The PCR-based STR system MBP-B (myelin basic protein locus B) has been reported to exhibit a high... more The PCR-based STR system MBP-B (myelin basic protein locus B) has been reported to exhibit a high rate of mutations. Using a newly designed pair of primers we present evidence that this is due to failed amplifications caused by a polymorphism in the annealing region of the reverse primer originally designed. With the new reverse primer described here no exclusions were found (out of 59 mother/child pairs analysed) while one was detected with the old set of primers. The results obtained with both pairs of primers in a random population sample (n = 112) from North Portugal are compared. In this sample 13 individuals typed as homozygotes with the pair of primers originally described, were found to be heterozygous when the amplifications were performed with the new reverse primer. By sequence analysis, a substitution in the reverse primer binding sequence originally described was determined. This substitution is located upstream from the repetition site and consists of G→A transition. This variation reaches polymorphic frequency and is responsible for the relatively frequent null alleles due to failed amplifications when the previously designed primers are used.
North African populations are considered genetically closer to Eurasians than to sub-Saharans. Ho... more North African populations are considered genetically closer to Eurasians than to sub-Saharans. However, they display a considerably high mtDNA heterogeneity among them, namely in the frequencies of the U6, East African, and sub-Saharan haplogroups. In this study, we describe and compare the female gene pools of two neighboring Tunisian populations, Kesra (Berber) and Zriba (non-Berber), which have contrasting historical backgrounds. Both populations presented lower diversity values than those observed for other North African populations, and they were the only populations not showing significant negative Fu's F(S) values. Kesra displayed a much higher proportion of typical sub-Saharan haplotypes (49%, including 4.2% of M1 haplogroup) than Zriba (8%). With respect to U6 sequences, frequencies were low (2% in Kesra and 8% in Zriba), and all belonged to the subhaplogroup U6a. An analysis of these data in the context of North Africa reveals that the emerging picture is complex, because Zriba would match the profile of a Berber Moroccan population, whereas Kesra, which shows twice the frequency of sub-Saharan lineages normally observed in northern coastal populations, would match a western Saharan population except for the low U6 frequency. The North African patchy mtDNA landscape has no parallel in other regions of the world and increasing the number of sampled populations has not been accompanied by any substantial increase in our understanding of its phylogeography. Available data up to now rely on sampling small, scattered populations, although they are carefully characterized in terms of their ethnic, linguistic, and historical backgrounds. It is therefore doubtful that this picture truly represents the complex historical demography of the region rather than being just the result of the type of samplings performed so far.
Molecular genetic data contain information on the history of populations. Evidence of prehistoric... more Molecular genetic data contain information on the history of populations. Evidence of prehistoric demographic expansions has been detected in the mitochondrial diversity of most human populations and in a Y-chromosome STR analysis, but not in a previous study of 11 Y-chromosome SNPs in Europeans. In this paper, we show that mismatch distributions and tests of mutation/drift equilibrium based on up to 166 Y-chromosome SNPs, in 46 samples from all continents, also fail to support an increase of the male effective population size. Computer simulations show that the low nuclear versus mitochondrial mutation rates cannot explain these results. However, ascertainment bias, i.e., when only highly variable SNP sites are typed, may be concealing any Y SNPs evidence for a recent, but not an ancient, increase in male effective population sizes. The results of our SNP analyses can be reconciled with the expansion of male effective population sizes inferred from STR loci, and with mitochondrial evidence, by admitting that humans were essentially polygynous during much of their history. As a consequence, until recently only a few men may have contributed a large fraction of the Y-chromosome pool at every generation. The number of breeding males may have increased, and the variance of their reproductive success may have decreased, through a recent shift from polygyny to monogamy, which is supported by ethnological data and possibly accompanied the shift from mobile to sedentary communities.
Goat is believed to be the first true livestock domesticated and, apart from its historical impor... more Goat is believed to be the first true livestock domesticated and, apart from its historical importance, keeps playing an essential economic role in very diverse human societies. We have analysed the female gene pool of all Portuguese autochthonous breeds (Bravia, Serrana, Charnequeira, Serpentina and Algarvia) through the mtDNA HVI sequencing of 288 unrelated animals sampled throughout the country. All breeds proved to be extremely diverse (average haplotype diversity of 0.977), in contrast with the Portuguese peripheral geographic situation in the distribution range of the species. Moreover, observed genetic distances between breeds do not correlate with microgeography inside Portugal. These observations are consistent with recurrent refreshment of the breeding stock through the introduction of exotic animals. Fitting the new data into the still loosely defined female genetic pool landscape of goats, all Portuguese animals, one sample excepted (belonging to Bravia), are classified into haplogroup A and haplotype sharing is geographically very sparse, including a Far East match. Our results confirm that goats stand out among most of domesticates as exceptionally diverse and showing an unparalleled degree of mobility of animals (at least females) used for reproductive purposes.
We report a simultaneous study of the VWA STR locus by the Perkin-Elmer Profiler Plus kit and the... more We report a simultaneous study of the VWA STR locus by the Perkin-Elmer Profiler Plus kit and the Promega GenePrint CTTv kit in a population sample from North Portugal and in 418 meiosis from family material and paternity cases. PCR amplification and genotyping were performed according to the manufacturer’s instructions using ABI 377 or ABI 310 automatic sequencers. Biological kinship in family material and paternity cases was validated by the use of the STR loci D3S1358, D5S818, D7S820, D8S1179, D13S317, D18S51, D21S11, FGA, CSF1PO, TH01 and TPOX. Out of 434 unrelated individuals we found 4 inconsistencies between the genotypes obtained using each kit. No exclusions were found in the meiotic analyses. In all cases, these inconsistencies were due to an annealing failure of the Perkin-Elmer forward primer resulting in false homozygotes. Sequencing analysis revealed an A-to-T substitution at position 1631 (GenBank sequence M25858), 52 bases upstream of the first TCTA motif of the repeat region. An estimate of the null allele frequency (s) in this study is thus obtainable from the expression s = 4/¶(2 × 434) = 0.46%. The relatively high frequency of these discrepancies in our population demonstrates the need for caution when comparing genotype or gene frequency estimates made from amplicons produced by different primers, when evaluating apparent exclusions in paternity testing and when searching for a match between individual genetic profiles in forensic databases. Our findings are also compared with those previously reported.
A sample of mitochondrial DNA (mtDNA) from the southeastern African population of Mozambique has ... more A sample of mitochondrial DNA (mtDNA) from the southeastern African population of Mozambique has been shown to have affinities with populations both to its north and south. From the north came sequences that may have been involved in the Bantu expansion (from western, through eastern, to southern Africa), such as members of haplogroups L3b, L3e1a and a subset of L1a. The dating of the major component of Mozambican mtDNAs, the subset L2a of haplogroup L2, displayed an age range compatible with the Bantu expansion. The southern influence was traced by the presence of sequence types from haplogroup L1d, a probable relict of Khoisan-speaking populations that inhabited the region prior to their displacement by the Bantu-speaking incomers. Within historical times, the forced displacement of Mozambicans as part of the slave trade, mainly documented as being to the Americas, generated a differential input of eastern African sequences into the mtDNA pools of the Americas and of Europe, as testified to by the greater number of sequence matches between Mozambique and the Americas, compared to those between Mozambique and Europe.
An enlarged mtDNA database (n=549) for the Portuguese population, comprising HVRI and HVRII regio... more An enlarged mtDNA database (n=549) for the Portuguese population, comprising HVRI and HVRII regions is reported. This database was used to test the effect of sample size on the estimation of relevant parameters such as haplotype diversity, number of different haplotypes, nucleotide diversity and number of polymorphic positions. Simulations were performed generating sets of random subsamples of variable sizes (n=50, 100, 200, 300 and 400). The results show that while haplotype and nucleotide diversities do not vary significantly with sample size, the numbers of haplotypes and polymorphic positions rise continuously inside the tested interval. These trends are interpretable by the evolution of the proportions of sequences that are found once or twice, which drop dramatically as sample size increases, with the corresponding rise in the frequency of those encountered 3 times or more. The generated data were also used to extrapolate saturation curves for the referred parameters. When considering for instance the number of haplotypes, it is shown that a sample size of 1,000 individuals is required for practical saturation (defined as the point where a sample size increase of 100 individuals corresponds to an increment in the diversity measure below 5%). For HVRII the same level is reached at n=900 and n=1,300 is needed when both regions are analysed simultaneously. Consequently, we can infer that currently used sample sizes are still rather inadequate for both anthropological and forensic purposes.
The PCR-based STR system MBP-B (myelin basic protein locus B) has been reported to exhibit a high... more The PCR-based STR system MBP-B (myelin basic protein locus B) has been reported to exhibit a high rate of mutations. Using a newly designed pair of primers we present evidence that this is due to failed amplifications caused by a polymorphism in the annealing region of the reverse primer originally designed. With the new reverse primer described here no exclusions were found (out of 59 mother/child pairs analysed) while one was detected with the old set of primers. The results obtained with both pairs of primers in a random population sample (n = 112) from North Portugal are compared. In this sample 13 individuals typed as homozygotes with the pair of primers originally described, were found to be heterozygous when the amplifications were performed with the new reverse primer. By sequence analysis, a substitution in the reverse primer binding sequence originally described was determined. This substitution is located upstream from the repetition site and consists of G→A transition. This variation reaches polymorphic frequency and is responsible for the relatively frequent null alleles due to failed amplifications when the previously designed primers are used.
North African populations are considered genetically closer to Eurasians than to sub-Saharans. Ho... more North African populations are considered genetically closer to Eurasians than to sub-Saharans. However, they display a considerably high mtDNA heterogeneity among them, namely in the frequencies of the U6, East African, and sub-Saharan haplogroups. In this study, we describe and compare the female gene pools of two neighboring Tunisian populations, Kesra (Berber) and Zriba (non-Berber), which have contrasting historical backgrounds. Both populations presented lower diversity values than those observed for other North African populations, and they were the only populations not showing significant negative Fu's F(S) values. Kesra displayed a much higher proportion of typical sub-Saharan haplotypes (49%, including 4.2% of M1 haplogroup) than Zriba (8%). With respect to U6 sequences, frequencies were low (2% in Kesra and 8% in Zriba), and all belonged to the subhaplogroup U6a. An analysis of these data in the context of North Africa reveals that the emerging picture is complex, because Zriba would match the profile of a Berber Moroccan population, whereas Kesra, which shows twice the frequency of sub-Saharan lineages normally observed in northern coastal populations, would match a western Saharan population except for the low U6 frequency. The North African patchy mtDNA landscape has no parallel in other regions of the world and increasing the number of sampled populations has not been accompanied by any substantial increase in our understanding of its phylogeography. Available data up to now rely on sampling small, scattered populations, although they are carefully characterized in terms of their ethnic, linguistic, and historical backgrounds. It is therefore doubtful that this picture truly represents the complex historical demography of the region rather than being just the result of the type of samplings performed so far.
Molecular genetic data contain information on the history of populations. Evidence of prehistoric... more Molecular genetic data contain information on the history of populations. Evidence of prehistoric demographic expansions has been detected in the mitochondrial diversity of most human populations and in a Y-chromosome STR analysis, but not in a previous study of 11 Y-chromosome SNPs in Europeans. In this paper, we show that mismatch distributions and tests of mutation/drift equilibrium based on up to 166 Y-chromosome SNPs, in 46 samples from all continents, also fail to support an increase of the male effective population size. Computer simulations show that the low nuclear versus mitochondrial mutation rates cannot explain these results. However, ascertainment bias, i.e., when only highly variable SNP sites are typed, may be concealing any Y SNPs evidence for a recent, but not an ancient, increase in male effective population sizes. The results of our SNP analyses can be reconciled with the expansion of male effective population sizes inferred from STR loci, and with mitochondrial evidence, by admitting that humans were essentially polygynous during much of their history. As a consequence, until recently only a few men may have contributed a large fraction of the Y-chromosome pool at every generation. The number of breeding males may have increased, and the variance of their reproductive success may have decreased, through a recent shift from polygyny to monogamy, which is supported by ethnological data and possibly accompanied the shift from mobile to sedentary communities.
Goat is believed to be the first true livestock domesticated and, apart from its historical impor... more Goat is believed to be the first true livestock domesticated and, apart from its historical importance, keeps playing an essential economic role in very diverse human societies. We have analysed the female gene pool of all Portuguese autochthonous breeds (Bravia, Serrana, Charnequeira, Serpentina and Algarvia) through the mtDNA HVI sequencing of 288 unrelated animals sampled throughout the country. All breeds proved to be extremely diverse (average haplotype diversity of 0.977), in contrast with the Portuguese peripheral geographic situation in the distribution range of the species. Moreover, observed genetic distances between breeds do not correlate with microgeography inside Portugal. These observations are consistent with recurrent refreshment of the breeding stock through the introduction of exotic animals. Fitting the new data into the still loosely defined female genetic pool landscape of goats, all Portuguese animals, one sample excepted (belonging to Bravia), are classified into haplogroup A and haplotype sharing is geographically very sparse, including a Far East match. Our results confirm that goats stand out among most of domesticates as exceptionally diverse and showing an unparalleled degree of mobility of animals (at least females) used for reproductive purposes.
We report a simultaneous study of the VWA STR locus by the Perkin-Elmer Profiler Plus kit and the... more We report a simultaneous study of the VWA STR locus by the Perkin-Elmer Profiler Plus kit and the Promega GenePrint CTTv kit in a population sample from North Portugal and in 418 meiosis from family material and paternity cases. PCR amplification and genotyping were performed according to the manufacturer’s instructions using ABI 377 or ABI 310 automatic sequencers. Biological kinship in family material and paternity cases was validated by the use of the STR loci D3S1358, D5S818, D7S820, D8S1179, D13S317, D18S51, D21S11, FGA, CSF1PO, TH01 and TPOX. Out of 434 unrelated individuals we found 4 inconsistencies between the genotypes obtained using each kit. No exclusions were found in the meiotic analyses. In all cases, these inconsistencies were due to an annealing failure of the Perkin-Elmer forward primer resulting in false homozygotes. Sequencing analysis revealed an A-to-T substitution at position 1631 (GenBank sequence M25858), 52 bases upstream of the first TCTA motif of the repeat region. An estimate of the null allele frequency (s) in this study is thus obtainable from the expression s = 4/¶(2 × 434) = 0.46%. The relatively high frequency of these discrepancies in our population demonstrates the need for caution when comparing genotype or gene frequency estimates made from amplicons produced by different primers, when evaluating apparent exclusions in paternity testing and when searching for a match between individual genetic profiles in forensic databases. Our findings are also compared with those previously reported.
A sample of mitochondrial DNA (mtDNA) from the southeastern African population of Mozambique has ... more A sample of mitochondrial DNA (mtDNA) from the southeastern African population of Mozambique has been shown to have affinities with populations both to its north and south. From the north came sequences that may have been involved in the Bantu expansion (from western, through eastern, to southern Africa), such as members of haplogroups L3b, L3e1a and a subset of L1a. The dating of the major component of Mozambican mtDNAs, the subset L2a of haplogroup L2, displayed an age range compatible with the Bantu expansion. The southern influence was traced by the presence of sequence types from haplogroup L1d, a probable relict of Khoisan-speaking populations that inhabited the region prior to their displacement by the Bantu-speaking incomers. Within historical times, the forced displacement of Mozambicans as part of the slave trade, mainly documented as being to the Americas, generated a differential input of eastern African sequences into the mtDNA pools of the Americas and of Europe, as testified to by the greater number of sequence matches between Mozambique and the Americas, compared to those between Mozambique and Europe.
An enlarged mtDNA database (n=549) for the Portuguese population, comprising HVRI and HVRII regio... more An enlarged mtDNA database (n=549) for the Portuguese population, comprising HVRI and HVRII regions is reported. This database was used to test the effect of sample size on the estimation of relevant parameters such as haplotype diversity, number of different haplotypes, nucleotide diversity and number of polymorphic positions. Simulations were performed generating sets of random subsamples of variable sizes (n=50, 100, 200, 300 and 400). The results show that while haplotype and nucleotide diversities do not vary significantly with sample size, the numbers of haplotypes and polymorphic positions rise continuously inside the tested interval. These trends are interpretable by the evolution of the proportions of sequences that are found once or twice, which drop dramatically as sample size increases, with the corresponding rise in the frequency of those encountered 3 times or more. The generated data were also used to extrapolate saturation curves for the referred parameters. When considering for instance the number of haplotypes, it is shown that a sample size of 1,000 individuals is required for practical saturation (defined as the point where a sample size increase of 100 individuals corresponds to an increment in the diversity measure below 5%). For HVRII the same level is reached at n=900 and n=1,300 is needed when both regions are analysed simultaneously. Consequently, we can infer that currently used sample sizes are still rather inadequate for both anthropological and forensic purposes.
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