Sree Kanthaswamy

Recommendations for animal DNA forensic and identity testing

Genetic analysis in animals has been used for many applications, such as kinship analysis, for determining the sire of an offspring when a female has been exposed to multiple males, determining parentage when an animal switches offspring with another dam, extended lineage reconstruction, estimating inbreeding, identification in breed registries, and speciation. It now also is being used increasingly to characterize animal materials in forensic cases. As such, it is important to operate under a set of minimum guidelines that assures that all service providers have a template to follow for quality practices. None have been delineated for animal genetic identity testing. Based on the model for human DNA forensic analyses, a basic discussion of the issues and guidelines is provided for animal testing to include analytical practices, data evaluation, nomenclature, allele designation, statistics, validation, proficiency testing, lineage markers, casework files, and reporting. These should provide a basis for professional societies and/or working groups to establish more formalized recommendations.

Hybridization and Stratification of Nuclear Genetic Variation in Macaca mulatta and M. fascicularis

We used genotypes for 13 short tandem repeats (STRs) to assess the genetic diversity within and differentiation among populations of rhesus macaques (Macaca mulatta) from mainland Asia and long-tailed macaques (M. fascicularis) from mainland and insular Southeast Asia. The subjects were either recently captured in the wild or derived from wild-caught founders maintained in captivity for biomedical research. A large number of alleles are shared between the 2 macaque species but a significant genetic division between them persists. The distinction is more clear-cut among populations that are not, or are unlikely to have recently been, geographically contiguous. Our results suggest there has been significant interspecific nuclear gene flow between rhesus macaques and long-tailed macaques on the mainland. Comparisons of mainland and island populations of long-tailed macaques reflect marked genetic subdivisions due to barriers to migration. Geographic isolation has restricted gene flow, allowing island populations to become subdivided and genetically differentiated. Indonesian long-tailed macaques show evidence of long-term separation and genetic isolation from the mainland populations, whereas long-tailed macaques from the Philippines and Mauritius both display evidence of founder effects and subsequent isolation, with the impact from genetic drift being more profound in the latter.

Pyrosequencing as a method for SNP identification in the rhesus macaque (Macaca mulatta)

BackgroundRhesus macaques (Macaca mulatta) are the primate most used for biomedical research, but phenotypic differences between Indian-origin and Chinese rhesus macaques have encouraged genetic methods for identifying genetic differences between these two populations. The completion of the rhesus genome has led to the identification of many single nucleotide polymorphisms (SNPs) in this species. These single nucleotide polymorphisms have many advantages over the short tandem repeat (STR) loci currently used to assay genetic variation. However, the number of currently identified polymorphisms is too small for whole genome analysis or studies of quantitative trait loci. To that end, we tested a combination of methods to identify large numbers of high-confidence SNPs, and screen those with high minor allele frequencies (MAF).ResultsBy testing our previously reported single nucleotide polymorphisms, we identified a subset of high-confidence, high-MAF polymorphisms. Resequencing revealed a large number of regionally specific SNPs not identified through a single pyrosequencing run. By resequencing a pooled sample of four individuals, we reliably identified loci with a MAF of at least 12.5%. Finally, we found that when applied to a larger, geographically variable sample of rhesus, a large proportion of our loci were variable in both populations, and very few loci were ancestry informative. Despite this fact, the SNP loci were more effective at discriminating Indian and Chinese rhesus than STR loci.ConclusionPyrosequencing and pooled resequencing are viable methods for the identification of high-MAF SNP loci in rhesus macaques. These SNP loci are appropriate for screening both the inter- and intra-population genetic variation.

Canine Population Data Generated from a Multiplex STR Kit for Use in Forensic Casework

Abstract:  Canine biological specimens are often part of the physical evidence from crime scenes. Until now, there have been no validated canine‐specific forensic reagent kits available. A multiplex genotyping system, comprising 18 short tandem repeats (STRs) and a sex‐linked zinc finger locus for gender determination, was developed for generating population genetic data assessing the weight of canine forensic DNA profiles. Allele frequencies were estimated for 236 pedigreed and 431 mixed breed dogs residing in the U.S. Average random match probability is 1 in 2 × 1033 using the regional database and 1 in 4 × 1039 using the breed dataset. Each pedigreed population was genetically distinct and could be differentiated from the mixed breed dog population but genetic variation was not significantly correlated with geographic transition. Results herein support the use of the allele frequency data with the canine STR multiplex for conveying the significance of identity testing for forensic casework, parentage testing, and breed assignments.

Genetic characterization of wild and captive rhesus macaques in China

The genetic structures of wild and captive rhesus macaque populations within China were compared by analyzing the mtDNA sequences of 203 captive‐bred Chinese rhesus macaques with 77 GenBank sequences from wild‐caught animals trapped throughout China. The genotypes of 22 microsatellites of captive Chinese rhesus macaques were also compared with those of captive Indian animals. The Chinese population is significantly differentiated from the Indian population and is more heterogeneous. Thus, compared with Indian rhesus macaques the phenotypic variance of traits with high heritability will be inflated in Chinese animals. Our data suggest that the western Chinese provinces have more subdivided populations than the eastern and southern Chinese provinces. The southern Chinese populations are the least structured and might have been more recently established. Human‐mediated interbreeding among captive Chinese populations has occurred, implying that Chinese breeding strategies can influence the interpretation of biomedical research in the USA.

Detecting signatures of inter-regional and inter-specific hybridization among the Chinese rhesus macaque Specific Pathogen-free (SPF) population using single nucleotide polymorphic (SNP) markers

Background While rates of gene flow between rhesus and longtail macaque populations near their hybrid zone in Indochina have been quantified elsewhere, this study demonstrates that the inter‐specific introgression is not limited to the Indochinese hybrid zone but is more geographically widespread.

The effect of SNP discovery method and sample size on estimation of population genetic data for Chinese and Indian rhesus macaques (Macaca mulatta).

This study was designed to address issues regarding sample size and marker location that have arisen from the discovery of SNPs in the genomes of poorly characterized primate species and the application of these markers to the study of primate population genetics. We predict the effect of discovery sample size on the probability of discovering both rare and common SNPs and then compare this prediction with the proportion of common and rare SNPs discovered when different numbers of individuals are sequenced. Second, we examine the effect of genomic region on estimates of common population genetic data, comparing markers from both coding and non-coding regions of the rhesus macaque genome and the population genetic data calculated from these markers, to measure the degree and direction of bias introduced by SNPs located in coding versus non-coding regions of the genome. We found that both discovery sample size and genomic region surveyed affect SNP marker attributes and population genetic estimates, even when these are calculated from an expanded data set containing more individuals than the original discovery data set. Although none of the SNP detection methods or genomic regions tested in this study was completely uninformative, these results show that each has a different kind of genetic variation that is suitable for different purposes, and each introduces specific types of bias. Given that each SNP marker has an individual evolutionary history, we calculated that the most complete and unbiased representation of the genetic diversity present in the individual can be obtained by incorporating at least 10 individuals into the discovery sample set, to ensure the discovery of both common and rare polymorphisms.

Genetic characterization of specific pathogen‐free rhesus macaque (Macaca mulatta) populations at the California National Primate Research Center (CNPRC)

A study based on 14 STRs was conducted to understand intergenerational genetic changes that have occurred within the California National Primate Research Centers (CNPRC) regular specific pathogen‐free (SPF) and super‐SPF captive rhesus macaque populations relative to their conventional founders. Intergenerational genetic drift has caused age cohorts of each study population, especially within the conventional population, to become increasingly differentiated from each other and from their founders. Although there is still only minimal stratification between the conventional population and either of the two SPF populations, separate derivation of the regular and super‐SPF animals from their conventional founders has caused the two SPF populations to remain marginally different from each other. The regular SPF and, especially, the super‐SPF populations have been influenced by the effects of differential ancestry, sampling, and lost rare alleles, causing a substantial degree of genetic divergence between these subpopulations. The country of origin of founders is the principal determinant of the MHC haplotype composition of the SPF stocks at the CNPRC. Selection of SPF colony breeders bearing desired genotypes of Mamu‐A*01 or ‐B*01 has not affected the overall genetic heterogeneity of the conventional and the SPF research stocks.

Single Nucleotide Polymorphisms Reveal Patterns of Allele Sharing across the Species Boundary between Rhesus (Macaca mulatta) and Cynomolgus (M. fascicularis) Macaques

Both phenotypic and genetic evidence for asymmetric hybridization between rhesus (Macaca mulatta) and cynomolgus (Macaca fascicularis) macaques has been observed in the region of Indochina where both species are sympatric. The large‐scale sharing of major histocompatibility complex (MHC) class II alleles between the two species in this region supports the hypothesis that genes, and especially genes involved in immune response, are being transferred across the species boundary. This differential introgression has important implications for the incorporation of cynomolgus macaques of unknown geographic origin in biomedical research protocols. Our study found that for 2,808 single‐nucleotide polymorphism (SNP) markers, the minor allele frequencies (MAF) and observed heterozygosity calculated from a sample of Vietnamese cynomolgus macaques was significantly different from those calculated from samples of both Chinese rhesus and Indonesian cynomolgus macaques. SNP alleles from Chinese rhesus macaques were overrepresented in a sample of Vietnamese cynomolgus macaques relative to their Indonesian conspecifics and located in genes functionally related to the primary immune system. These results suggest that Indochinese cynomolgus macaques represent a genetically and immunologically distinct entity from Indonesian cynomolgus macaques. Am. J. Primatol. 75:135‐144, 2013.

Development of a Nomenclature System for a Canine STR Multiplex Reagent Kit

Abstract:  Despite the popularity of dogs in US households, canine DNA evidence remains largely untapped in forensic investigations partially because of the absence of well‐defined forensic short tandem repeats (STRs), lack of standardized and validated PCR protocols, STR reagent kits, and poorly developed nomenclature. A nomenclature system was established based on internationally recognized recommendations for human forensic STRs for a recently developed canine STR reagent kit. Representative alleles were sequenced from each of the 18 STRs and the sex‐typing marker included in the kit. This study also reflects on the impact of point mutations, insertions, and deletions within and outside the STR core repeat structures. An understanding of the STRs’ sequence and repeat structures will enable development of a robust and reliable allele nomenclature and improve the accuracy and precision of allele fragment sizing in canine forensic profiling. The expected allele sizes have been calculated, and their repeat stuctures defined based on sequence information.