Polymorphic Nucleotide Positions Research Articles

Mountain- and brown hare genetic polymorphisms to survey local adaptations and conservation status of the heath hare (Lepus timidus sylvaticus, Nilsson 1831)

We provide the first whole genome sequences from three specimens of the mountain hare subspecies the heath hare (Lepus timidus sylvaticus), along with samples from two mountain hares (Lepus timidus timidus) and two brown hares (Lepus europaeus) from Sweden. The heath hare has a unique grey winter pelage as compared to other mountain hares (white) and brown hares (mostly brown), and face regional extinction, likely due to competitive exclusion from the non-native brown hare. Whole genome resequencing from the seven hare specimens were mapped to the Lepus timidus pseudoreference genome and used for detection of 11,363,883 polymorphic nucleotide positions. The data presented here could be useful for addressing local adaptations and conservation status of mountain hares and brown hares in Sweden, including unique subspecies.

Low Intrahost and Interhost Genetic Diversity of Carnivore Protoparvovirus 1 in Domestic Cats during a Feline Panleukopenia Outbreak.

Feline panleukopenia (FPL), a highly contagious and frequently fatal disease of cats, is caused by Feline parvovirus (FPV) and Canine parvovirus (CPV). We characterised the diversity of these Carnivore protoparvovirus 1 variants in 18 faecal samples collected from domestic cats with FPL during an outbreak, using targeted parvoviral DNA metagenomics to a mean depth of >10,000 × coverage per site. All samples comprised FPV alone. Compared with the reference FPV genome, isolated in 1967, 44 mutations were detected. Ten of these were nonsynonymous, including 9 in nonstructural genes and one in VP1/VP2 (Val232Ile), which was the only one to exhibit interhost diversity, being present in five sequences. There were five other polymorphic nucleotide positions, all with synonymous mutations. Intrahost diversity at all polymorphic positions was low, with subconsensus variant frequencies (SVF) of <1% except for two positions (2108 and 3208) in two samples with SVF of 1.1–1.3%. Intrahost nucleotide diversity was measured across the whole genome (0.7–1.5%) and for each gene and was highest in the NS2 gene of four samples (1.2–1.9%). Overall, intrahost viral genetic diversity was limited and most mutations observed were synonymous, indicative of a low background mutation rate and strong selective constraints.

Forensic Genetic Analysis of Mitochondrial DNA Hypervariable Region III Sequences in Muslims from South India

The absence of recombination, high rate of mutation, and maternal inheritance of the mitochondrial DNA (mtDNA) genome makes it a valuable tool in many fields including evolutionary anthropology, genetic genealogy, population history, and forensic science. The mtDNA genome can be separated into two parts: a large coding region and a smaller fragment called the control region or non-coding region that generally referred to as displacement loop (D loop). The mtDNA D-loop region was highly polymorphic and has proven a precious marker in forensic identification. The study aims to examine and suggests polymorphism of the HVRIII region as a power marker along with HVRI and HVRII in forensic investigations. Within the control region of the mtDNA genome, the sequences of hypervariable region III (HVR III) (nucleotide position 438-574) were obtained from 60 unrelated Muslims of Shrirangapattana town, located in Karnataka state of South India. The complete mtDNA control region was amplified and sequenced by the Sanger sequencing method. The study provided the identification of 18 different haplotypes and 17 polymorphic nucleotide positions. The most common haplotype (H.18) was consistent with the Anderson sequence which occurred fourteen times. The distribution of nucleotide substitutions, insertions, and deletions was computed and determined that transitions made up the majority of the variations (58%) in this region. The genetic diversity was estimated at 0.89939 and the random match probability at 0.1155. The power of discrimination was found to be 0.8844 and the rest of the statistical parameters such as the mean of pair-wise differences and nucleotide diversity were established as 2.255932 ± 1.25884 and 0.010071± 0.00623, respectively. Consequently, the discovery of high genetic, haplotype, and nucleotide diversity, and high power of discrimination impart the use of hypervariable region III (HVR III) as an important marker in forensic investigations.

Group-1 Grass Pollen Allergens with Near-Identical Sequences Identified in Species of Subtropical Grasses Commonly Found in Southeast Asia.

Background and objectives: Group-1 grass allergens or beta-expansins (EXPBs) are major allergens from pollen of all grass species. Previous studies showed that they are highly conserved (64–85%) in Pooideae species, which are found mostly in the temperate regions. However, the information about group-1 allergens from common grass species in subtropical areas is still lacking. This study aimed to assess the sequence diversity of group-1 grass pollen allergens in subtropical areas, especially in Southeast Asia. Materials and Methods: Group-1 allergens were cloned from pollen of eight grass species using a single set of primers. Sequences were analyzed and IgE and IgG4 binding regions were compared to the previously reported epitopes in homologous EXPBs. The phylogenetic analysis was used to assess the relationship between sequences of these species and previously characterized EXPBs. Moreover, three-dimensional structure of the EXPB was modeled based on homology to Zea m 1. Results: Sequences from eight grass species were nearly identical. It is conceivable that the primers used for cDNA amplification detected the same isoform in different species. In fact, the deduced amino acid sequences shared 97.79–100% identity with each other and 15/819 polymorphic nucleotide positions were identified. The predicted structure showed that the IgE and IgG4 epitopes and polymorphic residues were located in both domains 1 and 2. The dendrogram presents clustering of class A EXPBs into four groups corresponding to the grass subfamilies. Conclusions: This study identified the allergens with near-identical sequences from different grass species. This isoform could be the major cross-reacting allergenic protein from commonly found grass species.

Sequence diversity of MHC class-II DRB gene in gazelles (Gazella subgutturosa) raised in Sanliurfa of Turkey

In this study, we aimed to assess the sequence diversity of major histocompatibility complex (MHC) class-II DRB gene at exon 2 in gazelles raised in Sanliurfa Province of Turkey. Twenty DNA samples isolated from gazelles (Gazella subgutturosa) were used for sequencing exon 2 of MHC class-II DRB gene. Target region was amplified by polymerase chain reaction (PCR) and their products were directly sequenced. Nine of these 20 samples yielded unambiguously readable sequences. Three of the nine samples were homozygotes and each showed different sequences. A 262-bp sequence obtained from the three homozygote samples were submitted to GenBank (accession numbers: KC309405, KC309406 and KC309407). Using an allele specific PCR, we detected 10 additional haplotypes. Among 13 haplotypes, 45 nucleotide positions were polymorphic and most of the polymorphic nucleotide positions localized at peptide-binding region (PBR). Rates of nonsynonymous substitutions were significantly higher than synonymous substitutions at PBR. Phylogenetic analysis of the haplotypes showed that 10 haplotypes of the gazelles were clustered together while three were clustered with ovine and bovine haplotypes. The results indicated that at least 13 haplotypes at exon 2 of MHC class-II DRB gene were showing high degree of nucleotide and amino acid diversity, and certain haplotypes of G. subgutturosa were more similar to haplotypes from sheep or cattle than to each other. Rates of synonymous and nonsynonymous substitutions suggested that positive selection was a driving force for diversity at this locus in G. subgutturosa.

Genetic diversity of the toll-like receptor 2 (TLR2) in hare (Lepus capensis) populations from Tunisia

Toll-like receptors (TLRs) are a major group of proteins that recognize molecular components of infectious agents, known as pathogen associated molecular patterns (PAMPs). The structure of these genes is similar and characterized by the presence of an ectodomain, a signal transmembrane segment and a highly conserved cytoplasmic domain. The latter domain is homologous to the human interleukin-1 receptor (IL1R) and human IL-18 receptor (IL-18R) and designated TIR domain. The latter domain of the TLR genes was suggested to be very conservative and its evolution is driven by purifying selection. Variability and evolution of the TIR sequences of TLR2 gene were studied in three hare populations from Tunisia with different ecological characteristics (NT–North Tunisia with Mediterranean, CT–Central Tunisia with semi-arid, and ST–South Tunisia with arid climate). Sequencing of a 372bp fragment of TIR2 revealed 25 alleles among 110 hares. Twenty variable nucleotide positions were detected, of which 7 were non-synonymous. The highest variability was observed in CT, with 16 polymorphic positions. In ST, only 4 polymorphic nucleotide positions were detected with all diversity values lower than those recorded for the other two populations. By using several approaches, no positive selection was detected. However, evidence of purifying selection was found at two positions. The logistic models of the most common TIR2 protein variant that we run to examine whether its occurrence was affected by climatic variation independent of the geographic sample location suggested only a longitudinal effect. Finally, the mapping of the non-synonymous mutations to the inferred tertiary protein structure showed that they were all localized in the different loop regions. Among all non-synonymous substitutions, three were suggested to be deleterious as evidenced by PROVEAN analysis. The observed patterns of variability characterized by low genetic diversity in ST might suggest that the TIR region was more affected, than other markers, by genetic drift or/and that these patterns were shaped by different selective pressures under different ecological conditions. Notably, this low diversity was not detected by other (putatively neutral) microsatellite markers analysed in the course of other studies. But low diversity was also found for two MHC class II adaptive immune genes. As expected from functionally important regions, the evolution of the TIR2 domain is mainly driven by purifying selection. However, the occurrence of deleterious non-synonymous substitutions might highlight the flexible evolution of the TIR genes and/or their interactions with other proteins.

Genetic diversity of the human adenovirus species C DNA polymerase

BackgroundHuman Adenovirus (HAdV) are responsible for severe infections in hematopoietic stem cells transplant (HSCT) recipient, species C viruses being the most commonly observed in this population. There is no approved antiviral treatment yet. Cidofovir (CDV), a cytidine analog, is the most frequently used and its lipophilic conjugate, brincidofovir (BCV), is under clinical development. These drugs target the viral DNA polymerase (DNA pol). Little is known about the natural polymorphism of HAdV DNA pol in clinical strains. MethodsWe assessed the inter- and intra-species variability of the whole gene coding for HAdV DNA pol of HAdV clinical strains of species C. The study included 60 species C HAdV (21 C1, 27 C2 and 12 C5) strains isolated from patients with symptomatic infections who had never experienced CDV or BCV treatments and 20 reference strains. We also evaluated the emergence of mutations in thrirteen patients with persistent HAdV infection despite antiviral treatment. ResultsWe identified 356 polymorphic nucleotide positions (9.9% of the whole gene), including 102 positions with nonsynonymous mutations (28.0%) representing 8.7% of all amino acids. The mean numbers of nucleotide and amino acid mutations per strain were 23.1 (±6.2) and 5.2 (±2.4) respectively. Most of amino acid substitutions (60.6%) were observed in one instance only. A minority (13.8%) were observed in more than 10% of all strains. The most variable region was the NH2 terminal domain (44.2% of amino acid mutations). Mutations in the exonuclease domain accounted for 27.8%. The binding domains for the terminal protein (TPR), TPR1 and TPR2, presented a limited number of mutations, which were nonetheless frequently observed (62.5% and 58.8% of strains for TPR1 and TPR2, respectively). None of the mutations associated with CDV or BCV resistance were detected. In patients receieving antiviral drugs with persistent HAdV replication, we identified a new mutation in the NH2 terminal region. ConclusionsOur study shows a high diversity in HAdV DNA pol sequences in clinical species C HAdV and provides a comprehensive mapping of its natural polymorphism. These data will contribute to the interpretation of HAdV DNA pol mutations selected in patients receiving antiviral treatments.

P130 An algorithm to detect and quantify low frequency HLA-genotypes in NGS data

Aim Next-generation sequencing (NGS) provides a cost and time efficient approach for routine HLA typing. Analysis software typically presumes a diploid genotype with counts of sequencing reads from homozygous or heterozygous genotypes. However, chimeric samples with more than two alleles per locus and imbalanced read counts may occur, for instance, after partially HLA-mismatched hematopoietic stem cell transplantation. Detection of alleles at even very low allele frequencies can then become diagnostically relevant in the context of post-transplant relapse. Here, we describe and validate a novel algorithm for rare HLA allele detection using NGS data. Methods Sequencing reads are mapped against expected HLA-alleles in a chimeric DNA-sample. Only polymorphic nucleotide positions differentiating the expected allele sequences are considered for further analysis. Reads are classified based on the frequency of discriminatory di-nucleotides. The classification algorithm was validated using in vitro admixtures of DNA samples down to allelic frequencies of 0.5 percent based on NGS data targeting exons 2 and 3 for the HLA genes A, B, C, DQB1, DRB1, and DPB1. Results We found that sensitivity and specificity are allele and locus-specific and depend on the number of sequence differences between the alleles. In each of the analyzed admixture samples a subset of the 12 amplicons harbored sufficient sequence differences to detect genotypes at dilutions as low as 0.5 percent. Fig. 1 shows an example for 3 amplicons. Conclusions The proposed algorithm may support the application of cost effective NGS for sensitive detection of HLA microchimerism in different settings including transplantation and pregnancy. Download : Download high-res image (200KB) Download : Download full-size image

Tracking of the gene Fom2 and study on the genetic diversity of NB-ARC domain in the number of resistant and sensitive melon cultivars against Fusarium oxysporum f. sp. melonis (race 1)

Vascular wilt caused by Fusarium oxysporum f. sp. melonis (FOM) is one of the most important diseases in melon. Four pathogenic races named: 0, 1, 2, and 1.2 have been identified in population of this fungus based on the pathogenicity pattern on differential varieties. The gene Fom2 has been reported as a key gene for conferring resistance to the fungus race 1. The majority of Iranian melon cultivars are susceptible to race 1. Clarifying the occurrence pattern of Fom2 in the fungus race 1 in sensitive and resistant Iranian melon cultivars in comparison with two standard melon lines could be helpful for the investigation on the role of Fom2 in the cultivars reaction. Since, the full sequence of Fom2 from Melon and I2 from Tomato resistance sources were cloned; it has been shown that the LRR and NB-ARC domains in the coded proteins play critical roles in detection of Avr proteins and R proteins activation as well. Considering to the importance of NB-ARC domain and the lack of functional information in this region of the gene Fom2, this domain was selected for sequencing, identifying possible mutations and to study their possible relationship of mutations with the type of cultivars reaction against fungal races. Six melon cultivars including four Iranian local melon cultivars (Mashhadi, Shahde Shiraz, Khatooni and Khaghani) and two standard melon cultivars (Charentais Fom1 and Charentais Fom2) were used in this study. Two specific primer pairs (PSh20-F/R and PSh20.2-F/R) were designed and used for the tracking of partial and full-length sequence of Fom2. More investigation was performed on the diversity occurrence pattern among cultivars and its conformity with cultivars resistive pattern against the fungus race 1. Our results showed that the partial and the full length sequence of Fom2 had this potential to be tracked in sensitive as well as resistant cultivars. On the other hand, the cultivars reactions were not predictable based on the gene occurrence pattern. However, some studies have already been reported that the linked markers of Fom2 were not useful for the prediction of reaction pattern in Iranian melon cultivars. The partial segments of Fom2 amplified by PSh20-F/R primers were bi-directionally sequenced and the results were submitted in gene bank. Sequence analysis of NB-ARC domain in the six varieties led to detection of 16 polymorphic nucleotide positions, which nine of them appeared in protein levels. Six out of the nine mutations placed in AAA-ATPase subdomain. Although the polymorphic pattern of mutations in nucleotide and amino acid positions were not in accordance with the cultivars resistive pattern but the cultivars could be discriminated based on some other mutations. Due to the switching function of AAA-ATPase sub domain in ATP hydrolysis and activation of R proteins, variation in this region between the resistant and susceptible cultivars could be considerable. It seems the presence of identified mutations at AAA-ATPase subdomain of Fom2 could be considered in cultivars resistive pattern unconformity and it needs to be followed in future studies to clarify the action of detected mutations in this subdomain.

Molecular identification and quantification of tetracycline and erythromycin resistance genes in Spanish and Italian retail cheeses.

Large antibiotic resistance gene pools in the microbiota of foods may ultimately pose a risk for human health. This study reports the identification and quantification of tetracycline- and erythromycin-resistant populations, resistance genes, and gene diversity in traditional Spanish and Italian cheeses, via culturing, conventional PCR, real-time quantitative PCR (qPCR), and denaturing gradient gel electrophoresis (DGGE). The numbers of resistant bacteria varied widely among the antibiotics and the different cheese varieties; in some cheeses, all the bacterial populations seemed to be resistant. Up to eight antibiotic resistance genes were sought by gene-specific PCR, six with respect to tetracycline, that is, tet(K), tet(L), tet(M), tet(O), tet(S), and tet(W), and two with respect to erythromycin, that is, erm(B) and erm(F). The most common resistance genes in the analysed cheeses were tet(S), tet(W), tet(M), and erm(B). The copy numbers of these genes, as quantified by qPCR, ranged widely between cheeses (from 4.94 to 10.18log⁡10/g). DGGE analysis revealed distinct banding profiles and two polymorphic nucleotide positions for tet(W)-carrying cheeses, though the similarity of the sequences suggests this tet(W) to have a monophyletic origin. Traditional cheeses would therefore appear to act as reservoirs for large numbers of many types of antibiotic resistance determinants.

FCGR3B Gene Variants In Brazilians: New Alleles Should Be Considered?

Background FCGR3B gene encoding the receptor FcγRIIIb has three polymorphic forms (FCGR3B*01, FCGR3B*02 and FCGR3B*03) which represent the human neutrophil antigens HNA-1a, -1b and -1c, respectively. It has been postulated that variants of these alleles might represent new FCGR3B alleles, however there are few studies assessing the frequency of such variants in different populations. In this study we performed an analysis of the frequency of the FCGR3B alleles and its variant forms examining the polymorphic region of the FCGR3B gene in a large Brazilian population sample including healthy blood donors and patients with systemic lupus erythematosus (SLE). Study Design and Methods A segment containing the polymorphic nucleotide positions 141, 147, 227, 266 and 277 in exon 3 was sequenced from genomic DNA of 615 individuals including 310 blood donors and 305 patients with SLE. Additionally, nucleotide databases were screened for variant FCGR3B sequences. Results The allele frequencies for FCGR3B*01, FCGR3B*02 and FCGR3B*03 were 0.37, 0.59 and 0.04, respectively. In two patients with SLE (0.3%) there was no gene amplification characterizing the FCGR3B-null phenotype. Considering the 5 FCGR3B polymorphic sites, 4 variant forms were observed. One variant was linked to the FCGR3B*01 allele with SNPs occurring simultaneously in two positions: A227G and G277A (n=2; 0.3%). The other three variants, linked to the FCGR3B*02 allele were: A277G (n=8; 1.3%); C141G (n=3; 0.5%); and T147C (n=1; 0.2%). All mutations observed were missenses. The results are summarized in the Table. In 103/613 (16.8%) sequenced individuals we found 19 SNPs on 14 polymorphic sites distinct from those which characterize the FCGR3B alleles. Among the most frequent, T230A was observed in 36 cases (5.9%), T230G in 20 cases (3.3%), G249A in 6 cases (1%) and C337A in 6 cases (1%), all shown as heterozygous. The comparative analysis between the two groups of individuals revealed an association between the presence of the allele FCGR3B*01 and the HNA-1a/a and HNA-1a/b genotypes with increased susceptibility to SLE (p &lt;0.001; p = 0.028; and p = 0.012, respectively), however there was no significant correlation between these genotypes and disease severity. Discussion The observed frequencies of the FCGR3B*01, FCGR3B*02, and FCGR3B*03 alleles were similar to those reported by genotyping studies which included Europeans or North Americans individuals. The frequency of the null phenotype (0.3%) was similar to that reported by Europeans (0.2-0.8%). The variant FCGR3B*02A277G showed a frequency (1.3%) sufficiently high to be considered a new allele as proposed by the last Granulocyte Working Party/ISBT 2013. Interestingly, this variant was significantly more prevalent in patients with SLE than in blood donors (p=0.03). All the variants described in this study have been observed in different populations however we emphasize the presence of SNPs (T230A, T230G, G249A and C337A) in polymorphic sites different from those that characterize the FCGR3B gene alleles. We conclude that the FCGR3B gene is highly polymorphic and that the occurrence of new alleles must be considered, however the functional consequences of such changes has not yet been elucidated. Disclosures: No relevant conflicts of interest to declare.

The long polar fimbriae operon and its flanking regions in bovineEscherichia coliO157:H43 and STEC O136:H12 strains

Long polar fimbriae (Lpf) are intestinal adhesins and important virulence factors of pathogenic Escherichia coli strains. We cloned and sequenced the lpf2-1 operon (lpf2ABCD) and its flanking regions of an intimin- and Shiga toxin-negative atypical E.coli O157:H43 strain of bovine origin and also sequenced the lpf2-1 operon of six additional atypical O157 bovine E.coli strains of various serotypes. Nucleotide sequence comparison of these lpf operons showed sequence conservation as they contained only four polymorphic nucleotide positions. Investigation of these O157 strains as well as 13 E.coli Reference Collection (ECOR) strains carrying the lpf2-1 allele revealed high degree of sequence conservation in the lpf2 flanking regions. The lpf2-1 allele is also present in a bovine Shiga toxin-producing E.coli STEC O136:H12 strain, and in vitro adherence assays revealed that the absence of lpf2-1 in this strain did not affect its host cell-binding properties. Our data indicate that lpf2 loci are highly conserved in E.coli isolates; however, its role in adherence might be masked by other uncharacterized adhesins.

Increased level of intragenomic 16S rRNA gene heterogeneity in commensal strains closely related to Haemophilus influenzae

The 16S rRNA gene sequence of strains closely related to, but excluded from, Haemophilus influenzae was investigated and a conspicuously high number of polymorphic nucleotide positions due to intragenomic 16S rRNA gene heterogeneity was observed. The average frequency of 16S rRNA gene polymorphic nucleotide positions in 31 variant strains was 7.0×10(-3), which is approximately ten times the level observed in validated strains of H. influenzae. Sixty-seven polymorphic nucleotide positions in seven strains most likely originated from the simultaneous presence of two distinct types of helix 18 as a consequence of prior recombinatorial events. The increased level of 16S rRNA gene polymorphism in commensal taxa excluded from the pathogenic species H. influenzae is unexplained. The heterogeneity imposes difficulties on rRNA gene-based classification and systematics.

Intragenomic and intraspecific heterogeneity in rrs may surpass interspecific variability in a natural population of Veillonella

As well as intraspecific heterogeneity, intragenomic heterogeneity between 16S rRNA gene copies has been described for a range of bacteria. Due to the wide use of 16S rRNA gene sequence analysis for taxonomy, identification and metagenomics, evaluating the extent of these heterogeneities in natural populations is an essential prerequisite. We investigated inter- and intragenomic 16S rRNA gene heterogeneity of the variable region V3 in a population of 149 clinical isolates of Veillonella spp. of human origin and in 13 type or reference Veillonella strains using PCR-temporal temperature gel electrophoresis (TTGE). 16S rRNA gene diversity was high in the studied population, as 45 different banding patterns were observed. Intragenomic heterogeneity was demonstrated for 110 (74 %) isolates and 8 (61.5 %) type or reference strains displaying two or three different gene copies. Polymorphic nucleotide positions accounted for 0.5-2.5 % of the sequence and were scattered in helices H16 and H17 of the rRNA molecule. Some of them changed the secondary structure of H17. Phylotaxonomic structure of the population based on the single-copy housekeeping gene rpoB was compared with TTGE patterns. The intragenomic V3 heterogeneity, as well as recombination events between strains or isolates of different rpoB clades, impaired the 16S rRNA-based identification for some Veillonella species. Such approaches should be conducted in other bacterial populations to optimize the interpretation of 16S rRNA gene sequences in taxonomy and/or diversity studies.

Differences and changes in human papillomavirus type 16 variant status in human immunodeficiency virus-positive adults are not uncommon

Human papillomavirus type 16 (HPV-16) genotype variants have been the subject of several investigations, but study participants have rarely been sampled more than once. In this study, among a cohort of human immunodeficiency virus (HIV)-infected adults, HPV-16 variants were investigated in samples collected concurrently from the anus and cervix, as well as in serial samples collected from the same anatomical site at 12-month intervals. HPV-16 variants in stored extracts of cervical and anal samples were determined from subjects with multiple visits and at least one sample positive for HPV-16. Seven polymorphic nucleotide positions within the E6 region were analysed by pyrosequencing to determine genotype variants. Of 364 samples examined, 176 anal and 39 cervical swabs from 84 different subjects yielded unequivocal sequences of eight major HPV-16 variants. Eight samples contained probable novel HPV-16 variants and in one sample two variants were detected. In eight out of 29 (27.6%) anal-cervical sample pairs positive for HPV-16, discordant variants were found. From 57 anal and nine cervical sample series of HPV-16-positive samples, a change in HPV-16 variant status over time was seen in nine (13.6%) instances (seven anal and two cervical) from eight different participants. Changes in HPV-16 variants in HIV-infected adults were seen most frequently when different anatomical sites were sampled, but were also observed over time.

Mitochondrial DNA sequence analysis of a native Bolivian population

Mitochondrial DNA analysis is very useful for the interpretation of the history of human migration and to estimate the frequency of a haplotype in the forensic context. From a human settlement perspective, La Paz area is greatly interesting since the first planned city of the region is located there. Samples from 110 individuals from La Paz were studied analysing the polymorphisms in the D-loop, hypervariable region I (HVI) and hypervariable region II (HVII) in order to verify the genetic diversity. The aim of this study was to start the creation of a population database in order to obtain the genetic interpopulation variability and classify haplotypes into characteristic haplogroups of South America. A total of 97 different haplotypes were identified, 90 being unique, expressed by 122 polymorphic nucleotide positions. Nucleotide and sequence diversity were estimated to be 0.015 ± 0.0075 and 0.996, respectively. Haplogroup distribution in the samples was 57.27% B4, 19.09% C1, 10.00% A2, 3.64% D1, 2.73% D4h3, 1.82% H, and 0.91% for each of the haplogroups A4, B4c1a, CZ, D4J, M7a and M8/N9b. The rate of length heteroplasmy was 36.36% in HVI and 52.73% in HVII. Phylogenetic analysis reveals proximity to the Korean, Chilean aboriginal, Japanese and Australian populations. The estimated genetic variability of the studied population was high, suggesting an early settlement.

Evidence for gene recombination in FCGR3 gene variants

The genes encoding the Fcgamma receptors (FcgammaR) IIIa and IIIb (FCGR3A and FCGR3B) are clustered on chromosome 1 band q23-24 and exhibit allelic polymorphism. We investigated the molecular basis of additional new FCGR3 genomic variation. A segment shared by FCGR3A and FCGR3B containing the polymorphic nucleotide positions 141, 147, 227, 266, and 277 in exon 3 was cloned and sequenced from genomic DNA of 30 donors and 3 bacterial artificial chromosome (BAC) clones. A mixture consisting of isolated FCGR3B*2- and FCGR3A- plasmids was cloned and sequenced as well. Additionally, nucleotide databases were screened for clones with variant FCGR3 sequences. A total of 12 FCGR3 variants defined by the polymorphic positions were detected in whole blood genomic DNA from 23 of 24 FCGR3B*2 and/or FCGR3B*3 positive donors, the DNA from two of three BAC clones and in the DNA mixture of isolated FCGR3B*2- and FCGR3A- plasmids. Nucleotide exchanges of the variants were non-random and resulted from two alternative nucleotides present in one of the polymorphic position of the basic FCGR3 forms. Polymerase chain reaction (PCR) artefacts cannot be excluded as origin of new variants, but there is strong evidence that at least two variants are the result of a somatic recombination.

Variation in the gene encoding acetolactate‐synthase in Lolium species and proactive detection of mutant, herbicide‐resistant alleles

Summary Lolium species (ryegrasses) are genetically highly variable plants that are both forage crops and major weeds across the globe. As weeds, they rapidly evolve resistance under the selective pressure of acetolactate‐synthase (ALS) inhibitors, the most resistance‐prone herbicide group. Quick and accurate diagnosis is therefore of importance to prevent resistance spread in ryegrass. To develop proactive molecular tools for the detection of mutant, resistant ALS alleles, we assessed variation in the ryegrass ALS gene. Sequencing the full 1929‐bp ALS coding sequence in 59 plants from six distant locations revealed a total of 208 polymorphic nucleotide positions (one every 9.3 nucleotides). The heterogeneous distribution of synonymous and non‐synonymous substitutions along the ALS coding sequence suggested that nucleotide variation of ALS is shaped by purifying and background selection. Using regions of the ALS coding sequence with a low number of polymorphic nucleotide sites, five derived cleaved amplified polymorphic sequence (dCAPS) assays were developed targeting codons crucial for herbicide sensitivity. These enabled the first detection in ryegrass of a Pro‐197‐Thr substitution that confers herbicide resistance. Most assays could also be used to genotype Festuca and Vulpia plants. These dCAPS assays should prove powerful tools for both resistance diagnosis and population genetics studies.

Isolation and characterization of Treponema phagedenis-like spirochetes from digital dermatitis lesions in Swedish dairy cattle

BackgroundDigital dermatitis in cattle is an emerging infectious disease. Ulcerative lesions are typically located on the plantar skin between the heel bulbs and adjacent to the coronet. Spirochetes of the genus Treponema are found in high numbers in the lesions and are likely to be involved in the pathogenesis. The aim of this study was to obtain pure cultures of spirochetes from cattle with digital dermatitis and to describe them further.MethodsTissue samples and swabs from active digital dermatitis lesions were used for culturing. Pure isolates were subjected to, molecular typing through 16S rRNA gene sequencing, pulsed-field gel electrophoresis (PFGE), random amplified polymorphic DNA (RAPD) and an intergenic spacer PCR developed for Treponema spp. as well as API-ZYM and antimicrobial susceptibility tests. The antimicrobial agents used were tiamulin, valnemulin, tylosin, aivlosin, lincomycin and doxycycline.ResultsSeven spirochete isolates from five herds were obtained. Both 16S rRNA gene sequences, which were identical except for three polymorphic nucleotide positions, and the intergenic spacer PCR indicated that all isolates were of one yet unnamed species, most closely related to Treponema phagedenis. The enzymatic profile and antimicrobial susceptibility pattern were also similar for all isolates. However it was possible to separate the isolates through their PFGE and RAPD banding pattern.ConclusionThis is the first report on isolation of a Treponema sp. from cattle with digital dermatitis in Scandinavia. The phylotype isolated has previously been cultured from samples from cattle in the USA and the UK and is closely related to T. phagedenis. While very similar, the isolates in this study were possible to differentiate through PFGE and RAPD indicating that these methods are suitable for subtyping of this phylotype. No antimicrobial resistance could be detected among the tested isolates.

Mitochondrial DNA sequence analysis of native Bolivians population

Haplotypes of 111 individuals from La Paz have been studied by the analysis of the polymorphisms in mtDNA control region, HV1 and HV2 regions, in order to intend the genetic diversity. The aim was to created a population database, assess genetic interpopulation variability and classify haplogroups. We had identify 99 haplotypes with 125 polymorphic nucleotide positions, 93 haplotypes are unique. Nucleotide and sequence diversity are estimated in 0.016068 ± 0.008186 and 0.9988, respectively. Haplogroup distribution is as follows: 47.75% B; 14.41% C; 6.31% A; 4.5% D; 6.31% A, C, or D; 23.42% undetermined or compound haplogroups. Rate of heteroplasmy is 37.8% in HV1 and 53.2% in HV2.