Beyond barcoding: A mitochondrial genomics approach to molecular phylogenetics and diagnostics of blowflies (Diptera: Calliphoridae)

Members of the Calliphoridae (blowflies) are significant for medical and veterinary management, due to the ability of some species to consume living flesh as larvae, and for forensic investigations due to the ability of others to develop in corpses. Due to the difficulty of accurately identifying larval blowflies to species there is a need for DNA-based diagnostics for this family, however the widely used DNA-barcoding marker, cox1, has been shown to fail for several groups within this family. Additionally, many phylogenetic relationships within the Calliphoridae are still unresolved, particularly deeper level relationships. Sequencing whole mt genomes has been demonstrated both as an effective method for identifying the most informative diagnostic markers and for resolving phylogenetic relationships. Twenty-seven complete, or nearly so, mt genomes were sequenced representing 13 species, seven genera and four calliphorid subfamilies and a member of the related family Tachinidae. PCR and sequencing primers developed for sequencing one calliphorid species could be reused to sequence related species within the same superfamily with success rates ranging from 61% to 100%, demonstrating the speed and efficiency with which an mt genome dataset can be assembled. Comparison of molecular divergences for each of the 13 protein-coding genes and 2 ribosomal RNA genes, at a range of taxonomic scales identified novel targets for developing as diagnostic markers which were 117–200% more variable than the markers which have been used previously in calliphorids. Phylogenetic analysis of whole mt genome sequences resulted in much stronger support for family and subfamily-level relationships. The Calliphoridae are polyphyletic, with the Polleninae more closely related to the Tachinidae, and the Sarcophagidae are the sister group of the remaining calliphorids. Within the Calliphoridae, there was strong support for the monophyly of the Chrysomyinae and Luciliinae and for the sister-grouping of Luciliinae with Calliphorinae. Relationships within Chrysomya were not well resolved. Whole mt genome data, supported the previously demonstrated paraphyly of Lucilia cuprina with respect to L. sericata and allowed us to conclude that it is due to hybrid introgression prior to the last common ancestor of modern sericata populations, rather than due to recent hybridisation, nuclear pseudogenes or incomplete lineage sorting.     

26S rDNA-RFLP分析在非酿酒酵母菌分类研究中的应用

使用26S rDNA-RFLP分析,分析了分离自甘肃莫高葡萄酒厂的29株非酿酒酵母菌,被测菌株被分为10个类型。通过26S rDNA D1/D2区序列分析验证,证明此方法在葡萄酿酒酵母菌种多样性研究中良好的应用价值。     

Association of APOE, GCPII and MMP9 polymorphisms with common diseases and lipid levels in an older adult/elderly cohort

Background and aims

The characterization of candidate gene polymorphisms in elderly populations is an important tool for the identification of risk factors for age-related diseases and conditions. We aimed to genotype the APOE polymorphisms (rs429358 and rs7412), rs61886492 (1561C>T) and rs202720 of GCPII gene and rs3918242 (− 1562C>T) of MMP9 gene in an older-adult/elderly cohort from Cuiabá city, Mato Grosso Brazil as well as to characterize risk factors for morbidities and conditions affecting this cohort.

Methods

The studied population consisted of 570 subjects from Cuiabá city, Brazil, who were subjected to clinical interviews and blood collection for laboratory examinations and DNA extraction. Restriction Fragment Length Polymorphism Polymerase Chain Reaction (RFLP-PCR), sequence-specific primer PCR (SSP-PCR) and TaqMan® allelic discrimination assay were used for genotyping.

Results

The frequencies of APOE ε2 and ε4 were 6.6% and 14.8%, respectively, and the frequencies of GCPII rs61886492 T allele, GCPII rs202720 C allele and MMP9 rs3918242 T allele were, respectively, 3.0%, 26.6% and 10.1%. Significant associations between APOE ε2 allele with lower total cholesterol and LDL-cholesterol were found. In addition, MMP9 rs3918242 T allele was associated with higher LDL-cholesterol levels, suggesting a link between lipid metabolism alteration and cardiovascular disease.

Conclusions

The present findings contributed to characterize risk factors specific for the studied population and to better understand the molecular physiopathology of common morbidities and conditions affecting older-adult/elderly people.     

Impact of rifting and hydrography on the genetic structure of Clarias gariepinus in eastern Africa

In a study of the genetic structure of Clarias gariepinus at the ND5 and ND6 loci of mitochondrial DNA using RFLP–PCR in 16 populations (267 fish), a total of 18 haplotypes was detected. The most common haplotype was EA1 and represented 46% of all genotypes. Three phylogenetic groups characterized the region, with the East African clade occurring between the western rift in the west, Lake Baringo in the north, the Indian Ocean in the east and the Rufiji River in the south. The Lower Tana River (unlike the Upper Tana River) and Lake Kamnarok grouped with the North clade, while the Ruaha River (tributary of the Rufiji River) represented a contact zone between the East and the South-central African clade. Genetic structure was distinct ( F _ST=0·66), with the Lower Tana River and Lake Mtera forming specific units. Within the East clade, the Oloibortoto River, Upper Tana River and Lake Victoria each formed a distinct population. Lake Baringo grouped either with the eastern rift or the western rift depending on the analysis. Lake Jipe, the Pangani River, Rusizi River, Lake Edward, Nyabugogo River and the Luiche River clustered together. Among group variance explained 66·8% of the total variance; the impact of vicariance (rifting and uplifting) on the phylogeography was considerable. A distribution model of the catfish is proposed which combines the separation by rifting several million years ago with a high gene flow in the western rift due to historical connectivity among lakes and rivers.     

Rapid identification of wine yeast species based on RFLP analysis of the ribosomal internal transcribed spacer (ITS) region

In this study, we identified a total of 33 wine yeast species and strains using the restriction patterns generated from the region spanning the internal transcribed spacers (ITS 1 and 2) and the 5.8S rRNA gene. Polymerase chain reaction (PCR) products of this rDNA region showed a high length variation for the different species. The size of the PCR products and the restriction analyses with three restriction endonucleases (HinfI, CfoI, and HaeIII) yielded a specific restriction pattern for each species with the exception of the corresponding anamorph and teleomorph states, which presented identical patterns. This method was applied to analyze the diversity of wine yeast species during spontaneous wine fermentation. Received: 2 July 1997 / Accepted: 7 December 1997     

RAPD characterisation of two neotropical hybrid legumes

An investigation of randomly amplified polymorphic DNA (RAPD) and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) marker distribution was made for two well-characterised hybrids and their parents,Leucaena leucocephala andL. esculenta andParkinsonia aculeata andCercidium praecox. Three chloroplast DNA (cpDNA) markers identified the maternal parent of eachL. leucocephala ×L. esculenta hybrid. Fifteen species-diagnostic RAPD markers (invariant in one taxon and absent from the other) were always present in theLeucaena hybrid and assumed to be of nuclear origin, whilst three RAPD markers showed expression patterns identical to the cpDNA markers and were assumed to be of organellar origin. No RAPD or PCR-RFLP taxon-diagnostic markers were discovered for eitherP. aculeata orC. praecox. However, 21 RAPD markers were species-specific (polymorphic within one taxon but absent from the other) and Southern analysis indicated that none of the markers were of organellar origin. Only 67% additivity of markers specific toP. aculeata andC. praecox was demonstrated in the hybrids between these two species, whilst inLeucaena 97% additivity was demonstrated. Differences between the two hybridising situations were related to the behaviour of the molecular markers and the biology of the species.     

Promoter polymorphisms in DNA repair gene ERCC5 and susceptibility to gastric cancer in Chinese

Genetic variations in excision repair cross-complementing group 5 (ERCC5) might influence individual vulnerability to gastric cancer (GC). We investigated effects of two putatively functional polymorphisms in ERCC5 promoter region, rs751402 (+ 25A > G) and rs2296147 (+ 202C > T), and their potential interaction with environment factors on the risk of developing GC. We performed a sex- and age-matched case–control design with 400 GC cases and 400 healthy controls for rs751402 and 403 GC cases and 403 healthy controls for rs2296147. Our results showed that rs751402 were associated with increased GC risk (AA vs. GG: OR = 1.99, 95%CI: 1.20–3.31, P = 0.008; AG + AA vs. GG: OR = 1.41, 95%CI: 1.07–1.86, P = 0.016), and rs2296147 was also associated with increased cancer risk (CC vs. TT: OR = 2.17, 95%CI: 1.04–4.54, P = 0.039; CC vs. CT + TT: OR = 2.26, 95%CI: 1.09–4.69, P = 0.028). In a stratified analysis, rs751402 (AG + AA vs. GG: OR = 1.44, 95%CI: 1.02–2.02, P = 0.037) and rs2296147 (CC vs. CT + TT: OR = 2.33, 95%CI: 1.00–5.44, P = 0.050) were also found to be associated with diffuse-type GC risk. The most common GT haplotype (rs751402–rs2296147) showed protective effect for GC development (OR = 0.73, 95%CI: 0.58–0.91, P = 0.005), and especially for diffuse-type GC (OR = 0.68, 95%CI: 0.52–0.90, P = 0.006). Genetic effects on increased GC risk seemed to be enhanced by Helicobacter pylori infection, smoking and alcohol drinking, with corresponding adjusted ORs of 4.57, 2.42 and 2.50 for the rs751402 AG/AA variants, and of 5.32, 3.20 and 6.87 for the rs2296147 CC variant, but their interaction effects on GC risk didn't reach statistically significance. ERCC5 rs751402 and rs2296147 polymorphisms might alter the risk of developing GC and especially the diffuse subtype. Further validation of our results in larger populations and additional studies evaluating their function impact are required.     

New PCR-based methods for yeast identification

AIMS: To characterize reference yeast strains and identify indigenous strains isolated from wine fermentations by PCR methods. METHODS AND RESULTS: We compared several PCR techniques for yeast identification. We used oligonucleotide primers that are complementary to (i) intron splice sites, (ii) REP and (iii) ERIC elements to produce PCR fingerprints that display specific patterns between the different yeast species. These three techniques were used to characterize 41 reference yeast strains belonging to 15 different species and to identify 40 indigenous strains isolated from grape must and wine fermentations. Species-specific banding patterns were obtained with the three PCR-techniques with different degrees of intraspecific differentiation depending on the method. By comparing the PCR fingerprints of unknown isolates with those produced by reference strains, we identified yeast strains isolated from an industrial wine fermentation. CONCLUSIONS: All three PCR techniques are rapid, reliable and simple methods of yeast identification. As far as we know, this is the first time that the primers designed for amplifying repetitive elements in bacteria have been successfully used in yeast. SIGNIFICANCE AND IMPACT OF THE STUDY: Industry needs rapid, reliable and simple methods of yeast identification. The proposed PCR techniques will allow to achieve this objective.     

A survey of Echinococcus species in wild carnivores and livestock in East Africa

We examined 71 faecal samples of carnivores from Queen Elizabeth National Park (QENP), Uganda, for eggs of Echinococcus species. Thirty-nine faecal samples contained taeniid eggs. For species diagnosis, DNA was isolated from a total of 1984 individual taeniid eggs. To differentiate eggs of Echinococcus felidis from other taeniid taxa (including the closely related Echinococcus granulosus sensu stricto), a restriction fragment length polymorphism (RFLP)-PCR of the mitochondrial nad1 gene was developed. As the faecal samples were taken from the environment, the host species was determined for all samples, except for one, by RFLP-PCR of the cob gene. Seven hundred and ninety-one of the 1984 eggs yielded a suitable PCR product. E. felidis was present in 34 of 47 samples from lions, none of 18 samples from leopards, and one of five samples from spotted hyenas. No Echinococcus taxon other than E. felidis was found, but three samples from lions contained eggs of Taenia regis. Two hydatid cysts of warthog origin from QENP were available for this study; molecular examination showed that one belonged to E. felidis, the other to E. granulosus (G1 strain). As a comparison of methods demonstrated that molecular diagnostic tools used for previous surveys of Echinococcus isolates in eastern Africa are not suitable to discriminate between E. felidis and E. granulosus sensu stricto, we re-examined 412 hydatid cyst samples of human, sheep, cattle, camel and goat origin from Kenya. Previous results were confirmed, as E. granulosus sensu stricto and Echinococcus canadensis G6/7 strain, but no E. felidis was found among these samples. In conclusion, we provide evidence that E. felidis is a frequent parasite of lions in Uganda, and possibly also occurs in hyenas. Additionally, we show that warthogs interact as intermediate hosts for E. felidis. We did not find evidence that E. felidis is present in eastern Africa outside conservation areas.     

Genetic variation in DNA repair gene XRCC7 (G6721T) and susceptibility to breast cancer

The human XRCC7 is a DNA double-strand break (DSBs) repair gene, involved in non-homologous end joining (NHEJ). It is speculated that DNA DSBs repair have an important role during development of breast cancer. The human XRCC7 is a NHEJ DSBs repair gene. Genetic variation G6721T of XRCC7 (rs7003908) is located in the intron 8 of the gene. This polymorphism may regulate splicing and cause mRNA instability. In the present study, we specifically investigated whether common G6721T genetic variant of XRCC7 was associated with an altered risk of breast cancer. The present study included 362 females with breast cancer. Age frequency-matched controls (362 persons) were randomly selected from the healthy female blood donors, according to the age distribution of the cases. Using RFLP-PCR based method, the polymorphism of XRCC7 was determined. The TG (OR=1.20, 95% CI: 0.83-1.74, P=0.320) and TT (OR=1.01, 95% CI: 0.67-1.53, P=0.933) genotypes had no significant effect on risk of breast cancer, in comparison with the GG genotype. Our present findings indicate that the TT and TG genotypes were not associated with an altered breast cancer risk.