Comparison of spoligotyping, mycobacterial interspersed repetitive units typing and IS6110-RFLP in a study of genotypic diversity of Mycobacterium tuberculosis in Delhi, North India

The aim of the present study was to compare polymerase chain reaction (PCR)-based methods--spoligotyping and mycobacterial interspersed repetitive units (MIRU) typing--with the gold-standard IS6110 restriction fragment length polymorphism (RFLP) analysis in 101 isolates of Mycobacterium tuberculosis to determine the genetic diversity of M. tuberculosis clinical isolates from Delhi, North India. Spoligotyping resulted in 49 patterns (14 clusters); the largest cluster was composed of Spoligotype International Types (SITs)26 [Central-Asian (CAS)1-Delhi lineage], followed by SIT11 [East-African-Indian (EAI) 3-Indian lineage]. A large number of isolates (75%) belonged to genotypic lineages, such as CAS, EAI and Manu, with a high specificity for the Indian subcontinent, emphasising the complex diversity of the phylogenetically coherent M. tuberculosis in North India. MIRU typing, using 11 discriminatory loci, was able to distinguish between all but two strains based on individual patterns. IS6110-RFLP analysis (n = 80 strains) resulted in 67 unique isolates and four clusters containing 13 strains. MIRUs discriminated all 13 strains, whereas spoligotyping discriminated 11 strains. Our results validate the use of PCR-based molecular typing of M. tuberculosis using repetitive elements in Indian isolates and demonstrate the usefulness of MIRUs for discriminating low-IS6110-copy isolates, which accounted for more than one-fifth of the strains in the present study.     

SNP discovery by amplicon sequencing and multiplex SNP genotyping in the allopolyploid species Brassica napus

Oilseed rape (Brassica napus) is an allotetraploid species consisting of two genomes, derived from B. rapa (A genome) and B. oleracea (C genome). The presence of these two genomes makes single nucleotide polymorphism (SNP) marker identification and SNP analysis more challenging than in diploid species, as for a given locus usually two versions of a DNA sequence (based on the two ancestral genomes) have to be analyzed simultaneously during SNP identification and analysis. One hundred amplicons derived from expressed sequence tag (ESTs) were analyzed to identify SNPs in a panel of oilseed rape varieties and within two sister species representing the ancestral genomes. A total of 604 SNPs were identified, averaging one SNP in every 42 bp. It was possible to clearly discriminate SNPs that are polymorphic between different plant varieties from SNPs differentiating the two ancestral genomes. To validate the identified SNPs for their use in genetic analysis, we have developed Illumina GoldenGate assays for some of the identified SNPs. Through the analysis of a number of oilseed rape varieties and mapping populations with GoldenGate assays, we were able to identify a number of different segregation patterns in allotetraploid oilseed rape. The majority of the identified SNP markers can be readily used for genetic mapping, showing that amplicon sequencing and Illumina GoldenGate assays can be used to reliably identify SNP markers in tetraploid oilseed rape and to convert them into successful SNP assays that can be used for genetic analysis.     

Mesozoic origin and ‘out-of-India’ radiation of ricefishes (Adrianichthyidae)

The Indian subcontinent has an origin geologically different from Eurasia, but many terrestrial animal and plant species on it have congeneric or sister species in other parts of Asia, especially in the Southeast. This faunal and floral similarity between India and Southeast Asia is explained by either of the two biogeographic scenarios, ‘into-India’ or ‘out-of-India’. Phylogenies based on complete mitochondrial genomes and five nuclear genes were undertaken for ricefishes (Adrianichthyidae) to examine which of these two biogeographic scenarios fits better. We found that Oryzias setnai, the only adrianichthyid distributed in and endemic to the Western Ghats, a mountain range running parallel to the western coast of the Indian subcontinent, is sister to all other adrianichthyids from eastern India and Southeast–East Asia. Divergence time estimates and ancestral area reconstructions reveal that this western Indian species diverged in the late Mesozoic during the northward drift of the Indian subcontinent. These findings indicate that adrianichthyids dispersed eastward ‘out-of-India’ after the collision of the Indian subcontinent with Eurasia, and subsequently diversified in Southeast–East Asia. A review of geographic distributions of ‘out-of-India’ taxa reveals that they may have largely fuelled or modified the biodiversity of Eurasia.     

SNP/RD typing of Mycobacterium tuberculosis Beijing strains reveals local and worldwide disseminated clonal complexes

The Beijing strain is one of the most successful genotypes of Mycobacterium tuberculosis worldwide and appears to be highly homogenous according to existing genotyping methods. To type Beijing strains reliably we developed a robust typing scheme using single nucleotide polymorphisms (SNPs) and regions of difference (RDs) derived from whole-genome sequencing data of eight Beijing strains. SNP/RD typing of 259 M. tuberculosis isolates originating from 45 countries worldwide discriminated 27 clonal complexes within the Beijing genotype family. A total of 16 Beijing clonal complexes contained more than one isolate of known origin, of which two clonal complexes were strongly associated with South African origin. The remaining 14 clonal complexes encompassed isolates from different countries. Even highly resolved clonal complexes comprised isolates from distinct geographical sites. Our results suggest that Beijing strains spread globally on multiple occasions and that the tuberculosis epidemic caused by the Beijing genotype is at least partially driven by modern migration patterns. The SNPs and RDs presented in this study will facilitate future molecular epidemiological and phylogenetic studies on Beijing strains.     

Porcine Single Nucleotide Polymorphism (SNP) Development and Population Structure of Pigs Assessed by Validated SNPs

In this study, we identified porcine single nucleotide polymorphisms (SNPs) by aligning eight sequences generated with two approaches: amplification of 665 intronic regions using one sample from each of eight breeds, including three East Asian pigs, and amplification of 289 3'-UTR regions using two samples from each of four major commercial breeds. The 1,760 and 599 SNPs were validated using two 384-sample DNA panels by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The phylogenetic tree and Structure analyses classified the pigs into two large clusters: Euro-American and East Asian populations. The membership proportions, however, differed between inferred clusters for K = 2 generated by the two approaches. With intronic SNPs, Euro-American breeds constituted about 100% of the Euro-American cluster, but with 3'-UTR SNPs, about 17% of the East Asian cluster comprised five Euro-American breeds. The differences in the SNP discovery panels may affect population structure found in study panels of large samples.     

A promoter mutation causes differential nitrate reductase activity of Mycobacterium tuberculosis and Mycobacterium bovis

The recent publication of the genome sequence of Mycobacterium bovis showed >99.95% identity to M. tuberculosis. No genes unique to M. bovis were found. Instead numerous single-nucleotide polymorphisms (SNPs) were identified. This has led to the hypothesis that differential gene expression due to SNPs might explain the differences between the human and bovine tubercle bacilli. One phenotypic distinction between M. tuberculosis and M. bovis is nitrate reduction, which not only is an essential diagnostic tool but also contributes to mycobacterial pathogenesis. We previously showed that narGHJI encodes a nitrate reductase in both M. tuberculosis and M. bovis and that NarGHJI-mediated nitrate reductase activity was substantially higher in the human tubercle bacillus. In the present study we used a genetic approach to demonstrate that an SNP within the promoter of the nitrate reductase gene cluster narGHJI is responsible for the different nitrate reductase activity of M. tuberculosis and M. bovis. This is the first example of an SNP that leads to differential gene expression between the human and bovine tubercle bacilli.     

Balinese Y-chromosome perspective on the peopling of Indonesia: genetic contributions from pre-neolithic hunter-gatherers, Austronesian farmers, and Indian traders

The island of Bali lies near the center of the southern chain of islands in the Indonesian archipelago, which served as a stepping-stone for early migrations of hunter-gatherers to Melanesia and Australia and for more recent migrations of Austronesian farmers from mainland Southeast Asia to the Pacific. Bali is the only Indonesian island with a population that currently practices the Hindu religion and preserves various other Indian cultural, linguistic, and artistic traditions (Lansing 1983). Here, we examine genetic variation on the Y chromosomes of 551 Balinese men to investigate the relative contributions of Austronesian farmers and pre-Neolithic hunter-gatherers to the contemporary Balinese paternal gene pool and to test the hypothesis of recent paternal gene flow from the Indian subcontinent. Seventy-one Y-chromosome binary polymorphisms (single nucleotide polymorphisms, SNPs) and 10 Y-chromosome-linked short tandem repeats (STRs) were genotyped on a sample of 1,989 Y chromosomes from 20 populations representing Indonesia (including Bali), southern China, Southeast Asia, South Asia, the Near East, and Oceania. SNP genotyping revealed 22 Balinese lineages, 3 of which (O-M95, O-M119, and O-M122) account for nearly 83.7% of Balinese Y chromosomes. Phylogeographic analyses suggest that all three major Y-chromosome haplogroups migrated to Bali with the arrival of Austronesian speakers; however, STR diversity patterns associated with these haplogroups are complex and may be explained by multiple waves of Austronesian expansion to Indonesia by different routes. Approximately 2.2% of contemporary Balinese Y chromosomes (i.e., K-M9*, K-M230, and M lineages) may represent the pre-Neolithic component of the Indonesian paternal gene pool. In contrast, eight other haplogroups (e.g., within H, J, L, and R), making up approximately 12% of the Balinese paternal gene pool, appear to have migrated to Bali from India. These results indicate that the Austronesian expansion had a profound effect on the composition of the Balinese paternal gene pool and that cultural transmission from India to Bali was accompanied by substantial levels of gene flow.     

Characterization of M. tuberculosis strains from west African patients by spoligotyping

Analysis of Mycobacterium tuberculosis strains was carried out using isolates collected from 69 Senegalese and 20 Ivory Coast tuberculosis patients. These 89 isolates were typed by means of the spoligotyping technique, showing clusterized populations of bacterial strains. In the Senegalese patients, 35 genetic profiles were observed with 10 clusters of spoligotypes from 44 isolates. Among Ivory Coast patients, 11 spoligotypes were found for 20 isolates. A particular cluster of isolates was evident both in Senegalese (10) and Ivory Coast (11) patients. These results show the existence of polymorphism of the direct repeat region for African M. tuberculosis strains. However they suggest that additionnal markers are needed for accurate epidemiological studies in areas that are highly endemic for tuberculosis.     

Modern and ancestral genotypes of Mycobacterium tuberculosis from Andhra Pradesh, India

Traditionally, the distribution of the Mycobacterium tuberculosis genotypes in India has been characterized by widespread prevalence of ancestral lineages (TbD1+ strains and variants) in the south and the modern forms (TbD1(-) CAS and variants) predominating in the north of India. The pattern was, however, not clearly known in the south-central region such as Hyderabad and the rest of the state of Andhra Pradesh where the prevalence of both tuberculosis (TB) and human immunodeficiency virus (HIV) infection is one of the highest in the country; this area has been the hotspot of TB vaccine trials. Spoligotyping of 101 clinical isolates obtained from Hyderabad and rural Andhra Pradesh confirmed the occurrence of major genogroups such as the ancestral (or the TbD1+ type or the East African Indian (EAI) type), the Central Asian (CAS) or Delhi type and the Beijing lineage in Andhra Pradesh. Sixty five different spoligotype patterns were observed for the isolates included in this study; these were further analyzed based on specific genetic signatures/mutations. It was found that the major genogroups, CAS and "ancestral," were almost equally prevalent in our collection but followed a north-south compartmentalization as was also reported previously. However, we observed a significant presence of MANU lineage in south Andhra Pradesh, which was earlier reported to be overwhelmingly present in Mumbai. This study portrays genotypic diversity of M. tuberculosis from the Indian state of Andhra Pradesh and provides a much needed snapshot of the strain diversity that will be helpful in devising effective TB control programs in this part of the world.     

Austro-Asiatic tribes of Northeast India provide hitherto missing genetic link between South and Southeast Asia

Northeast India, the only region which currently forms a land bridge between the Indian subcontinent and Southeast Asia, has been proposed as an important corridor for the initial peopling of East Asia. Given that the Austro-Asiatic linguistic family is considered to be the oldest and spoken by certain tribes in India, Northeast India and entire Southeast Asia, we expect that populations of this family from Northeast India should provide the signatures of genetic link between Indian and Southeast Asian populations. In order to test this hypothesis, we analyzed mtDNA and Y-Chromosome SNP and STR data of the eight groups of the Austro-Asiatic Khasi from Northeast India and the neighboring Garo and compared with that of other relevant Asian populations. The results suggest that the Austro-Asiatic Khasi tribes of Northeast India represent a genetic continuity between the populations of South and Southeast Asia, thereby advocating that northeast India could have been a major corridor for the movement of populations from India to East/Southeast Asia.     

The identification of SNPs with indeterminate positions using the Equine SNP50 BeadChip

We have used linkage disequilibrium (LD) to identify single nucleotide polymorphisms (SNPs) on the Illumina Equine SNP50 BeadChip, which may be incorrectly positioned on the genome map. A total of 1201 Thoroughbred horses were genotyped using the Illumina Equine SNP50 BeadChip. LD was evaluated in a pairwise fashion between all autosomal SNPs, both within and across chromosomes. Filters were then applied to the data, firstly to identify SNPs that may have been mapped to the wrong chromosome and secondly to identify SNPs that may have been incorrectly positioned within chromosomes. We identified a single SNP on ECA28, which showed low LD with neighbouring SNPs but considerable LD with a group of SNPs on ECA10. Furthermore, a cluster of SNPs on ECA5 showed unusually low LD with surrounding SNPs. A total of 39 SNPs met the criteria for unusual within-chromosome LD. The results of this study indicate that some SNPs may be misplaced. This finding is significant, as misplaced SNPs may lead to difficulties in the application of genomic methods, such as homozygosity mapping, for which SNP order is important.     

MIRU-VNTR genotyping of Mycobacterium tuberculosis strains from East Siberian: Beijing family versus Kilimanjaro family

Seventy-two M. tuberculosis strains isolated from patients in different regions of East Siberia were studied by MIRU-VNTR repeats. Phylogenetic trees of relationship between the studied strains and of those from other world regions were investigated by phylogenetic analysis, i.e. NJ, UPGMA and ML. Phylogenetic evidence is provided for the prevalence of Beijing and Beijing-like strains (71%) in the above region. Besides, there was found a group of strains (115%) with the robust genotype which was given the name of ASU (African-Siberian-Ural). A linkage between MIRU10 (5-10 repeat) and MIRU26 (single repeat) was established as a predominant ASU sign. According to preliminary results, apart from Siberia, ASU strains can be encountered in the Urals and Africa (Tanzania, Kilimanjaro genotype). MIRU-VNTR consensus of ASU for East Siberia is 22( > 6)225113233.     

Features of SNP and SSR diversity in a set of ICARDA barley germplasm collection

Detection and utilization of genetic variation available in the germplasm collection for crop improvement have been the prime activities of breeders. Here a set of ICARDA barley germplasm collection comprising of 185 cultivated (Hordeum vulgare L.) and 38 wild (H. spontaneum L.) genotypes originated from 30 countries of four continents was genotyped with 68 single nucleotide polymorphism (SNP) and 45 microsatellite or simple sequence repeat (SSR) markers derived from genes (expressed sequence tags, ESTs). As two SNP markers provided 2 and 3 datapoints, a total of 71 SNPs were surveyed that yielded a total of 143 alleles. The number of SSR alleles per locus ranged from 3 to 22 with an average of 7.9 per marker. Average PIC (polymorphism information content) value for SSR and SNP markers were recorded as 0.63 and 0.38, respectively. Heterogeneity was recorded at both SNP and SSR loci in an average of 5.72 and 12.42% accessions, respectively. Genetic similarity matrices for SSR and SNP allelic data were highly correlated (r = 0.75, P < 0.005) and therefore allelic data for both markers were combined and analyzed for understanding the genetic relationships among the germplasm surveyed. Majority of clusters/subclusters were found to contain genotypes from the same geographic origins. While comparing the genetic diversity, the accessions coming from Middle East Asia and North East Asia showed more diversity as compared to that of other geographic regions. Majority of countries representing Africa, Middle East Asia, North East Asia and Arabian Peninsula included the genotypes that contained rare alleles. As expected, spontaneum accessions, as compared to vulgare accessions, showed a higher number of total alleles, higher number of alleles per locus, higher effective number of alleles and higher allelic richness and a higher number of rare alleles were observed. In summary, the examined ICARDA germplasm set showed ample natural genetic variation that can be harnessed for future breeding of barley as climate change and sustainability have become important throughout all growing areas of the world, drought/heat tolerance being the most important ones.     

Genomic Diversity and Evolution of Mycobacterium ulcerans Revealed by Next-Generation Sequencing

Mycobacterium ulcerans is the causative agent of Buruli ulcer, the third most common mycobacterial disease after tuberculosis and leprosy. It is an emerging infectious disease that afflicts mainly children and youths in West Africa. Little is known about the evolution and transmission mode of M. ulcerans, partially due to the lack of known genetic polymorphisms among isolates, limiting the application of genetic epidemiology. To systematically profile single nucleotide polymorphisms (SNPs), we sequenced the genomes of three M. ulcerans strains using 454 and Solexa technologies. Comparison with the reference genome of the Ghanaian classical lineage isolate Agy99 revealed 26,564 SNPs in a Japanese strain representing the ancestral lineage. Only 173 SNPs were found when comparing Agy99 with two other Ghanaian isolates, which belong to the two other types previously distinguished in Ghana by variable number tandem repeat typing. We further analyzed a collection of Ghanaian strains using the SNPs discovered. With 68 SNP loci, we were able to differentiate 54 strains into 13 distinct SNP haplotypes. The average SNP nucleotide diversity was low (average 0.06–0.09 across 68 SNP loci), and 96% of the SNP locus pairs were in complete linkage disequilibrium. We estimated that the divergence of the M. ulcerans Ghanaian clade from the Japanese strain occurred 394 to 529 thousand years ago. The Ghanaian subtypes diverged about 1000 to 3000 years ago, or even much more recently, because we found evidence that they evolved significantly faster than average. Our results offer significant insight into the evolution of M. ulcerans and provide a comprehensive report on genetic diversity within a highly clonal M. ulcerans population from a Buruli ulcer endemic region, which can facilitate further epidemiological studies of this pathogen through the development of high-resolution tools.     

The impact of natural selection on an ABCC11 SNP determining earwax type

A nonsynonymous single nucleotide polymorphism (SNP), rs17822931-G/A (538G>A; Gly180Arg), in the ABCC11 gene determines human earwax type (i.e., wet or dry) and is one of most differentiated nonsynonymous SNPs between East Asian and African populations. A recent genome-wide scan for positive selection revealed that a genomic region spanning ABCC11, LONP2, and SIAH1 genes has been subjected to a selective sweep in East Asians. Considering the potential functional significance as well as the population differentiation of SNPs located in that region, rs17822931 is the most plausible candidate polymorphism to have undergone geographically restricted positive selection. In this study, we estimated the selection intensity or selection coefficient of rs17822931-A in East Asians by analyzing two microsatellite loci flanking rs17822931 in the African (HapMap-YRI) and East Asian (HapMap-JPT and HapMap-CHB) populations. Assuming a recessive selection model, a coalescent-based simulation approach suggested that the selection coefficient of rs17822931-A had been approximately 0.01 in the East Asian population, and a simulation experiment using a pseudo-sampling variable revealed that the mutation of rs17822931-A occurred 2006 generations (95% credible interval, 1,023-3,901 generations) ago. In addition, we show that absolute latitude is significantly associated with the allele frequency of rs17822931-A in Asian, Native American, and European populations, implying that the selective advantage of rs17822931-A is related to an adaptation to a cold climate. Our results provide a striking example of how local adaptation has played a significant role in the diversification of human traits.     

基于高密度SNP数据的东亚人群遗传结构研究

目的 东亚疆域辽阔,民族众多,有着广泛多样的语言。中国34个省级行政区可划分为7个地理分区,人群主要分属世界七大语系。已有研究主要集中在东亚人群的起源、迁徙、融合等遗传历史。本文基于5 147份世界人群个体的高密度单核苷酸多态性（SNP）数据,从地域及语言两个角度研究东亚人群尤其是中国人群与世界其他人群的遗传关系,研究中国人群的遗传关系和遗传结构。方法 收集了5 147份世界人群个体的高密度SNP数据,并对其进行质控、合并。通过频率差异分析方法对最终获得的32 789个SNP进行统计学检验,并进一步使用主成分分析、系统发育树、祖先成分分析和D检验统计等方法,对东亚人群与世界其他人群的遗传关系,以及中国人群的遗传关系和遗传结构进行研究。结果 研究发现东亚人群与非洲、美洲和欧洲人群存在显著差异。中国人群可分为7个亚群,不同人群间的遗传聚类与其地理分布、语系语族和族源历史有很强的相关性。结论 本文研究了中国人群与世界人群的遗传关系和差异,并系统研究了中国人群的遗传亚结构。这将丰富东亚人群的群体遗传学、法医遗传学等研究基础,为个体化医疗等工作提供数据支撑。     

Performance of different SNP panels for parentage testing in two East Asian cattle breeds

The International Society for Animal Genetics (ISAG) proposed a panel of single nucleotide polymorphisms (SNPs) for parentage testing in cattle (a core panel of 100 SNPs and an additional list of 100 SNPs). However, markers specific to East Asian taurine cattle breeds were not included, and no information is available as to whether the ISAG panel performs adequately for these breeds. We tested ISAG's core (100 SNP) and full (200 SNP) panels on two East Asian taurine breeds: the Korean Hanwoo and the Japanese Wagyu, the latter from the Australian herd. Even though the power of exclusion was high at 0.99 for both ISAG panels, the core panel performed poorly with 3.01% false‐positive assignments in the Hanwoo population and 3.57% in the Wagyu. The full ISAG panel identified all sire–offspring relations correctly in both populations with 0.02% of relations wrongly excluded in the Hanwoo population. Based on these results, we created and tested two population‐specific marker panels: one for the Wagyu population, which showed no false‐positive assignments with either 100 or 200 SNPs, and a second panel for the Hanwoo, which still had some false‐positive assignments with 100 SNPs but no false positives using 200 SNPs. In conclusion, for parentage assignment in East Asian cattle breeds, only the full ISAG panel is adequate for parentage testing. If fewer markers should be used, it is advisable to use population‐specific markers rather than the ISAG panel.     

Development of EST derived SSRs and SNPs as a genomic resource in Indian catfish, <Emphasis Type="Italic">Clarias batrachus</Emphasis>

Clarias batrachus, an Indian catfish species, is endemic to the Indian subcontinent and potential cultivable species. The genomic resources in C. batrachus in the form of ESTs containing microsatellite repeats (EST-SSR) and single nucleotide polymorphisms (SNPs) that are associated with the expressed genes from spleen were mined. From a total of 1,937 ESTs generated, 1,698 unique sequences were obtained, out of which 221 EST-SSRs were identified and 54% could be functionally annotated by similarity searches. A total of 23 contigs containing 3 or more ESTs were found to contain 31 SNP loci, out of which 8 ESTs showed similarity to genes of known function and 1 for hypothetical protein. Nine ESTs with SSRs and/or SNPs identified in this study were reported to be associated with diseases in human and animals. These identified loci can be developed into markers in C. batrachus, which can be useful in linkage mapping, comparative genomics studies and for its genetic improvement programmes.     

SNP ID-info: SNP ID searching and visualization platform

Many association studies provide the relationship between single nucleotide polymorphisms (SNPs), diseases and cancers, without giving a SNP ID, however. Here, we developed the SNP ID-info freeware to provide the SNP IDs within inputting genetic and physical information of genomes. The program provides an "SNP-ePCR" function to generate the full-sequence using primers and template inputs. In "SNPosition," sequence from SNP-ePCR or direct input is fed to match the SNP IDs from SNP fasta-sequence. In "SNP search" and "SNP fasta" function, information of SNPs within the cytogenetic band, contig position, and keyword input are acceptable. Finally, the SNP ID neighboring environment for inputs is completely visualized in the order of contig position and marked with SNP and flanking hits. The SNP identification problems inherent in NCBI SNP BLAST are also avoided. In conclusion, the SNP ID-info provides a visualized SNP ID environment for multiple inputs and assists systematic SNP association studies. The server and user manual are available at http://bio.kuas.edu.tw/snpid-info.     

Evidence for recombination in Mycobacterium tuberculosis

Due to its mostly isolated living environment, Mycobacterium tuberculosis is generally believed to be highly clonal, and thus recombination between different strains must be rare and is not critical for the survival of the species. To investigate the roles recombination could have possibly played in the evolution of M. tuberculosis, an analysis was conducted on previously determined genotypes of 36 synonymous single nucleotide polymorphisms (SNPs) in 3,320 M. tuberculosis isolates. The results confirmed the predominant clonal structure of the M. tuberculosis population. However, recombination between different strains was also suggested. To further resolve the issue, 175 intergenic SNPs and 234 synonymous SNPs were genotyped in 37 selected representative strains. A clear mosaic polymorphic pattern ahead of the MT0105 locus encoding a PPE (Pro-Pro-Glu) protein was obtained, which is most likely a result of recombination hot spot. Given that PPE proteins are thought to be critical in host-pathogen interactions, we hypothesize that recombination has been influential in the history of M. tuberculosis and possibly a major contributor to the diversity observed ahead of the MT0105 locus.