The complete mitochondrial genome study and phylogenetic analysis of Asiatic Water Snake (Fowlea piscator) using 'Next-Generation Sequencing' technology.

The first complete mitochondrial genome sequencing of Asiatic Water snake or Checkered Keelback or Fowlea piscator (=Xenochrophis piscator) was carried out using Next-Generation Sequencing technology. The complete mitochondrial genome of Asiatic Water snake is 16,999bp long with a base composition of 33% A, 28% T, 12% G and 27% C, with a GC content of 39%. Like the typical snake mitochondrial genome, F. piscator also shows relatively similar mitogenome arrangement comprising 37 genes including 13 protein-coding genes, 22 tRNA genes, two rRNA genes and two non-coding regions or a duplicated control region (D-Loop) along with an origin of replication. Nine genes including eight tRNAs and NAD6 were encoded on the Light or L-strand. Phylogenetic analyses using the complete mitochondrial genome of F. piscator demonstrate a close relationship with the family Colubridae and sub family Natricinae.

An evaluation of inter and intra population structure of Uttar Pradesh, inferred from 24 autosomal STRs.

AIM: The present study was designed to explore the STR diversity and genomic history of the inhabitants of the most populous subdivision of the country. A set of 24 hypervariable autosomal STRs was used to estimate the genetic diversity within the studied population. A panel of 15 autosomal STRs, which is most common in the previously reported data sets, was used to estimate the genetic diversity between the studied population, and obtained unique relations were reported here. METHOD: The genetic diversity and polymorphism among 636 individuals of different ethnic groups, residing in Bareilly, Pilibhit, Shahjahanpur, Gorakhpur, Jhansi, and Varanasi regions of Uttar Pradesh, India, was investigated. This investigation was carried out via 24 autosomal STRs. RESULT: The 24 loci studied showed the highest value of combined power of discrimination (CPD = 1), combined power of exclusion (CPE = 0.99999999985), combined paternity index (CPI = 6.10 × 109) and lowest combined matching probability (CPM = 7.90 × 10-31). CONCLUSION: The studied population showed genetic closeness with the population of Uttarakhand, the Jats of Delhi,the Jat Sikh (Punjab), and the population of Rajasthan. Among the tested loci, SE33 and Penta E were found to be most useful in terms of the highest discrimination power, lowest matching probability, the highest power of exclusion, and highest polymorphism information content for the Uttar Pradesh population .

Identification of Indian crocodile species through DNA barcodes.

The biodiversity of India includes three crocodile species, Crocodylus palustris, Crocodylus porosus, and Gavialis gangeticus, whose status is threatened due to bushmeat crisis and illegal hunting. The crocodilian conservation management requires novel techniques to help forensic analysts to reveal species identity. DNA barcoding is a species identification technique, where a partial cytochrome c oxidase subunit 1 gene is used as a marker for species identification. Herein, the DNA barcoding technique is evaluated for three Indian crocodiles by analyzing an approximately 750-bp barcode region. The alignment result shows interspecific variations between sequences for discrimination of the three Indian crocodiles leading to species identification. The phylogenetic analyses also substantiate the established crocodilian relationships, which add further advantage to use this DNA barcoding approach for Indian crocodiles. This study provides preliminary evidences for the use of DNA barcoding technique in the identification of Indian crocodile species.

Complete mitochondrial genome sequence from an endangered Indian snake, Python molurus molurus (Serpentes, Pythonidae).

This paper reports the complete mitochondrial genome sequence of an endangered Indian snake, Python molurus molurus (Indian Rock Python). A typical snake mitochondrial (mt) genome of 17258 bp length comprising of 37 genes including the 13 protein coding genes, 22 tRNA genes, and 2 ribosomal RNA genes along with duplicate control regions is described herein. The P. molurus molurus mt. genome is relatively similar to other snake mt. genomes with respect to gene arrangement, composition, tRNA structures and skews of AT/GC bases. The nucleotide composition of the genome shows that there are more A-C % than T-G% on the positive strand as revealed by positive AT and CG skews. Comparison of individual protein coding genes, with other snake genomes suggests that ATP8 and NADH3 genes have high divergence rates. Codon usage analysis reveals a preference of NNC codons over NNG codons in the mt. genome of P. molurus. Also, the synonymous and non-synonymous substitution rates (ka/ks) suggest that most of the protein coding genes are under purifying selection pressure. The phylogenetic analyses involving the concatenated 13 protein coding genes of P. molurus molurus conformed to the previously established snake phylogeny.

Validation of a multiplex PCR assay for the forensic identification of Indian crocodiles.

A dependable and efficient wildlife species identification system is essential for swift dispensation of the justice linking wildlife crimes. Development of molecular techniques is befitting the need of the time. The forensic laboratories often receive highly ill-treated samples for identification purposes, and thus, validation of any novel methodology is necessary for forensic usage. We validate a novel multiplex polymerase chain reaction assay, developed at this laboratory for the forensic identification of three Indian crocodiles, Crocodylus palustris, Crocodylus porosus, and Gavialis gangeticus, following the guidelines of Scientific Working Group on DNA Analysis Methods. The multiplex PCR was tested for its specificity, reproducibility, sensitivity, and stability. This study also includes the samples treated with various chemical substances and exposed to various environmental regimes. The result of this validation study promises this technique to be an efficient identification tool for Indian crocodiles and therefore is recommended for forensic purposes.

Complete mitochondrial genome sequences of three Crocodylus species and their comparison within the Order Crocodylia.

Crocodylus is the largest genus within the Order Crocodylia consisting of eleven species. This paper reports the complete mitochondrial genome sequences of three Crocodylus species, Crocodylus moreletii, Crocodylus johnstoni and Crocodylus palustris, and compares the newly obtained mitochondrial DNA sequences with other crocodilians, available in the public databases. The mitochondrial genomes of C. moreletii, C. johnstoni and C. palustris are 16,827 bp, 16,851 bp and 16,852 bp in length, respectively. These mitochondrial genomes consist of 13 protein coding genes, two ribosomal RNA genes, 22 transfer RNA genes and a non-coding region. The mitochondrial genomes of all the Crocodylus species, studied herein show identical characteristics in terms of nucleotide composition and codon usage, suggestive of the existence of analogous evolutionary patterns within the genus, Crocodylus. The synonymous and non-synonymous substitution rates for all the protein coding genes of Crocodylus were observed in between 0.001 and 0.275 which reveal the prevalence of purifying selection in these genes. The phylogenetic analyses based on complete mitochondrial DNA data substantiate the previously established crocodilian phylogeny. This study provides a better understanding of the crocodilian mitochondrial genome and the data described herein will prove useful for future studies concerning crocodilian mitochondrial genome evolution.

DNA mini-barcoding: an approach for forensic identification of some endangered Indian snake species.

Illegal trade of snake skin and uncontrolled hunting have instigated the extermination of many endangered snake species. Efforts to check illegal trade are often impeded due to lack of proper species identification methods. Hence, conservation strategies demand for authentic and quick identification techniques to trace the origin of the seized samples. This study employs DNA mini-barcoding as a method to identify some endangered snake species of India. We have designed two sets of novel primers for targeting regions within the mitochondrial Cytochrome Oxidase I gene to produce 175 bp and 245 bp amplicons. 175 bp fragment was amplified in all 11 snake species studied while the 245 bp amplicon was obtained in 10 species. DNA mini-barcodes recovered from these amplicons enabled the identification of snake species by retrieving the sequences available in public databases. The similarity scores ranging from 98 to 100% (98% taken as threshold value for species identification) signify the consistency of these mini-barcodes in snake species identification. Moreover, the results of the validation study confirm the effectiveness of the technique in forensic perspective, where the diagnostic morphological features of the seized sample are often missing.

Molecular phylogenetic analyses of genus Crocodylus (Eusuchia, Crocodylia, Crocodylidae) and the taxonomic position of Crocodylus porosus.

The genus Crocodylus consists of 11 species including the largest living reptile, Crocodylus porosus. The current understanding of the intrageneric relationships between the members of the genus Crocodylus is sparse. Even though members of this genus have been included in many phylogenetic analyses, different molecular approaches have resulted in incongruent trees leaving the phylogenetic relationships among the members of Crocodylus unresolved inclusive of the placement of C. porosus. In this study, the complete mitochondrial genome sequences along with the partial mitochondrial gene sequences and a nuclear gene, C-mos were utilized to infer the intrageneric relationships among Crocodylus species with a special emphasis on the phylogenetic position of C. porosus. Four different phylogenetic methods, Neighbour Joining, Maximum Parsimony, Maximum Likelihood and Bayesian inference, were utilized to reconstruct the crocodilian phylogeny. The uncorrected pairwise distances computed in the study, show close proximity of C. porosus to C. siamensis and the tree topologies thus obtained, also consistently substantiated this relationship with a high statistical support. In addition, the relationship between C. acutus and C. intermedius was retained in all the analyses. The results of the current phylogenetic study support the well established intergeneric crocodilian phylogenetic relationships. Thus, this study proposes the sister relationship between C. porosus and C. siamensis and also suggests the close relationship of C. acutus to C. intermedius within the genus Crocodylus.

Molecular identification of three Indian snake species using simple PCR-RFLP method.

Three endangered Indian snake species, Python molurus, Naja naja, and Xenochrophis piscator are known to be significantly involved in illegal trade. Effective authentication of species is required to curb this illegal trade. In the absence of morphological features, molecular identification techniques hold promise to address the issue of species identification. We present an effective PCR-restriction fragment length polymorphism method for easy identification of the three endangered snake species, Python molurus, Naja naja, and Xenochrophis piscator. A 431-bp amplicon from cytochrome b gene was amplified using novel snake-specific primers following restriction digestion with enzymes Mbo II and Fok I. The species-specific reference fragment patterns were obtained for the target species, which enabled successful identification of even highly degraded shed skin sample confirming the utility of the technique in case of poor-quality DNA. The assay could be effectively used for forensic authentication of three Indian snake species and would help strengthen conservation efforts.

A novel multiplex PCR assay for the identification of Indian crocodiles.

Illegal hunting has been a major threat for the survival of wildlife fauna, including the three crocodile species that India harbours: Crocodylus palustris, Crocodylus porosus and Gavialis gangeticus. Although law prevents trade on these species, illicit hunting for trade continues to threaten the survival of these endangered species; conservation strategies therefore require a rapid molecular identification technique for Indian crocodiles. A multiplex polymerase chain reaction (PCR) assay with species-specific primers, considered as one of the most effective molecular techniques, is described herein. The primers were designed to yield species-specific sized amplicons. The assay discriminates the three Indian crocodile species unambiguously within a short time period using only simple agarose gel electrophoresis. We recommend this multiplex PCR assay to be used in the identification of Indian crocodile species.

Traces of sub-Saharan and Middle Eastern lineages in Indian Muslim populations.

Islam is the second most practiced religion in India, next to Hinduism. It is still unclear whether the spread of Islam in India has been only a cultural transformation or is associated with detectable levels of gene flow. To estimate the contribution of West Asian and Arabian admixture to Indian Muslims, we assessed genetic variation in mtDNA, Y-chromosomal and LCT/MCM6 markers in 472, 431 and 476 samples, respectively, representing six Muslim communities from different geographical regions of India. We found that most of the Indian Muslim populations received their major genetic input from geographically close non-Muslim populations. However, low levels of likely sub-Saharan African, Arabian and West Asian admixture were also observed among Indian Muslims in the form of L0a2a2 mtDNA and E1b1b1a and J(*)(xJ2) Y-chromosomal lineages. The distinction between Iranian and Arabian sources was difficult to make with mtDNA and the Y chromosome, as the estimates were highly correlated because of similar gene pool compositions in the sources. In contrast, the LCT/MCM6 locus, which shows a clear distinction between the two sources, enabled us to rule out significant gene flow from Arabia. Overall, our results support a model according to which the spread of Islam in India was predominantly cultural conversion associated with minor but still detectable levels of gene flow from outside, primarily from Iran and Central Asia, rather than directly from the Arabian Peninsula.

Allele frequency distribution for 15 autosomal STR loci in Afridi Pathan population of Uttar Pradesh, India.

Allele frequencies of the 15 autosomal short tandem repeat (STR) loci D8S1179, D21S11, D7S820, CSF1PO D19S433, vWA, TPOX, D18S51, D3S1358, THO1, D13S317, D16S539, D2S1338, D5S818 and FGA were determined in Afridi Pathan population of Uttar Pradesh, India. All the 15 STR loci studied were found to be highly polymorphic with respect to observed heterozygosity values. Adherence to the expectations of the Hardy-Weinberg equilibrium (HWE) was confirmed for all the loci with an exception of TPOX and FGA. The allele 12 in CSF1PO was found to be most frequent. The power of discrimination was found to be high ranging from a minimum of 0.858 for the locus CSFIPO to maximum of 0.962 for the locus FGA, thereby facilitating the validation and efficiency of these STR markers in human identification. Population differentiation test between the studied and neighboring populations revealed significant differences at several loci suggesting the endogamous nature of the studied population. To the best of our knowledge, Afridi Pathan population has not been explored genetically for generating forensic data on STR markers. Therefore, STR allele frequency data of this unique population is a valuable contribution to the existing DNA database on Indian populations.

Molecular identification of Indian crocodile species: PCR-RFLP method for forensic authentication*.

South East Asian countries are known for illegal poaching and trade of crocodiles clandestinely, to be used in skin, medicinal, and cosmetic industries. Besides crocodiles being listed in the Convention on International Trade in Endangered Species of Wild Fauna and Flora, India has its Wildlife Protection Act, 1972 for conservation of crocodile species. Hitherto, lack of any rapid and reliable technique for examinations of crocodile-based crime exhibits such as skin, bones, etc. has been a major problem for an effective promulgation of law on illegal trade. DNA-based identification of species using PCR-RFLP technique for an apt identification of all the three Indian crocodile species namely, Crocodylus porosus, Crocodylus palustris and Gavialis gangeticus is presented here. A 628 bp segment of cytochrome b gene was amplified using novel primers followed by restriction digestion with three enzymes i.e., HaeIII, MboI, and MwoI, separately and in combination. The technique has produced a species-specific pattern for identifying the three crocodile species individually, which fulfills the requirement for its forensic application. It is expected that the technique will prove handy in identification of all the three Indian crocodile species and strengthen conservation efforts.

Diverse genetic origin of Indian Muslims: evidence from autosomal STR loci.

The origin and relationships of Indian Muslims is still dubious and are not yet genetically well studied. In the light of historically attested movements into Indian subcontinent during the demic expansion of Islam, the present study aims to substantiate whether it had been accompanied by any gene flow or only a cultural transformation phenomenon. An array of 13 autosomal STR markers that are common in the worldwide data sets was used to explore the genetic diversity of Indian Muslims. The austere endogamy being practiced for several generations was confirmed by the genetic demarcation of each of the six Indian Muslim communities in the phylogenetic assessments for the markers examined. The analyses were further refined by comparison with geographically closest neighboring Hindu religious groups (including several caste and tribal populations) and the populations from Middle East, East Asia and Europe. We found that some of the Muslim populations displayed high level of regional genetic affinity rather than religious affinity. Interestingly, in Dawoodi Bohras (TN and GUJ) and Iranian Shia significant genetic contribution from West Asia, especially Iran (49, 47 and 46%, respectively) was observed. This divulges the existence of Middle Eastern genetic signatures in some of the contemporary Indian Muslim populations. Our study reveals that the spread of Islamic faith in the Indian subcontinent was predominantly cultural transformation associated with minor gene flow from West Asia.

Microsatellite diversity delineates genetic relationships of Shia and Sunni Muslim populations of Uttar Pradesh, India.

In this study we characterize the genetic diversity and relationships between the Shia and Sunni Muslim populations of North India and geographically targeted neighboring and global populations. We examined a number of parameters of population genetic and forensic interest based on the allele frequencies from 15 autosomal STR loci (D8S1179, D21S11, D7S820, CSF1PO, D19S433, VWA, TPOX, D18S51, D3S1358, THO1, D13S317, D16S539, D2S1338, D5S818, and FGA). All the studied loci were consistent with Hardy-Weinberg equilibrium, except loci D18S51 and FGA for both Muslim populations, even after applying the Bonferroni correction. The combined power of exclusion and combined power of discrimination values for all 15 STR loci were 0.9999 and >0.99999, respectively, in both Muslim populations. Gene diversity values ranged from 0.6784 (TPOX) to 0.9027 (FGA) for Shia Muslims and from 0.7152 (CSF1PO) to 0.9120 (D18S51) for Sunni Muslims. The observed heterozygosity (H(o)) ranged from 0.5833 (D18S51) to 0.8595 (VWA) in Shia Muslims and from 0.6818 (CSF1PO) to 0.8333 (D21S11) in Sunni Muslims and was lower than the expected heterozygosity (H(e)) for 11 out of the 15 STRs typed. We analyzed the genetic affinities of the Shia and Sunni Muslim populations with their geographically closest neighboring North Indian, Middle Eastern, East Asian, and European populations using distance-based methods, including neighbor-joining trees and multidimensional scaling. In addition, we estimated the genetic contribution of the putative parental populations included in the analysis to the Shia and Sunni Muslim gene pool using admixture analysis. Although we observed a certain degree of genetic contribution from Iran to both Muslim populations, the results of the phylogenetic analyses based on autosomal STRs suggest genetic relatedness with some of the geographically closest neighboring Hindu religious populations.

Forensic STR profile of two endogamous populations of Madhya Pradesh, India.

Genotypic polymorphism studies at 15 highly polymorphic short tandem repeat (STR) loci were carried out in two populations belonging to one caste and one tribal group of Madhya Pradesh, in central region of India. These include 110 individuals from Brahmin caste (Kanyakubj) and 89 from Gond tribe (Ojha). The 15 loci studied are: 13 CODIS STR core markers, i.e., D8S1179, D3S1358, D21S11, D7S820, CSF1PO, vWA, TPOX, D18S51, THO1, D13S317, D16S539, D5S818, FGA and 2 other loci D19S433 and D2S1338. The results show departure from the Hardy-Weinberg equilibrium with respect to two loci, viz., D3S1358 and FGA in Gond tribe and at seven loci, viz., D21S11, D19S433, TPOX, D18S51, THO1, D5S818, and FGA in Brahmin caste. Population differentiation tests between the two studied populations and with seven neighboring populations (4 tribes and 3 castes - two middle castes and one Deshasth Brahmin) revealed significant differences at several loci. The power of discrimination of the microsatellite markers used was found to be high for both the populations. The data thereof is of immense significance for forensic result interpretation and is an addition to the existing autosomal STR database on Indian population.

Genetic affinity between diverse ethnoreligious communities of Tamil Nadu, India: a microsatellite study.

Historically, a number of local Hindu caste groups have converted to Islam and formed religious endogamous groups. Therefore the local caste groups and religious communities in a region are expected to show genetic relatedness. In this study we investigate the genetic relationship between Tamil-speaking (Dravidian language) Muslims (Sunni), six endogamous Hindu castes, and a tribal ethnic group (Irulars) using 13 CODIS (Combined DNA Index System) autosomal microsatellite markers. Muslims show the highest average heterozygosity (0.405) compared to the other groups. The neighbor-joining tree and the multidimensional-scaling plot show clustering of Tamil-speaking Muslims with three caste groups (Gounder, Paraiyar, and Vanniyar), whereas the Irular tribe is separated out of the cluster.

Allele frequency distribution for 15 autosomal STR loci in two Muslim populations of Tamilnadu, India.

Allele frequencies of the 15 autosomal STR loci: D8S1179, D21S11, D7S820, CSF1PO, D19S433, vWA, TPOX, D18S51, D3S1358, THO1, D13S317, D16S539, D2S1338, D5S818, and FGA were determined in two endogamous Muslim populations (Dawoodi Bohra Muslims from Shiite Muslims and Sunni Muslims) residing in Tamilnadu, India. The Loci D7S820, CSF1PO, D19S433, vWA, TPOX, D13S317, D16S539, D5S818, and FGA in Dawoodi Bohra Muslims from Shiite Muslims, and CSF1PO, D19S433, TPOX, and D16S539 in Sunni Muslims were found to deviate significantly from Hardy-Weinberg equilibrium. The power of discrimination of the analyzed markers was found to be high for the populations, thereby facilitating the validation and efficiency of these STR markers in human identification.