancock represent a closely related sibling species pair for which the biological species limits are unclear; i.e., it is uncertain if they are truely two biological species, or one biological species which has been incorrectly taxonomically split. The geographic ranges of the two taxa are thought to abut or overlap on or around the Isthmus of Kra, a recognised biogeographic barrier located on the narrowest portion of the Thai Peninsula. We collected fresh material of B. dorsalis sensu lato (i.e., B. dorsalis sensu stricto + B. papayae) in a north-south transect down the Thai Peninsula, from areas regarded as being exclusively B. dorsalis s.s., across the Kra Isthmus, and into regions regarded as exclusively B. papayae. We carried out microsatellite analyses and took measurements of male genitalia and wing shape. Both the latter morphological tests have been used previously to separate these two taxa. No significant population structuring was found in the microsatellite analysis and results were consistent with an interpretation of one, predominantly panmictic population. Both morphological datasets showed consistent, clinal variation along the transect, with no evidence for disjunction. No evidence in any tests supported historical vicariance driven by the Isthmus of Kra, and none of the three datasets supported the current taxonomy of two species. Rather, within and across the area of range overlap or abutment between the two species, only continuous morphological and genetic variation was recorded. Recognition that morphological traits previously used to separate these taxa are continuous, and that there is no genetic evidence for population segregation in the region of suspected species overlap, is consistent with a growing body of literature that reports no evidence of biological differentiation between these taxa.

Sex-linked microsatellite marker detected in the female gametophytes of Undaria pinnatifida (Phaeophyta)

P>In our microsatellite analysis of three male and three female gametophytes of Undaria pinnatifida (Harv.) Suringar, a microsatellite marker (part of the locus Up-AC-2A8, GenBank accession no. AY738602.1) was only polymerase chain reaction-amplified in three female gametophytes. This putative female-specific marker was further tested by the use of 32 male and 21 female gametophytes maintained in the Marine Biological Culture Collection Centre, China. In addition, three sporophytes were included for confirmation. Results showed that the marker was present in all of the female gametophytes and sporophyte cultures, but absent in all of the male gametophytes. To our knowledge, this is the first sex-related marker ever reported in U. pinnatifida. The discovery of this marker will accelerate gender identification and shed light on our understanding of the mechanisms of sex determination at a molecular level in this commercially important seaweed.

Whole-genome genotyping of grape using a panel of microsatellite

The use of microsatellite markers in large-scale genetic studies is limited by its low throughput and high cost and labor requirements. Here, we provide a panel of 45 multiplex PCRs for fast and cost-efficient genome-wide fluorescence-based microsatellite analysis in grapevine. The developed multiplex PCRs panel (with up to 15-plex) enables the scoring of 270 loci covering all the grapevine genome (9 to 20 loci/chromosome) using only 45 PCRs and sequencer runs. The 45 multiplex PCRs were validated using a diverse grapevine collection of 207 accessions, selected to represent most of the cultivated Vitis vinifera genetic diversity. Particular attention was paid to quality control throughout the whole process (assay replication, null allele detection, ease of scoring). Genetic diversity summary statistics and features of electrophoretic profiles for each studied marker are provided, as are the genotypes of 25 common cultivars that could be used as references in other studies.

CDKN2A/p16 is inactivated in most melanoma cell lines

The CDKN2A gene maps to chromosome 9p21-22 and is responsible for melanoma susceptibility in some families. Its product, p16, binds specifically to CDK4 and CDK6 in vitro and in vivo, inhibiting their kinase activity. CDKN2A is homozygously deleted or mutated in a large proportion of tumor cell lines and some primary tumors, including melanomas. The aim of this study was to investigate the involvement of CDKN2A and elucidate the mechanisms of p16 inactivation in a panel of 60 cell lines derived from sporadic melanomas. Twenty-six (43%) of the melanoma lines were homozygously deleted for CDKN2A, and an additional 15 (25%) lines carried missense, nonsense, or frameshift mutations. All but one of the latter group were shown by microsatellite analysis to be hemizygous for the region of 9p surrounding CDKN2A. p16 was detected by Western blotting in only five of the cell lines carrying mutations. Immunoprecipitation of p16 in these lines, followed by Western blotting to detect the coprecipitation of CDK4 and CDK6, revealed that p16 was functionally compromised in all cell lines but the one that carried a heterozygous CDKN2A mutation. In the remaining 19 lines that carried wild-type CDKN2A alleles, Western blot analysis and immunoprecipitation indicated that 11 cell lines expressed a wild-type protein. Northern blotting was performed on the remaining eight cell lines and revealed that one cell line carried an aberrantly sized RNA transcript, and two other cell lines failed to express RNA. The promoter was found to be methylated in five cell lines that expressed CDKN2A transcript but not p16. Presumably, the message seen by Northern blotting in these cell lines is the result of cross-hybridization of the total cDNA probe with the exon 1beta transcript. Microsatellite analysis revealed that the majority of these cell lines were hemi/homozygous for the region surrounding CDKN2A, indicating that the wild-type allele had been lost. In the 11 cell lines that expressed functional p16, microsatellite analysis revealed loss of heterozygosity at the markers immediately surrounding CDKN2A in five cases, and the previously characterized R24C mutation of CDK4 was identified in one of the remaining 6 lines. These data indicate that 55 of 60 (92%) melanoma cell lines demonstrated some aberration of CDKN2A or CDK4, thus suggesting that this pathway is a primary genetic target in melanoma development.

Genetic diversity in three redclaw crayfish (Cherax quadricarinatus, von Martens) lines developed in culture in China

The redclaw crayfish Cherax quadricarinatus (von Martens) accounts for the entire commercial production of freshwater crayfish in Australia. Two forms have been recognized, an 'Eastern' form in northern Queensland and a 'Western' form in the Northern Territory and far northern Western Australia. To date, only the Eastern form has been exported overseas for culture (including to China). The genetic structure of three Chinese redclaw crayfish culture lines from three different geographical locations in China (Xiamen in Fujian Province, Guangzhou in Guangdong Province and Chongming in Shanghai) were investigated for their levels and patterns of genetic diversity using microsatellite markers. Twenty-eight SSR markers were isolated and used to analyse genetic diversity levels in three redclaw crayfish culture lines in China. This study set out to improve the current understanding of the molecular genetic characteristics of imported strains of redclaw crayfish reared in China. Microsatellite analysis revealed moderate allelic and high gene diversity in all three culture lines. Polymorphism information content estimates for polymorphic loci varied between 0.1168 and 0.8040, while pairwise F ST values among culture lines were moderate (0.0020-0.1244). The highest estimate of divergence was evident between the Xiamen and Guangzhou populations.

Confirmation that Xq27 and Xq28 are susceptibility loci for migraine in independent pedigrees and a case-control cohort

Investigations into migraine genetics have suggested that susceptibility loci exist on the X chromosome. These reports are supported by evidence that demonstrates male probands as having a higher proportion of affected first-degree relatives as well as the female preponderance of 3:1 that the disorder displays. We have previously implicated the Xq24-28 locus in migraine using two independent multigenerational Australian pedigrees that demonstrated excess allele sharing at the Xq24, Xq27 and Xq28 loci. Here, we expand this work to investigate a further six independent migraine pedigrees using 11 microsatellite markers spanning the Xq27–28 region. Furthermore, 11 candidate genes are investigated in an Australian case-control cohort consisting of 500 cases and 500 controls. Microsatellite analysis showed evidence of excess allele sharing to the Xq27 marker DXS8043 (LOD* 1.38 P = 0.005) in MF879 whilst a second independent pedigree showed excess allele sharing to DXS8061 at Xq28 (LOD* 1.5 P = 0.004). Furthermore, analysis of these key markers in a case control cohort showed significant association to migraine in females at the DXS8043 marker (T1 P = 0.009) and association with MO at DXS8061 (T1 P = 0.05). Further analysis of 11 key genes across these regions showed significant association of a three-marker risk haplotype in the NSDHL gene at Xq28 (P = 0.0082). The results of this study add further support to the presence of migraine susceptibility loci on chromosome Xq27 and Xq28 as well as point to potential candidate genes in the regions that warrant further investigation.

Common origins of MDA-MB-435 cells from various sources with those shown to have melanoma properties

Recently, the tissue origin of MDA-MB-435 cell line has been the subject of considerable debate. In this study, we set out to determine whether MDA-MB-435-DTP cells shown to express melanoma-specific genes were identical to various other MDA-MB-435 cell stocks worldwide. CGH-microarray, genetic polymorphism genotyping, microsatellite fingerprint analysis and/or chromosomal number confirmed that the MDA-MB-435 cells maintained at the Lombardi Comprehensive Cancer Center (MDA-MB-435-LCC) are almost identical to the MDA-MB-435-DTP cells, and showed a very similar profile to those obtained from the same original source (MD Anderson Cancer Center) but maintained independently (MDA-MB-435-PMCC). Gene expression profile analy-sis confirmed common expression of genes among different MDA-MB-435-LCC cell stocks, and identified some unique gene products in MDA-MB-435-PMCC cells. RT-PCR analysis confirmed the expression of the melanoma marker tyrosinase across multiple MDA-MB-435 cell stocks. Collectively, our results show that the MDA-MB-435 cells used widely have identical origins to those that exhibit a melanoma-like gene expression signature, but exhibit a small degree of genotypic and phenotypic drift.

LCC15-MB cells are MDA-MB-435 : a review of misidentified breast and prostate cell lines

Current stocks of the LCC15-MB cell line, which we originally isolated from a human breast-bone metastasis, were found to be genetically matched to the MDA-MB-435 cell line from the Lombardi Cancer Center (MDA-MB-435-LCC) using comparative genomic hybridisation, DNA microsatellite analysis and chromosomal number. LCC15-MB stocks used for our previously published studies as well as the earliest available LCC15-MB cells also showed identity to MDA-MB-435-LCC cells. The original karyotype reported for LCC15-MB cells was considerably different to that of MDA-MB-435 cells, indicating that the original LCC15-MB cells were lost to contamination by MDA-MB-435-LCC cells. Chromosome number is the simplest test to distinguish original LCC 15-MB cells (n ∼ 75) from MDA-MB-435 (n ∼ 52). Collectively, our results prove that LCC15-MB cells currently available are MDA-MB-435 cells and we suggest their re-designation as MDA-MB-435-LCC15 cells. We also review the known misclassification of breast and prostate cancer cell lines to date and have initiated a register maintained at http://www.svi.edu.au/cell_lines_registry.doc.

Molecular Alterations in Asbestos-Related Lung Cancer

Background: Asbestos is a well known cancer-causing mineral fibre, which has a synergistic effect on lung cancer risk in combination with tobacco smoking. Several in vitro and in vivo experiments have demonstrated that asbestos can evoke chromosomal damage and cause alterations as well as gene expression changes. Lung tumours, in general, have very complex karyotypes with several recurrently gained and lost chromosomal regions and this has made it difficult to identify specific molecular changes related primarily to asbestos exposure. The main aim of these studies has been to characterize asbestos-related lung cancer at a molecular level. Methods: Samples from asbestos-exposed and non-exposed lung cancer patients were studied using array comparative genomic hybridization (aCGH) and fluorescent in situ hybridization (FISH) to detect copy number alterations (CNA) as well as microsatellite analysis to detect allelic imbalance (AI). In addition, asbestos-exposed cell lines were studied using gene expression microarrays. Results: Eighteen chromosomal regions showing differential copy number in the lung tumours of asbestos-exposed patients compared to those of non-exposed patients were identified. The most significant differences were detected at 2p21-p16.3, 5q35.3, 9q33.3-q34.11, 9q34.13-q34.3, 11p15.5, 14q11.2 and 19p13.1-p13.3 (p<0.005). The alterations at 2p and 9q were validated and characterized in detail using AI and FISH analysis in a larger study population. Furthermore, in vitro studies were performed to examine the early gene expression changes induced by asbestos in three different lung cell lines. The results revealed specific asbestos-associated gene expression profiles and biological processes as well as chromosomal regions enriched with genes believed to contribute to the common asbestos-related responses in the cell lines. Interestingly, the most significant region enriched with asbestos-response genes was identified at 2p22, close to the previously identified region showing asbestos-related CNA in lung tumours. Additionally, in this thesis, the dysregulated biological processes (Gene Ontology terms) detected in the cell line experiment were compared to dysregulated processes identified in patient samples in a later study (Ruosaari et al., 2008a). Commonly affected processes such as those related to protein ubiquitination, ion transport and surprisingly sensory perception of smell were identified. Conclusions: The identification of specific CNA and dysregulated biological processes shed some light on the underlying genes acting as mediators in asbestos-related lung carcinogenesis. It is postulated that the combination of several asbestos-specific molecular alterations could be used to develop a diagnostic method for the identification of asbestos-related lung cancer.