Standardized Index Of Association Research Articles

Attack of the clones: Population genetics reveals clonality of Colletotrichum lupini, the causal agent of lupin anthracnose.

Colletotrichum lupini, the causative agent of lupin anthracnose, affects lupin cultivation worldwide. Understanding its population structure and evolutionary potential is crucial to design successful disease management strategies. The objective of this study was to employ population genetics to investigate the diversity, evolutionary dynamics, and molecular basis of the interaction of this notorious lupin pathogen with its host. A collection of globally representative C. lupini isolates was genotyped through triple digest restriction site-associated DNA sequencing, resulting in a data set of unparalleled resolution. Phylogenetic and structural analysis could distinguish four independent lineages (I-IV). The strong population structure and high overall standardized index of association (r̅d ) indicates that C. lupini reproduces clonally. Different morphologies and virulence patterns on white lupin (Lupinus albus) and Andean lupin (Lupinus mutabilis) were observed between and within clonal lineages. Isolates belonging to lineage II were shown to have a minichromosome that was also partly present in lineage III and IV, but not in lineage I isolates. Variation in the presence of this minichromosome could imply a role in host-pathogen interaction. All four lineages were present in the South American Andes region, which is suggested to be the centre of origin of this species. Only members of lineage II have been found outside South America since the 1990s, indicating it as the current pandemic population. As a seedborne pathogen, C. lupini has mainly spread through infected but symptomless seeds, stressing the importance of phytosanitary measures to prevent future outbreaks of strains that are yet confined to South America.

Another advantage of multi-locus variable-number tandem repeat analysis that can putatively subdivide enterohemorrhagic Escherichia coli O157 strains into clades by maximum a posteriori estimation.

Enterohemorrhagic Escherichia coli O157 (O157) strains can be subdivided into clades based on their single-nucleotide polymorphisms, but such analysis using conventional methods requires intense effort by laboratories. Although multi-locus variable-number tandem repeat analysis (MLVA), which can be performed with low laboratory burden, has been used as a molecular epidemiological tool, it has not been evaluated whether MLVA can be used the clade subdivision of O157 strains like it can for that of other pathogenic bacteria. This study aimed to establish a method for subdividing O157 strains into clades using MLVA data. The standardized index of association, ISA, for O157 strains isolated in Chiba prefecture, Japan (Chiba isolates) revealed the presence of unique tandem repeat patterns in each major clade (clades 2, 3, 7, 8, and 12). A likelihood database of tandem repeats for these clades was then constructed using the Chiba isolates, and a formula for maximum a posteriori (MAP) estimation was constructed. The ratio of the number of O157 strains putatively subdivided into a clade by MAP estimation from MLVA data relative to the number of O157 strains subdivided using single-nucleotide polymorphism analysis (designated as the concordance ratio [CR]) was calculated using the Chiba isolates and O157 strains isolated in Yamagata prefecture (Yamagata isolates). The CRs for the major Chiba and Yamagata isolate clades, other than clade 2, were 89%-100%. Although the CR for clade 2 Chiba isolates was >95%, that of the Yamagata isolates was only 78.9%. However, these clade 2 CRs were not significantly different from one another, indicating that clade 2 strains can be subdivided correctly by MAP estimation. In conclusion, this study expands the utility of MLVA, previously applied predominantly for molecular epidemiological analysis, into a low-laboratory-burden tool for subdividing O157 strains into phylogenetic groups.

Genome-wide analysis identified candidate variants and genes associated with heat stress adaptation in Egyptian sheep breeds.

Heat stress caused by climatic changes is one of the most significant stresses on livestock in hot and dry areas. It has particularly adverse effects on the ability of the breed to maintain homeothermy. Developing countries are advised to protect and prepare their animal resources in the face of potential threats such as climate change. The current study was conducted in Egypt’s three hot and dry agro-ecological zones. Three local sheep breeds (Saidi, Wahati, and Barki) were studied with a total of 206 ewes. The animals were exercised under natural heat stress. The heat tolerance index of the animals was calculated to identify animals with high and low heat tolerance based on their response to meteorological and physiological parameters. Genomic variation in these breeds was assessed using 64,756 single nucleotide polymorphic markers (SNPs). From the perspective of comparative adaptability to harsh conditions, our objective was to investigate the genomic structure that might control the adaptability of local sheep breeds to environmental stress under hot and dry conditions. In addition, indices of population structure and diversity of local breeds were examined. Measures of genetic diversity showed a significant influence of breed and location on populations. The standardized index of association (rbarD) ranged from 0.0012 (Dakhla) to 0.026 (Assuit), while for the breed, they ranged from 0.004 (Wahati) to 0.0103 (Saidi). The index of association analysis (Ia) ranged from 1.42 (Dakhla) to 35.88 (Assuit) by location and from 6.58 (Wahati) to 15.36 (Saidi) by breed. The most significant SNPs associated with heat tolerance were found in the MYO5A, PRKG1, GSTCD, and RTN1 genes (p ≤ 0.0001). MYO5A produces a protein widely distributed in the melanin-producing neural crest of the skin. Genetic association between genetic and phenotypic variations showed that OAR1_18300122.1, located in ST3GAL3, had the greatest positive effect on heat tolerance. Genome-wide association analysis identified SNPs associated with heat tolerance in the PLCB1, STEAP3, KSR2, UNC13C, PEBP4, and GPAT2 genes.

Elucidating the Population Structure and Genetic Diversity of Indian Puccinia striiformis f. sp. tritici Pathotypes Based on Microsatellite Markers

In India, systematic wheat yellow rust survey and pathotype (race) analysis work began in 1930. However, information on population structure and genetic diversity of yellow rust pathogen has not been available. To address this, we conducted studies on population structure and genetic diversity of Puccinia striiformis f. sp. tritici (Pst) pathotypes using 38 simple sequence repeat primer-pairs. Bayesian assignment and discriminant analysis of principal components indicated the presence of two distinct Pst subpopulations (Pop1 and Pop2) along with 37.9% admixed pathotypes. The unweighted pair-group method with arithmetic mean also categorized these pathotypes into two major clusters. Principal coordinates analysis explained 20.06 and 12.50% variance in horizontal and vertical coordinates, respectively. Index of association (IA) and the standardized index of association ([Formula: see text]) values showed that Pst subpopulations reproduced asexually (clonally). In total, 102 alleles were detected, with the expected heterozygosity (Hexp) per locus ranging from 0.13 to 0.73, with a mean of 0.47. The average polymorphic information content value of 0.40 indicated high genetic diversity among pathotypes. Analysis of molecular variance revealed 12% of the total variance between subpopulations, 11% among the pathotypes of each subpopulation, and 77% within pathotypes. A significant moderate level of genetic differentiation (FST = 0.122, P < 0.001) and gene flow (Nm = 1.80) were observed between subpopulations. The Pst virulence phenotypes showed a weak positive correlation (R2 = 0.027, P < 0.02) with molecular genotypes.

Identification of Novel Microsatellite Markers to Assess the Population Structure and Genetic Differentiation of Ustilago hordei Causing Covered Smut of Barley.

Barley covered smut (CS) pathogen Ustilago hordei genome was mined for microsatellite distribution and their application in defining population structure and genetic variation. To dissect the molecular variation and genetic structure of U. hordei, 59 fungal isolates representing two distinct agro-ecological zones of India were analyzed by employing simple sequence repeats (SSRs). Using bioinformatic approaches, a total of 100,239 and 137,442 microsatellites were identified from 20.13 and 26.94 Mb of assembled genomic sequences of Uh364 and Uh4857-4 isolates of U. hordei, respectively. Penta-nucleotides (31.29 and 29.75%) followed by tri-nucleotide (28.27 and 29.88%) were most prevalent in both the genomes. Out of them, 15 polymorphic microsatellites showing conservancies in both the genomes were selected for exploring population genetic structure of U. hordei. An average of two alleles per microsatellite marker was generated with band size ranging from 180 to 850 bp. Polymorphic information content (PIC) varied between 0.095 and 0.37. Fifty-nine isolates were distributed in two distinct groups with about 65% genetic similarity according to UPGMA clustering and population structure analysis (K = 2). Gene flow analysis (Nm = 1.009) reflected moderate gene flow among the analyzed population. An analysis of molecular variance (AMOVA) displayed high level of genetic variation within population (87%) and low variation among populations (13%). Linkage disequilibrium (LD) analysis indicated positively significant but relatively low standardized index of association (SIA) value in both the population sets (SIA = 0.181), advocating a state of LD with epidemic population structure. In conclusion, the newly developed neutral SSR markers are highly polymorphic within U. hordei and will be useful for revealing evolutionary history and providing deep insight into the population dynamics of U. hordei in India as well as facilitating developing management strategies for CS of barley.

Phylogeography and Population Structure Analysis Reveal Diversity by Gene Flow and Mutation in Ustilago segetum (Pers.) Roussel tritici Causing Loose Smut of Wheat.

Ustilago segetum (Pers.) Roussel tritici (UST) causes loose smut of wheat account for considerable grain yield losses globally. For effective management, knowledge of its genetic variability and population structure is a prerequisite. In this study, UST isolates sampled from four different wheat growing zones of India were analyzed using the second largest subunit of the RNA polymerase II (RPB2) and a set of sixteen neutral simple sequence repeats (SSRs) markers. Among the 112 UST isolates genotyped, 98 haplotypes were identified. All the isolates were categorized into two groups (K = 2), each consisting of isolates from different sampling sites, on the basis of unweighted paired-grouping method with arithmetic averages (UPGMA) and the Bayesian analysis of population structure. The positive and significant index of association (IA = 1.169) and standardized index of association (rBarD = 0.075) indicate population is of non-random mating type. Analysis of molecular variance showed that the highest variance component is among isolates (91%), with significantly low genetic differentiation variation among regions (8%) (Fst = 0.012). Recombination (Rm = 0) was not detected. The results showed that UST isolates have a clonal genetic structure with limited genetic differentiation and human arbitrated gene flow and mutations are the prime evolutionary processes determining its genetic structure. These findings will be helpful in devising management strategy especially for selection and breeding of resistant wheat cultivars.

Multilocus sequence typing detects new Piscirickettsia salmonis hybrid genogroup in Chilean fish farms: Evidence for genetic diversity and population structure.

Piscirickettsia salmonisis the causative bacterial pathogen of piscirickettsiosis, a salmonid disease that causes notable mortalities in the worldwide aquaculture industry. Published research describes the phenotypic traits, virulence factors, pathogenicity and antibiotic-resistance potential for various P.salmonisstrains. However, evolutionary and genetic information is scarce for P.salmonis. The present study used multilocus sequence typing (MLST) to gain insight into the population structure and evolution of P.salmonis. Forty-two Chilean P.salmonisisolates, as well as the type strain LF-89T , were recovered from diseased Salmo salar, Oncorhynchus kisutchand Oncorhynchus mykissfrom two Chilean Regions. MLST assessed the loci sequences of dnaK, efp, fumC, glyA, murG, rpoD and trpB. Bioinformatics analyses established the genetic diversity among P.salmonis isolates (H=0.5810). A total of 23 sequence types (ST) were identified, 53.48% of which were represented by ST1, ST5 and ST2. Population structure analysis through polymorphism patterns showed few polymorphic sites (218 nucleotides from 4,010bp), while dN/dS ratio analysis indicated purifying selection for dnaK, epf, fumC, murG, and rpoD but neutral selection for the trpB loci. The standardized index of association indicated strong linkage disequilibrium, suggesting clonal population structure. However, recombination events were detected in a group of seven isolates. Findings included genogroups homologous to the LF-89T and EM-90 strains, as well as a seven-isolate hybrid genogroup recovered from both assessed regions (three O.mykiss and four S.salar isolates). The presented MLST scheme has comparative potential, with promising applications in studying distinct P.salmonis isolates (e.g., from different hosts, farms, geographical areas) and in understanding the epidemiology of this pathogen.

Prevalence and genetic diversity of human diarrheagenic Escherichia coli isolates by multilocus sequence typing

BackgroundThe population structure of human diarrheagenic Escherichia coli (DEC) isolates derived from worldwide collections remains undefined. MethodsA total of 1196 clinical isolates were obtained from a multilocus sequence typing (MLST) database. Genetic diversity analysis, MLST analysis, and phylogenetic analysis combined with different pathotypes were performed through a variety of calculation software applications. ResultsAll isolates were categorized as one of 579 different sequence types (STs). The eBURST algorithm resolved these 579 STs into 27 clonal complexes (CCs), 37 concatemers, and 210 singletons, revealing a high level of genetic diversity in the population structure of DEC. CC10 was the most prevalent CC, comprising 276 (23.08%, 276/1196) isolates with 85 (14.68%, 85/579) STs widely distributed in 20 countries. The population structure of five common pathotypes was highly diversified, and isolates with the same ST or CC were heterogeneous for different pathotypes. Sequence variations were more abundant in fumC and gyrB than in the other five genes, and these exhibited the highest degree of nucleotide diversity (0.03886 and 0.03075, respectively) and the greatest number of polymorphic nucleotide sites (137 and 139, respectively). The dN/dS ratios of seven analyzed loci varied from 0.0083 (recA) to 0.0434 (purA), and the ratio for the concatenated sequence was 0.2518, revealing the effects of purifying selection on housekeeping genes during the evolutionary process. Significant allele linkage disequilibrium was detected when the standardized index of association (ISA) was calculated both for the entire collection of isolates (0.3174, p<0.001) and for the 579 STs (0.1475, p<0.001). ConclusionsThis study facilitated a comprehensive understanding of the genetic diversity of human DEC distributed across the global population. The results provide genetic evidence that will allow us to uncover the microevolutionary relationships among different pathogenic isolates of DEC.

Population genetics of Enterocytozoon bieneusi in captive giant pandas of China

BackgroundMost studies on Enterocytozoon bieneusi are conducted based on the internal transcribed spacer (ITS) region of the rRNA gene, whereas some have examined E. bieneusi population structures. Currently, the population genetics of this pathogen in giant panda remains unknown. The objective of this study was to determine the E. bieneusi population in captive giant pandas in China.ResultsWe examined 69 E. bieneusi-positive specimens from captive giant pandas in China using five loci (ITS, MS1, MS3, MS4 and MS7) to infer E. bieneusi population genetics. For multilocus genotype (MLG) analysis of E. bieneusi-positive isolates, the MS1, MS3, MS4, and MS7 microsatellite and minisatellite loci were amplified and sequenced in 48, 45, 50 and 47 specimens, respectively, generating ten, eight, nine and five types. We successfully amplified 36 specimens and sequenced all five loci, forming 24 MLGs. Multilocus sequence analysis revealed a strong and significant linkage disequilibrium (LD), indicating a clonal population. This result was further supported by measurements of pairwise intergenic LD and a standardized index of association (ISA) from allelic profile data. The analysis in STRUCTURE suggested three subpopulations in E. bieneusi, further confirmed using right’s fixation index (FST). Subpopulations 1 and 2 exhibited an epidemic structure, whereas subpopulation 3 had a clonal structure.ConclusionsOur results describe E. bieneusi population genetics in giant pandas for the first time, improving the current understanding E. bieneusi epidemiology in the studied region. These data also benefit future studies exploring potential transmission risks from pandas to other animals, including humans.

The cacao pathogen Moniliophthora roreri (Marasmiaceae) possesses biallelic A and B mating loci but reproduces clonally.

The cacao pathogen Moniliophthora roreri belongs to the mushroom-forming family Marasmiaceae, but it has never been observed to produce a fruiting body, which calls to question its capacity for sexual reproduction. In this study, we identified potential A (HD1 and HD2) and B (pheromone precursors and pheromone receptors) mating genes in M. roreri. A PCR-based method was subsequently devised to determine the mating type for a set of 47 isolates from across the geographic range of the fungus. We developed and generated an 11-marker microsatellite set and conducted association and linkage disequilibrium (standardized index of association, IA(s)) analyses. We also performed an ancestral reconstruction analysis to show that the ancestor of M. roreri is predicted to be heterothallic and tetrapolar, which together with sliding window analyses support that the A and B mating loci are likely unlinked and follow a tetrapolar organization within the genome. The A locus is composed of a pair of HD1 and HD2 genes, whereas the B locus consists of a paired pheromone precursor, Mr_Ph4, and receptor, STE3_Mr4. Two A and B alleles but only two mating types were identified. Association analyses divided isolates into two well-defined genetically distinct groups that correlate with their mating type; IA(s) values show high linkage disequilibrium as is expected in clonal reproduction. Interestingly, both mating types were found in South American isolates but only one mating type was found in Central American isolates, supporting a prior hypothesis of clonal dissemination throughout Central America after a single or very few introductions of the fungus from South America.

Population Structure, Pathogenicity, and Mating Type Distribution of Magnaporthe oryzae Isolates from East Africa

Rice blast, caused by Magnaporthe oryzae, is one of the emergent threats to rice production in East Africa (EA), where little is known about the population genetics and pathogenicity of this pathogen. We investigated the genetic diversity and mating type (MAT) distribution of 88 isolates of M. oryzae from EA and representative isolates from West Africa (WA) and the Philippines (Asia) using amplified fragment length polymorphism markers and mating-type-specific primer sets. In addition, the aggressiveness of each isolate was evaluated by inoculating on the susceptible Oryza sativa indica 'Co39', scoring the disease severity and calculating the disease progress. Hierarchical analysis of molecular variance revealed a low level of genetic differentiation at two levels (FST 0.12 and FCT 0.11). No evidence of population structure was found among the 65 isolates from EA, and gene flow among EA populations was high. Moreover, pairwise population differentiation (GST) in EA populations ranged from 0.03 to 0.04, suggesting that >96% of genetic variation is derived from within populations. However, the populations from Asia and WA were moderately differentiated from EA ones. The spatial analysis of principal coordinates and STRUCTURE revealed overlapping between individual M. oryzae isolates from EA, with limited distinctness according to the geographic origin. All the populations were clonal, given the positive and significant index of association (IA) and standardized index of association (rd), which indicates a significant (P<0.001) departure from panmixia (IA and rd=0). Both MAT1-1 and MAT1-2 were detected. However, MAT1-1 was more prevalent than MAT1-2. Pathogenicity analysis revealed variability in aggressiveness, suggesting a potential existence of different races. Our data suggest that either M. oryzae populations from EA could be distributed as a single genetic population or gene flow is exerting a significant influence, effectively swamping the action of selection. This is the first study of genetic differentiation of rice-infecting M. oryzae strains from EA, and may guide further studies on the pathogen as well as resistance breeding efforts.

The evolution and population structure of Lactobacillus fermentum from different naturally fermented products as determined by multilocus sequence typing (MLST).

BackgroundLactobacillus fermentum is economically important in the production and preservation of fermented foods. A repeatable and discriminative typing method was devised to characterize L. fermentum at the molecular level. The multilocus sequence typing (MLST) scheme developed was based on analysis of the internal sequence of 11 housekeeping gene fragments (clpX, dnaA, dnaK, groEL, murC, murE, pepX, pyrG, recA, rpoB, and uvrC).ResultsMLST analysis of 203 isolates of L. fermentum from Mongolia and seven provinces/ autonomous regions in China identified 57 sequence types (ST), 27 of which were represented by only a single isolate, indicating high genetic diversity. Phylogenetic analyses based on the sequence of the 11 housekeeping gene fragments indicated that the L. fermentum isolates analyzed belonged to two major groups. A standardized index of association (IAS) indicated a weak clonal population structure in L. fermentum. Split decomposition analysis indicated that recombination played an important role in generating the genetic diversity observed in L. fermentum. The results from the minimum spanning tree strongly suggested that evolution of L. fermentum STs was not correlated with geography or food-type.ConclusionsThe MLST scheme developed will be valuable for further studies on the evolution and population structure of L. fermentum isolates used in food products.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-015-0447-z) contains supplementary material, which is available to authorized users.

Development of a multilocus sequence typing scheme for Ureaplasma

Ureaplasma is a commensal of the human urogenital tract but is always associated with invasive diseases such as non-gonococcal urethritis and infertility adverse pregnancy outcomes. To better understand the molecular epidemiology and population structure of Ureaplasma, a multilocus sequence typing (MLST) scheme based on four housekeeping genes (ftsH, rpL22, valS, thrS) was developed and validated using 283 isolates, including 14 serovars of reference strains and 269 strains obtained from clinical patients. A total of 99 sequence types (STs) were revealed: the 14 type strains of the Ureaplasma serovars were assigned to 12 STs, and 87 novel and special STs appeared among the clinical isolates. ST1 and ST22 were the predominant STs, which contained 68 and 70 isolates, respectively. Two clonal lineages (CC1 and CC2) were shown by eBURST analysis, and linkage disequilibrium was revealed through a standardized index of association (I A (S)). The neighbor-joining tree results of 14 Ureaplasma serovars showed two genetically significantly distant clusters, which was highly congruent with the species taxonomy of ureaplasmas [Ureaplasma parvum (UPA) and Ureaplasma urealyticum (UUR)]. Analysis of the biotypes of 269 clinical isolates revealed that all the isolates of CC1 were UPA and those of CC2 were UUR. Additionally, CC2 was found more often in symptomatic patients with vaginitis, tubal obstruction, and cervicitis. In conclusion, this MLST scheme is adequate for investigations of molecular epidemiology and population structure with highly discriminating capacity.

Population Structure of Pseudomonas aeruginosa from Five Mediterranean Countries: Evidence for Frequent Recombination and Epidemic Occurrence of CC235

Several studies in recent years have provided evidence that Pseudomonas aeruginosa has a non-clonal population structure punctuated by highly successful epidemic clones or clonal complexes. The role of recombination in the diversification of P. aeruginosa clones has been suggested, but not yet demonstrated using multi-locus sequence typing (MLST). Isolates of P. aeruginosa from five Mediterranean countries (n = 141) were subjected to pulsed-field gel electrophoresis (PFGE), serotyping and PCR targeting the virulence genes exoS and exoU. The occurrence of multi-resistance (≥3 antipseudomonal drugs) was analyzed with disk diffusion according to EUCAST. MLST was performed on a subset of strains (n = 110) most of them had a distinct PFGE variant. MLST data were analyzed with Bionumerics 6.0, using minimal spanning tree (MST) as well as eBURST. Measurement of clonality was assessed by the standardized index of association (IA S). Evidence of recombination was estimated by ClonalFrame as well as SplitsTree4.0. The MST analysis connected 70 sequence types, among which ST235 was by far the most common. ST235 was very frequently associated with the O11 serotype, and frequently displayed multi-resistance and the virulence genotype exoS −/exoU +. ClonalFrame linked several groups previously identified by eBURST and MST, and provided insight to the evolutionary events occurring in the population; the recombination/mutation ratio was found to be 8.4. A Neighbor-Net analysis based on the concatenated sequences revealed a complex network, providing evidence of frequent recombination. The index of association when all the strains were considered indicated a freely recombining population. P. aeruginosa isolates from the Mediterranean countries display an epidemic population structure, particularly dominated by ST235-O11, which has earlier also been coupled to the spread of ß-lactamases in many countries.

Biased distribution of IS 629 among strains in different lineages of enterohemorrhagic Escherichia coli serovar O157

The distribution of insertion sequence (IS) 629 among strains of enterohemorrhagic Escherichia coli serovar O157 (O157) was investigated and compared with the strain lineages defined by lineage specific polymorphism assay-6 (LSPA-6) to demonstrate the effectiveness of IS 629 analysis for population genetics analysis. Using pulsed-field gel electrophoresis and variable-number tandem repeat typing, 140 strains producing both VT1 and VT2 and 98 strains producing only VT2 were selected from a total of 592 strains isolated from patients and asymptomatic carriers in Chiba Prefecture, Japan, during 2003–2008. By LSPA-6 analysis, six strains had atypical amplicon sizes in their Z5935 loci and five strains had atypical amplicon sizes in their arp- iclR intergenic regions. Sequence analyses of PCR amplified DNAs showed that five of the six loci used for LSPA-6 analysis had tandem repeats and the allele changes were due to changes in the number of tandem repeats. Subculturing and long-term incubation was found to have no detectable effect on the lineages defined by LSPA-6 analysis, demonstrating the robustness of LSPA-6 analysis. Minimum spanning tree analysis reconstruction revealed that strains in lineage I, I/II, and II clustered on separate branches, indicating that the distribution of IS 629 was biased among O157 strains in different lineages. Strains with LSPA-6 codes 231111, 211113, and 211114 had atypical amplicon sizes and were clustered in lineage I/II branch, and strains with LSPA-6 codes 212114, 221123, 221223, 222123, 222224, 242123, 252123, and 242222 had atypical amplicon sizes and clustered in lineage II branches. Linkage disequilibrium was observed in strains in every lineage when the standardized index of association was calculated using IS 629 distribution data. Therefore, the distribution analysis of IS 629 may be effective for population genetics analysis of O157 due to the biased IS 629 distribution among strains in the three O157 lineages.

Concordance of variable-number tandem repeat (VNTR) and large sequence polymorphism (LSP) analyses of Mycobacterium tuberculosis strains

Variable-number tandem repeat (VNTR) and large sequence polymorphism (LSP) analyses were compared to determine whether VNTR analysis was effective for population genetic analysis of Mycobacterium tuberculosis strains. A total of 682 strains, 510 Beijing genotype and 172 non-Beijing genotype strains, were studied. The number of repeats was investigated for 24 VNTR loci: the 15 loci of “optimized miru”, the 8 loci of “Beijing option”, and 1 locus for “JATA12”. Six loci (miru31, Mtub4, QUB4156c, QUB3232, VNTR3820, and VNTR4120) showed significantly different median numbers of repeats in strains belonging to different lineages defined by LSP ( P < 0.01, Mann–Whitney U test). When a minimum-spanning tree (MST) was reconstructed using these 6 loci, most strains clustered in the expected branches in the MST branches. However, topology of the MST was not congruent with the evolutional hypothesis of M. tuberculosis, indicating that MST analysis using VNTR data should not use for phylogeny of the organism. When the standardized index of association (s I A) was calculated using data for the 6 VNTR loci, the value of s I A was significantly different from zero (Monte Carlo simulation with 10,000 resamplings) in every lineage, indicating the linkage disequilibrium in different lineage strains of M. tuberculosis. These results were consistent with the hypothesis that clonal evolution of lineages of the organism has occurred. Therefore, the 6 loci identified in this study would be effective for M. tuberculosis population genetic analysis due to their significantly different median numbers of repeat and linkage disequilibrium though VNTR data was not effective for phylogeny of the organism.

Widespread homologous recombination within and between Streptomyces species

Horizontal gene transfer (HGT) is widespread in the microbial world, but its impact on the origin and persistence of microbial species remains poorly defined. HGT can result in either acquisition of new genetic material or homologous replacement of existing genes. The evolutionary significance of homologous recombination in a population can be quantified by examining the relative rates at which polymorphisms are introduced from recombination (rho) and mutation (theta(w)). We used multilocus sequence analysis (MLSA) to quantify both intraspecies and interspecies homologous recombination among streptomycetes, multicellular Gram-positive bacteria ubiquitous in soil, which are an important source of antibiotics and bioactive compounds. Intraspecies recombination was examined using strains of Streptomyces flavogriseus isolated from soils at five locations spanning 1000 km. The strains had >99.8% nucleotide identity across the loci examined. We found remarkable levels of gene exchange within S. flavogriseus (rho/theta(w)=27.9), and found that the population was in linkage equilibrium (standardized index of association=0.0018), providing evidence for a freely recombining sexual population structure. We also examined interspecies homologous recombination among different Streptomyces species in an MLSA data set and found that 40% of the species had housekeeping genes acquired through HGT. The recombination rate between these named species (rho/theta(w)=0.21) exceeds that observed within many species of bacteria. Despite widespread gene exchange in the genus, the intraspecies recombination rate exceeded the interspecies rate by two orders of magnitude suggesting that patterns of gene exchange and recombination may shape the evolution of streptomycetes.

Identification of Streptococcus uberis Multilocus Sequence Types Highly Associated with Mastitis

Multilocus sequence typing analysis of Streptococcus uberis has identified a cluster of isolates associated with clinical and subclinical mastitis and a cluster associated with cows with low somatic cell counts in their milk. Specific groups of genotypes (global clonal complex [GCC] sequence type 5s [ST5s] and GCC ST143s) were highly associated (P = 0.006) with clinical and subclinical mastitis and may represent a lineage of virulent isolates, whereas isolates belonging to GCC ST86 were associated with low-cell-count cows. This study has, for the first time, demonstrated the occurrence of identical sequence types (ST60 and ST184) between different continents (Australasia and Europe) and different countries (Australia and New Zealand). The standardized index of association and the empirical estimation of the rate of recombination showed substantial recombination within the S. uberis population in Australia, consistent with previous multilocus sequence type analyses.