A single multiplex PCR and SNaPshot minisequencing reaction of 42 SNPs to classify admixture populations into mitochondrial DNA haplogroups

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

A single multiplex SNaPshot reaction of 42 SNPs to classify admixture populations into mitochondrial DNA haplogroups

SNaPshot minisequencing reaction is in increasing use because of its fast detection of many polymorphisms in a single assay. In this work we described a highly sensitive single nucleotide polymorphisms (SNPs) typing method with detection of 42 mitochondrial DNA (mtDNA) SNPs in a single PCR and SNaPshot multiplex reaction in order to allow haplogroup classification in Latin American admixture population. We validated the panel typing 160 Brazilian individuals. DNA was extracted from blood spotted on filter paper using Chelex protocol. Forty SNPs were selected targeting haplogroup-specific mutations in Europeans, Africans and Asians (only precursors of Native Americans haplogroups A2, B2, C1, and D1) and two non-coding SNPs were chosen to increase the power of discrimination between individuals (SNPs positions 16,519 and 16,362). It was done using a modified version of a previously published multiplex SNaPshot minisequencing reaction established to resolve European haplogroups, adding SNPs targeting Africans (L0, L1, L2, L3, and L*) and Asians (A, B, C, and D) haplogroups based on SNPs described at PhyloTree.org build 2. PCR primers were designed using PerlPrimer software and checked with the Autodimer program. Thirty-three primer-pairs were used to amplify 42 SNPs. Using this panel, we were able to successfully classify 160 individuals into their correct haplogroups. Complete SNP profiles were obtained from 10. pg of total DNA. We conclude that it is possible to build and genotype more than 40 mtDNA SNPs in a single multiplex PCR and SNaPshot reaction, with sensitivity and reliability, resolving haplogroup classification in admixture populations. © 2011.

Mitochondrial DNA mapping of social-biological interactions in Brazilian Amazonian African-descendant populations

ABSTRACT: The formation of the Brazilian Amazonian population has historically involved three main ethnic groups, Amerindian, African and European. This has resulted in genetic investigations having been carried out using classical polymorphisms and molecular markers. To better understand the genetic variability and the micro-evolutionary processes acting in human groups in the Brazilian Amazon region we used mitochondrial DNA to investigate 159 maternally unrelated individuals from five Amazonian African-descendant communities. The mitochondrial lineage distribution indicated a contribution of 50.2% from Africans (L0, L1, L2, and L3), 46.6% from Amerindians (haplogroups A, B, C and D) and a small European contribution of 1.3%. These results indicated high genetic diversity in the Amerindian and African lineage groups, suggesting that the Brazilian Amazonian African-descendant populations reflect a possible population amalgamation of Amerindian women from different Amazonian indigenous tribes and African women from different geographic regions of Africa who had been brought to Brazil as slaves. The present study partially mapped the historical biological and social interactions that had occurred during the formation and expansion of Amazonian African-descendant communities.

Genetic diversity of the Brazilian Creole cattle Pe-duro assessed by microsatellites and mitochondrial DNA

The objective of this study was to describe the genetic diversity and structure of the largest Pe-duro population by assessing variation at ten autosomal microsatellite (STR) loci and mitochondrial DNA (mtDNA) sequences. The mean expected heterozygosity was 0.755, the mean observed heterozygosity was 0.600 and significant inbreeding coefficient (Fis) and deviations from the Hardy-Weinberg equilibrium in most of analyzed loci demonstrate the impact of inbreeding and homozygosis on this population. A more in-depth genetic analysis could be achieved by expanding the STR list. The analysis of mtDNA provided evidence of ancestral African taurine haplotypes in Pe-duro and excluded maternal Zebuine introgression. In this report, the main Pe-duro population is genetically portrayed by sampling approximately 40% of it. As this herd represents the core of the Pe-duro conservation program, these findings are of outstanding value for the management and preservation of this Brazilian 'native' cattle breed.

Genetic diversity of the Brazilian Creole cattle Pé-duro assessed by microsatellites and mitochondrial DNA

The objective of this study was to describe the genetic diversity and structure of the largest Pé-duro population by assessing variation at ten autosomal microsatellite (STR) loci and mitochondrial DNA (mtDNA) sequences. The mean expected heterozygosity was 0.755, the mean observed heterozygosity was 0.600 and significant inbreeding coefficient (Fis) and deviations from the Hardy-Weinberg equilibrium in most of analyzed loci demonstrate the impact of inbreeding and homozygosis on this population. A more in-depth genetic analysis could be achieved by expanding the STR list. The analysis of mtDNA provided evidence of ancestral African taurine haplotypes in Pé-duro and excluded maternal Zebuine introgression. In this report, the main Pé-duro population is genetically portrayed by sampling approximately 40% of it. As this herd represents the core of the Pé-duro conservation program, these findings are of outstanding value for the management and preservation of this Brazilian 'native' cattle breed.

Genetic variation and demographic history of the Haplochromis laparogramma group of Lake Victoria-An analysis based on SINEs and mitochondrial DNA

More than 500 endemic haplochromine cichlid species inhabit Lake Victoria. This striking species diversity is a classical example of recent explosive adaptive radiation thought to have happened within the last similar to 15,000 years. In this study, we examined the population structure and historical demography of 3 pelagic haplochromine cichlid species that resemble in morphology and have similar niche, Haplochromis (Yssichromis) laparogramma, Haplochromis (Y.) pyrrhocephalus, and Haplochromis (Y.) sp. "glaucocephalus". We investigated the sequences of the mitochondrial DNA control region and the insertion patterns of short interspersed elements (SINEs) of 759 individuals. We show that sympatric forms are genetically differentiated in 4 of 6 cases, but we also found apparent weakening of the genetic differentiation in areas with turbid water. We estimated the timings of population expansion and species divergence to coincide with the refilling of the lake at the Pleistocene/Holocene boundary. We also found that estimates can be altered significantly by the choice of the shape of the molecular clock. If we employ the nonlinear clock model of evolutionary rates in which the rates are higher towards the recent, the population expansion was dated at around the event of desiccation of the lake ca. 17,000 YBP. Thus, we succeeded in clarifying the species and population structure of closely related Lake Victoria cichlids and in showing the importance of applying appropriate clock calibrations in elucidating recent evolutionary events. (C) 2009 Elsevier B.V. All rights reserved.

Philopatry of male marine turtles inferred from mitochondrial DNA markers

Recent studies of mitochondrial DNA (mtDNA) variation among marine turtle populations are consistent with the hypothesis that females return to beaches in their natal region to nest as adults. In contrast, less is known about breeding migrations of male marine turtles and whether they too are philopatric to natal regions. Studies of geographic structuring of restriction fragment and microsatellite polymorphisms at anonymous nuclear loci in green turtle (Chelonia mydas) populations indicate that nuclear gene flow is higher than estimates from mtDNA analyses. Regional populations from the northern and southern Great Barrier Reef were distinct for mtDNA but indistinguishable at nuclear loci, whereas the Gulf of Carpentaria (northern Australia) population was distinct for both types of marker. To assess whether this result was due to reduced philopatry of males across the Great Barrier Reef, we determined the mtDNA haplotypes of breeding males at courtship areas for comparison with breeding females from the same three locations. We used a PCR-restriction fragment length polymorphism approach to determine control region haplotypes and designed mismatch primers for the identification of specific haplotypes. The mtDNA haplotype frequencies were not significantly different between males and females at any of the three areas and estimates of Fst among the regions were similar for males and females (Fst = 0.78 and 0.73, respectively). We conclude that breeding males, like females, are philopatric to courtship areas within their natal region. Nuclear gene flow between populations is most likely occurring through matings during migrations of both males and females through nonnatal courtship areas.

Trans-Pacific migrations of the loggerhead turtle (Caretta caretta) demonstrated with mitochondrial DNA markers.

Juvenile loggerhead turtles (Caretta caretta) have recently been documented in the vicinity of Baja California and thousands of these animals have been captured in oceanic fisheries of the North Pacific. The presence of loggerhead turtles in the central and eastern North Pacific is a prominent enigma in marine turtle distribution because the nearest documented nesting concentrations for this species are in Australia and Japan, over 10,000 km from Baja California. To determine the origin of the Baja California feeding aggregate and North Pacific fishery mortalities, samples from nesting areas and pelagic feeding aggregates were compared with genetic markers derived from mtDNA control region sequences. Overall, 57 of 60 pelagic samples (95%) match haplotypes seen only in Japanese nesting areas, implicating Japan as the primary source of turtles in the North Pacific Current and around Baja California. Australian nesting colonies may contribute the remaining 5% of these pelagic feeding aggregates. Juvenile loggerhead turtles apparently traverse the entire Pacific Ocean, approximately one-third of the planet, in the course of developmental migrations, but mortality in high-seas fisheries raises concern over the future of this migratory population.

Conservation genetics of North Atlantic loggerhead sea turtles: analysis of nuclear and mitochondrial DNA

[EN] Complex population structure has been described for the loggerhead sea turtle (Caretta caretta), revealing lower levels of population genetic structure in nuclear compared to mitochondrial DNA assays. This may result from mating during spatially overlapping breeding migrations, or male-biased dispersal as previously found for the green turtle (Chelonia mydas). To further investigate these multiple possibilities, we carried out a comparative analysis from twelve newly developed microsatellite loci and the mitochondrial DNA control region (~804 bp) in adult females of the Cape Verde Islands (n=158), and Georgia, USA (n=17).

Assessing the mitochondrial DNA diversity of the Chagas disease vector Triatoma sordida (Hemiptera: Reduviidae)

Triatoma sordida is a species that transmits Trypanosoma cruzi to humans. In Brazil, T. sordida currently deserves special attention because of its wide distribution, tendency to invade domestic environments and vectorial competence. For the planning and execution of control protocols to be effective against Triatominae, they must consider its population structure. In this context, this study aimed to characterise the genetic variability of T. sordida populations collected in areas with persistent infestations from Minas Gerais, Brazil. Levels of genetic variation and population structure were determined in peridomestic T. sordida by sequencing a polymorphic region of the mitochondrial cytochrome b gene. Low nucleotide and haplotype diversity were observed for all 14 sampled areas; π values ranged from 0.002-0.006. Most obtained haplotypes occurred at low frequencies, and some were exclusive to only one of the studied populations. Interpopulation genetic diversity analysis revealed strong genetic structuring. Furthermore, the genetic variability of Brazilian populations is small compared to that of Argentinean and Bolivian specimens. The possible factors related to the reduced genetic variability and strong genetic structuring obtained for studied populations are discussed in this paper.

Nuclear and mitochondrial DNA markers in traceability of retail beef samples

Several characteristics are important in a traceability system of animal products, such as age at slaughter, breed composition, besides information of the productive chain. In general, the certification agent records information about the animals and the system which it came from, although cannot guarantee that the slaughtering, meat processing and distribution are error proof. Besides, there is a differential price, at least at the international market, based on sex and breed composition of the animals. Genetic markers allow identification of characteristics controlled in the beef cattle traceability program, as sex and breed composition, in order to correctly identify and appraise the final product for the consumer. The hypothesis of this study was that the majority beef samples retailed in the local market originate from female with a great participation of zebu breeds. Therefore, the objective of this work was to characterize retail beef samples with DNA markers that identify cattle sex and breed composition. Within 10 beef shops localized in Pirassununga, SP, Brazil, 61 samples were collected, all were genotyped as harboring Bos taurus mitochondrial DNA and 18 were positive for the Y chromosome amplification (male). For the marker sat1711b-Msp I the frequency of the allele A was 0.278 and for the marker Lhr-Hha I the frequency of the allele T was 0.417. The results of sat1711b-Msp I and Lhr-Hha I allelic frequencies are suggestive that the proportion of indicus genome compared with the taurine genome in the market meat is smaller than the observed in the Nellore breed. The procedure described in this study identified sex and subspecies characteristics of beef meat samples, with potential application in meat products certification in special as an auxiliary tool in beef cattle traceability programs.

Species determination of Brazilian mammals implicated in the epidemiology of rabies based on the control region of mitochondrial DNA

Identification of animals that are decomposing or have been run over or burnt and cannot be visually identified is a problem in the surveillance and control of infectious diseases. Many of these animals are wild and represent a valuable source of information for epidemiologic research as they may be carriers of an infectious agent. This article discusses the results obtained using a method for identifying mammals genetically by sequencing their mitochondrial DNA control region. Fourteen species were analyzed and identified. These included the main reservoirs and transmitters of rabies virus, namely, canids, chiroptera and primates. The results prove that this method of genetic identification is both efficient and simple and that it can be used in the surveillance of infectious diseases which includes mammals in their epidemiologic cycle, such as rabies.

No relationship found between point heteroplasmy in mitochondrial DNA control region and age range, sex and haplogroup in human hairs

The analysis of heteroplasmy (presence of more than one type of mitochondrial DNA in an individual) is used as a tool in human identification studies, anthropology, and most currently in studies that relate heteroplasmy with longevity. The frequency of heteroplasmy and its correlation with age has been analyzed using different tissues such as blood, muscle, heart, bone and brain and in different regions of mitochondrial DNA, but this analysis had never been performed using hair samples. In this study, samples of hair were sequenced in order to ascertain whether the presence or not of heteroplasmy varied according to age, sex and origin of haplogroup individuals. The samples were grouped by age (3 groups), gender (male and female) and haplogroup of origin (European, African and Native American), and analyzed using the chi-square statistical test (chi(2)). Based in statistical results obtained, we conclude that there is no relationship between heteroplasmy and sex, age and haplogroup origin using hair samples.

Geographic variation of sex pheromone and mitochondrial DNA in Diatraea saccharalis (Fab., 1794) (Lepidoptera: Crambidae)

(9Z,11E)-hexadecadienal and (Z11)-hexadecenal, the main sex pheromone components of the sugarcane borer, Diatraea saccharalis, were identified and quantified from four Brazilian and one Colombian populations using GC-EAD, GC-MS and GC analyses. Three different ratios were observed, 9:1,6:1, and 3:1. The pheromone concentration for the major component, (9Z,11E)-hexadecadienal, varied from 6.8 ng/gland to 21.9 ng/gland and from 1.7 ng/gland to 6.5 to the minor component, (Z11)-hexadecenal. The 25 D. saccharalis cytochrome oxidase II sequences that were analyzed showed low intra-specific variation and represented only 11 haplotypes, with the most frequent being the one represented by specimens from Sao Paulo, Parana, and Pernambuco states. Specimens from Colombia showed the highest genetic divergence from the others haplotypes studied. Data on the genetic variability among specimens, more than their geographic proximity, were in agreement with data obtained from analyses of the pheromone extracts. Our data demonstrate a variation in pheromone composition and a covariation in haplotypes of the D. saccharalis populations studied. (C) 2010 Elsevier Ltd. All rights reserved.