David Gamarra

New cyt b gene universal primer set for forensic analysis

Analysis of mitochondrial DNA, and in particular the cytochrome b gene (cyt b), has become an essential tool for species identification in routine forensic practice. In cases of degraded samples, where the DNA is fractionated, universal primers that are highly efficient for the amplification of the target region are necessary. Therefore, in the present study a new universal cyt b primer set with high species identification capabilities, even in samples with highly degraded DNA, has been developed. In order to achieve this objective, the primers were designed following the alignment of complete sequences of the cyt b from 751 species from the Class of Mammalia listed in GenBank. A highly variable region of 148bp flanked by highly conserved sequences was chosen for placing the primers. The effectiveness of the new pair of primers was examined in 63 animal species belonging to 38 Families from 14 Orders and 5 Classes (Mammalia, Aves, Reptilia, Actinopterygii, and Malacostraca). Species determination was possible in all cases, which shows that the fragment analyzed provided a high capability for species identification. Furthermore, to ensure the efficiency of the 148bp fragment, the intraspecific variability was analyzed by calculating the concordance between individuals with the BLAST tool from the NCBI (National Center for Biotechnological Information). The intraspecific concordance levels were superior to 97% in all species. Likewise, the phylogenetic information from the selected fragment was confirmed by obtaining the phylogenetic tree from the sequences of the species analyzed. Evidence of the high power of phylogenetic discrimination of the analyzed fragment of the cyt b was obtained, as 93.75% of the species were grouped within their corresponding Orders. Finally, the analysis of 40 degraded samples with small-size DNA fragments showed that the new pair of primers permits identifying the species, even when the DNA is highly degraded as it is very common in forensic samples.

New Q lineage found in bovine (Bos taurus) of Iberian Peninsula.

Abstract The northern Iberian Peninsula is home to a variety of autochthonous cattle breeds, such as the Terreña and Pirenaica. With the objective of characterizing the matrilineal lineages of these breeds, a study of mitochondrial DNA was performed. The D-loop of 155 individuals was analyzed and most of the individuals were carriers of the T3 haplogroup, while haplogroups T and T1 were much less frequent. A Pirenaica individual belonging to the Q haplogroup was found. To verify the presence of the Q haplogroup individual, the entire mitochondrial DNA was sequenced and compared with two descendants. The individuals were assigned to the Q1 sub-haplogroup. These findings extend the geographic distribution of the Q haplogroup to the south west of the European continent. This new Q1 lineage has seven polymorphisms in the coding region, so this lineage is probably as old as the Q lineages described to date.

Population genetic data of 38 insertion-deletion markers in six populations of the northern fringe of the Iberian Peninsula

Insertion-deletions have been reported very useful markers for forensic purposes. To further deepen in this matter, 38 non-coding bi-allelic autosomal indels were analyzed in 575 individuals representing six populations from the northern fringe of the Iberian Peninsula. Autochthonous populations from the Basque Country, northern Navarre, the Pas Valley in Cantabria and Aragon were analyzed, together with non-autochthonous populations from the Basque Country and northern Navarre. At the intra-population level, all loci analyzed were in Hardy-Weinberg equilibrium except for marker rs33917182 in autochthonous Basques. Linkage disequilibrium (LD) test did not reveal statistically significant allelic association between the different loci pairs in all six populations. Forensic parameters proved to be highly informative in the six populations analyzed, even if a scenario with population substructure and local inbreeding was considered for match probability calculations, and the potential of this indels set to be used in combination with other genetic markers is remarkable. As for inter-population analyses, in general terms the six populations showed low but statistically significant genetic distances. However, though this indels set efficiently differentiate between main ancestries, it does not allow an accurate separation at a local level and, for the time being, their combination with other informative markers is needed to maximize the power to accurately differentiate populations with close genetic ancestry.

Association of the C47T Polymorphism in SOD2 with Amnestic Mild Cognitive Impairment and Alzheimer's Disease in Carriers of the APOEε4 Allele.

Oxidative stress plays an important part in amnestic mild cognitive impairment (aMCI), the prodromal phase of Alzheimers disease (AD). Recent evidence shows that polymorphisms in the SOD2 gene affect the elimination of the reactive oxygen species (ROS) generated in mitochondria. The aim of this study was to determine whether the functional rs4880 SNP in the SOD2 gene is a risk factor associated with aMCI and sporadic AD. 216 subjects with aMCI, 355 with AD, and 245 controls have been studied. The SNP rs4880 of the SOD2 gene was genotyped by RT-PCR and the APOE genotype was determined by PCR and RFLPs. Different multinomial logistic regression models were used to determine the risk levels for aMCI and AD. Although the T allele of the SOD2 rs4880 SNP gene (rs4880-T) is not an independent risk for aMCI or AD, this allele increases the risk to aMCI patients carrying at least one APOEε4 allele. Moreover, rs4880-T allele and APOEε4 allele combination has been found to produce an increased risk for AD compared to aMCI reference patients. These results suggest that APOEε4 and rs4880-T genotype may be a risk for aMCI and a predictor of progression from aMCI to AD.

Distinct correlations between lipogenic gene expression and fatty acid composition of subcutaneous fat among cattle breeds

BackgroundThe fatty acid (FA) composition of adipose tissue influences the nutritional quality of meat products. The unsaturation level of FAs is determined by fatty acid desaturases such as stearoyl-CoA desaturases (SCDs), which are under control of the transcription factor sterol regulatory element-binding protein (SREBP). Differences in SCD genotype may thus confer variations in lipid metabolism and FA content among cattle breeds. This study investigated correlations between FA composition and lipogenic gene expression levels in the subcutaneous adipose tissue of beef cattle breeds of different gender from the Basque region of northern Spain. Pirenaica is the most important beef cattle breed in northern Spain, while Salers cattle and Holstein-Friesian cull cows are also an integral part of the regional beef supply.ResultsPirenaica heifers showed higher monounsaturated FA (MUFA) and conjugated linoleic acid (CLA) contents in subcutaneous adipose tissue than other breeds (P < 0.001). Alternatively, Salers bulls produced the highest oleic acid content, followed by Pirenaica heifers (P < 0.001). There was substantial variability in SCD gene expression among breeds, consistent with these differences in MUFA and CLA content. Correlations between SCD1 expression and most FA desaturation indexes (DIs) were positive in Salers (P < 0.05) and Pirenaica bulls, while, in general, SCD5 expression showed few significant correlations with DIs. There was a significant linear correlation between SCD1 and SRBEP1 in all breeds, suggesting strong regulation of SCD1 expression by SRBEP1. Pirenaica heifers showed a stronger correlation between SCD1 and SREBP1 than Pirenaica bulls. We also observed a opposite relationship between SCD1 and SCD5 expression levels and opposite associations of isoform expression levels with the ∆9 desaturation indexes.ConclusionsThese results suggest that the relationships between FA composition and lipogenic gene expression are influenced by breed and sex. The opposite relationship between SCD isoforms suggests a compensatory regulation of total SCD activity, while opposite relationships between SCD isoforms and desaturation indexes, specially 9c-14:1 DI, previously reported as an indicator of SCD activity, may reflect distinct activities of SCD1 and SCD5 in regulation of FA content. These findings may be useful for beef/dairy breeding and feeding programs to supply nutritionally favorable products.

Species identification in meat products: A new screening method based on high resolution melting analysis of cyt b gene

Meat adulteration by substitution with lower value products and/or mislabeling involves economic, health, quality and socio-religious issues. Therefore, identification and traceability of meat species has become an important subject to detect possible fraudulent practices. In the present study the development of a high resolution melt (HRM) screening method for the identification of eight common meat species is reported. Samples from Bos taurus, Ovis aries, Sus scrofa domestica, Equus caballus, Oryctolagus cuniculus, Gallus gallus domesticus, Meleagris gallopavo and Coturnix coturnix were analyzed through the amplification of a 148 bp fragment from the cyt b gene with a universal primer pair in HRM analyses. Melting profiles from each species, as well as from several DNA mixtures of these species and blind samples, allowed a successful species differentiation. The results demonstrated that the HRM method here proposed is a fast, reliable, and low-cost screening technique.

Two ovine mitochondrial DNAs harboring a fifth 75/76 bp repeat motif without altered gene expression in Northern Spain

The Basque Country is home to the Latxa sheep breed, which is divided in several varieties such as Latxa Black Face (LBKF) and Latxa Blonde Face (LBLF). Mitochondrial DNA control region analysis of 174 male sheep (97 LBKF and 77 LBLF) was performed with the objective of characterizing the maternal lineages of these two varieties that are the basis to produce the cheese with Idiazabal quality label. The percentage of unique haplotypes was 77.32% in LBKF and 67.53% in LBLF. Most of the individuals were classified into B haplogroup (98.85%), while A haplogroup was much less frequent. Two Latxa individuals (one LBKF and one LBLF), both belonging to B haplogroup, displayed an additional 75/76 bp tandem repeat motif. Only 33 other sequences with this repeat motif were found among 11 061 sheep sequences included in the GenBank database. Gene expression was analyzed in peripheral blood leukocytes since the additional 75/76 bp repeat motif falls within ETAS1, a domain with a possible function in regulation of replication and transcription. The mRNA expression from four mitochondrial genes (COI, cyt b, ND1, and ND2) was analyzed in the two individuals of this study with a fifth repeat motif and in four without it. Although lower transcription was observed when the additional 75/76 bp repeat motif was present, no statistically significant differences were observed. Therefore, the variation in the number of the 75/76 repeat motif does not seem to modify the gene expression rate in mitochondrial genes.