Population genetic structure of three major river Populations of rohu,Labeo rohita (Cyprinidae: Cypriniformes) using microsatellite DNA markers

Determining the genetic structure is essential for developing conservation and stock improvement plans. Four dinucleotide microsatellite loci were analysed to reveal population genetic structure of the Indian major carp,Labeo rohita collected from three major rivers namely the Halda, the Jamuna, and the Padma in Bangladesh. The four loci were polymorphic (P ₉₅) in all the populations. The populations varied in the number and frequencies of alleles as well as heterozygosities in the loci analyzed. Population differentiation (F _ST) value between the Halda and the Jamuna population was significant (P<0.05). Relatively high level of gene flow and low level ofF _ST values were found between the Padma and the Jamuna population. The unweighted pair group method with averages (UPGMA) dendrogram based on genetic distance resulted in two clusters: the Halda population was alone in one cluster whereas the Jamuna and the Padma made another cluster. The results revealed a relatively low level of genetic variability in the river populations ofL. rohita in Bangladesh.     

Population genetic analysis among five Indian population groups using six microsatellite markers

Genetic variation at six tetranucleotide microsatellites (HUMTHO1, HUMVWA, F13A01, D3S1359, D12S66, and D12S67) has heen determined in five endogamous ethnic population groups of India belonging to two major linguistic families. The populations analyzed were Konkanastha Brahmins and Marathas (Maharashtra state) from the Indo-Aryan linguistic family and Nairs, Ezhavas, and Muslims (Kerala state) from the Dravidian family. All six loci show high gene diversity, ranging from 0.63 +/- 0.04 to 0.84 +/- 0.02. The average GST value observed was 1.7%, indicating that the differences between the populations account for less than 2% of the diversity, while the genetic variation is high within the five population groups studied (>98%). The phylogenetic tree fails to show any clear cluster. The absence of any cluster along with low average GST is suggestive of substantial genetic similarity among the studied populations, in spite of clear geographical, linguistic, and cultural barriers. This similarity indicates either a greater gene flow between these groups or, alternatively, may reflect a recent evolution for them, considering that the Indian caste system evolved only about 3000 years ago.     

Identification of microsatellite DNA markers for population structure analysis in Indian major carp, Cirrhinus mrigala (Hamilton-Buchanan, 1882)

Forty‐four primer sequences available for four cyprinid fishes were tested to amplify microsatellite loci in Indian major carp, Cirrhinus mrigala. A total of 12 loci were successfully amplified with clear scorable patterns and five thereof were polymorphic. Suitability of the identified polymorphic loci in population structure analysis of C. mrigala was assessed. Genetic variation was examined in 76 specimens collected from five different rivers. The mean observed heterozygosity ranged from 0.247 to 0.333. Significant heterogeneity in allele frequencies was detected, indicating that the samples analysed did not belong to homogenous populations. The identified microsatellite markers are promising for the analysis of intraspecific divergence in C. mrigala across its distribution range.     

Multiplexed microsatellite markers for the genetic analysis of Eucalyptus leucoxylon (Myrtaceae) and their utility for ecological and breeding studies in other eucalyptus species

Eucalyptus leucoxylon is a widespread woodland tree species found in southeastern Australia that has suffered from, and continues to be, threatened by the impacts of habitat clearance and degradation. Populations now consist predominantly of scattered individuals, and their conservation status is of increasing concern. We report the development and characterization of a set of eight highly polymorphic microsatellite loci for E. leucoxylon. The loci can be amplified in three PCR multiplexes and electrophoresed in a single lane, allowing rapid throughput of large numbers of samples. A total of 111 alleles were detected in 68 individuals with an average of 12.3 alleles per locus, a mean expected heterozygosity of 0.83, and a mean observed heterozygosity of 0.72. The combined probabilities of identity and probabilities of paternity exclusion allow an extremely precise level of individual identification, indicating that these microsatellite markers will be ideal for population genetic and parentage-type studies in E. leucoxylon. The markers also exhibited an average of 76% conservation within the subgenus Symphyomyrtus, to which E. leucoxylon belongs, and 53% conservation across other subgenera of Eucalyptus, demonstrating the potential of these markers in ecological and breeding studies in a wide range of Eucalyptus species.     

Cross-species microsatellite markers for elucidating population genetic structure in Arabidopsis and Arabis (Brassicaeae)

Species closely related to model organisms present the opportunity to efficiently apply molecular and functional tools developed by a large research community to taxa with different ecological and evolutionary histories. We complied 42 microsatellite loci that amplify under common conditions in four closely related Arabidopsis: A. thaliana; A. halleri; A. lyrata ssp. lyrata; and A. lyrata ssp. petraea, as well as in one more distantly related crucifer; Arabis drummondii. Variation at these loci is amenable to a diversity of applications including population genetics, phylogeographical analyses, mapping of inter and intraspecific crosses, and recombination mapping. Our analysis of microsatellite variation illustrates significant differences in population genetic parameters among three Arabidopsis species. A population of A. thaliana, an inbreeding annual plant associated with disturbed habitats, was highly monomorphic (P = 8% percent polymorphic loci) and only 0.2% heterozygous for 648 locus-by-individual combinations. A population of the self-incompatible perennial herb, A. halleri, was more genetically variable (P = 71%) and had an excess of heterozygosity that may reflect a recent population bottleneck associated with human-mediated founder events. A population of the self-incompatible perennial herb, A. lyrata ssp. petraea, was even more genetically variable (P = 86%) and appeared to be at mutation-drift equilibrium. Population structure estimated from neutrally evolving loci provides an empirical expectation against which hypotheses of adaptive evolution at functional loci can be tested.     

Identification of a new Indian camel germplasm by microsatellite markers based genetic diversity and population structure of three camel populations

Camel invokes fascinating chapter of Indian desert history and is integral component of its ecosystem. Camel population has reached a crisis point after three decades of decline (75%) causing major concern to the policy makers. >28% of Indian camel is not yet characterized. It is imperative to describe country’s camel germplasm and its existing diversity for designing conservation plan. One such population is Sindhi, distributed along border with Pakistan. Twenty five microsatellite markers being valuable tool for estimating genetic diversity were selected to elucidate genetic variability and relationship of Sindhi with two registered camel breeds of India- Marwari and Kharai. The standard metrics of genomic diversity detected moderate variability in all the three populations. A total of 303 alleles with a mean of 8.116 ± 0.587 alleles per locus were found in total of 143 animals. Sindhi population had intermediate allelic diversity with 8.522 ± 1.063 alleles per locus. Corresponding values in Marwari and Kharai were 8.783 ± 0.962 and 7.043 ± 1.030, respectively. Genetic variability within the breeds was moderate as evidenced by the mean observed heterozygosity of 0.556 ± 0.025. Sindhi camel population harbors higher genetic variability (Ho = 0.594) as compared to the two registered camel breeds (Marwari, 0.543 and Kharai, 0.531). Mean expected heterozygosity under Hardy-Weinberg equilibrium was higher than the observed values across the three camel groups, indicating deviations from assumptions of this model. In fact, average positive F value of 0.084 to 0.206 reflected heterozygote deficiency in these populations. These Indian camel populations have not experienced serious demographic bottlenecks in the recent past. Differences among populations were medium and accounted for 7.3% of total genetic variability. Distinctness of three camel populations was supported by all the approaches utilized to study genetic relationships such as genetic distances, phylogenetic relationship, correspondence analysis, clustering method based on Bayesian approach and individual assignment. Sindhi camel population was clearly separated from two registered breeds of Indian camel. Results conclude Sindhi to be a separate genepool. Moderate genetic diversity provides an optimistic viewpoint for the survival of severely declining indigenous camel populations with appropriate planning strategies for conserving the existing genetic variation and to avoid any escalation of inbreeding.     

Isolation and cross‐species amplification of microsatellite loci in the freshwater minnow Zacco pachycephalus (Teleostei: Cyprinidae) for diversity and conservation genetic analysis

Asian minnows of the genus Zacco are dominant fish in various freshwater ecosystems. Two species, Zacco pachycephalus and Z. platypus, occur in Taiwan and are favourite targets for local sport anglers. The introduction of Zacco fish into nonindigenous habitats in Taiwan has become a conservation issue. We developed eight polymorphic microsatellites for Z. pachycephalus (average H_E = 0.779) and these microsatellites were applicable to Z. platypus, which showed a comparable polymorphic level (average H_E = 0.784). These loci can be used as genetic markers for identifying conservation units and studying population differentiation for both Zacco species.     

Isolation and characterization of microsatellite loci from chub Leuciscus cephalus (Pisces: Cyprinidae)

Twelve microsatellite markers were isolated from chub Leuciscus cephalus Linné, 1766 (Cyprinidae), a freshwater fish widely distributed in Europe. We assessed the level of genetic diversity for these loci in 24 individuals sampled in the Rhone river watershed. Nine loci were polymorphic, displaying from two to 13 alleles per locus with expected heterozygosity ranging from 0.160 to 0.887. Simultaneous migration of different polymerase chain reaction products was developed for routine analysis of L. cephalus populations.     

Inheritance of amplified fragment length polymorphism markers and their utility in population genetic analysis of Plecoglossus altivelis

The reproducibility, mode of inheritance and polymorphism of amplified fragment length polymorphism (AFLP) markers were examined in ayu Plecoglossus altivelis (Salmoniformes: Plecoglossidae). The AFLP markers were highly reproducible, their inheritance following Mendelian expectations. The number of fragments amplified (34–134), polymorphic ratio (0·15–0·78) and average heterozygosity (0·02–0·25) of the AFLP markers showed significant variation among six primer pairs and among ayu populations, including a landlocked Lake‐Biwa population, two amphidromous populations ( P. a. altivelis ) and two Ryukyu‐ayu populations ( P. a. ryukyuensis ). Although AFLP analysis provided similar results in intra‐population diversity and relationships among populations to those found by analyses of allozymes, microsatellites and mitochondrial DNA sequences, AFLPs showed higher polymorphisms and hence greater distinction between genetically close populations.     

Analysis of 22 heterologous microsatellite markers for genetic variability in Indian goats

The genetic variability of 22 heterologous microsatellite markers was analyzed in two Indian goat breeds, namely Bengal and Chegu. The heterozygosity, polymorphism information content (PIC), and probability of identity of two individuals were calculated for all microsatellite loci in both the breeds. The observed number of alleles varied between 4 and 13 at the studied microsatellite loci. The evaluated microsatellite loci exhibited high mean heterozygosity of 0.69 +/- 0.11 and 0.66 +/- 0.07 in Bengal and Chegu goats, respectively. The mean PIC values of the studied loci in these breeds were 0.79 +/- 0.08 and 0.78 +/- 0.05, respectively. The probability of identity of two random individuals from different breeds, taking into account, all the 22 microsatellite loci was as low as 5.523 x 10(-40). On the basis of these results, we propose that these microsatellite markers may be used with reliability for studying genetic diversity and for identification of individuals in Indian goat breeds.     

Development of microsatellite markers for parentage analysis in the great tinamou (Tinamus major)

Eighteen microsatellite loci were isolated from great tinamous (Tinamus major), which are large terrestrial birds found in the Neotropics. These are the first primers developed for the Order Tinamiformes. Paternity analyses are possible because the levels of heterozygosity are sufficiently high (0.29-0.90).     

Applicability of bovine microsatellite markers for population genetic studies on African buffalo (Syncerus caffer)

The applicability of bovine autosomal microsatellite markers for population genetic studies on African buffalo was investigated. A total of 168 microsatellite markers were tested for PCR amplification on a test panel of seven African buffalo. Amplification was observed for 139 markers (83%), and 101 markers were studied further with 91 (90%) being polymorphic. The mean number of alleles per marker was 5.0 (SE = 0.2) and the mean heterozygosity per marker was 0.61 (SE = 0.03). Considering the overall high level of polymorphism, it was concluded that most bovine microsatellite markers are applicable in African buffalo.     

Global population genetic structure and male-mediated gene flow in the green sea turtle (Chelonia mydas): analysis of microsatellite loci

We assessed the degree of population subdivision among global populations of green sea turtles, Chelonia mydas, using four microsatellite loci. Previously, a single-copy nuclear DNA study indicated significant male-mediated gene flow among populations alternately fixed for different mitochondrial DNA haplotypes and that genetic divergence between populations in the Atlantic and Pacific Oceans was more common than subdivisions among populations within ocean basins. Even so, overall levels of variation at single-copy loci were low and inferences were limited. Here, the markedly more variable microsatellite loci confirm the presence of male-mediated gene flow among populations within ocean basins. This analysis generally confirms the genetic divergence between the Atlantic and Pacific. As with the previous study, phylogenetic analyses of genetic distances based on the microsatellite loci indicate a close genetic relationship among eastern Atlantic and Indian Ocean populations. Unlike the single-copy study, however, the results here cannot be attributed to an artifact of general low variability and likely represent recent or ongoing migration between ocean basins. Sequence analyses of regions flanking the microsatellite repeat reveal considerable amounts of cryptic variation and homoplasy and significantly aid in our understanding of population connectivity. Assessment of the allele frequency distributions indicates that at least some of the loci may not be evolving by the stepwise mutation model.     

Homoplasy and mutation model at microsatellite loci and their consequences for population genetics analysis

Homoplasy has recently attracted the attention of population geneticists, as a consequence of the popularity of highly variable stepwise mutating markers such as microsatellites. Microsatellite alleles generally refer to DNA fragments of different size (electromorphs). Electromorphs are identical in state (i.e. have identical size), but are not necessarily identical by descent due to convergent mutation(s). Homoplasy occurring at microsatellites is thus referred to as size homoplasy. Using new analytical developments and computer simulations, we first evaluate the effect of the mutation rate, the mutation model, the effective population size and the time of divergence between populations on size homoplasy at the within and between population levels. We then review the few experimental studies that used various molecular techniques to detect size homoplasious events at some microsatellite loci. The relationship between this molecularly accessible size homoplasy size and the actual amount of size homoplasy is not trivial, the former being considerably influenced by the molecular structure of microsatellite core sequences. In a third section, we show that homoplasy at microsatellite electromorphs does not represent a significant problem for many types of population genetics analyses realized by molecular ecologists, the large amount of variability at microsatellite loci often compensating for their homoplasious evolution. The situations where size homoplasy may be more problematic involve high mutation rates and large population sizes together with strong allele size constraints.     

Transferability and utility of white oat (Avena sativa) microsatellite markers for genetic studies in black oat (Avena strigosa)

Preservation and use of wild oat species germplasm are essential for further improvement of cultivated oats. We analyzed the transferability and utility of cultivated (white) oat Avena sativa (AACCDD genome) microsatellite markers for genetic studies of black oat A. strigosa (A(s)A(s) genome) genotypes. The DNA of each black oat genotype was extracted from young leaves and amplified by PCR using 24 microsatellite primers developed from white oat. The PCR products were separated on 3% agarose gel. Eighteen microsatellite primer pairs amplified consistent products and 15 of these were polymorphic in A. strigosa, demonstrating a high degree of transferability. Microsatellite primer pairs AM3, AM4, AM21, AM23, AM30, and AM35 consistently amplified alleles only in A. sativa, which indicates that they are putative loci for either the C or D genomes of Avena. Using the data generated by the 15 polymorphic primer pairs, it was possible to separate 40 genotypes of the 44 that we studied. The four genotypes that could not be separated are probably replicates. We conclude that A. sativa microsatellites have a high transferability index and are a valuable resource for genetic studies and characterization of A. strigosa genotypes.     

Isolation and characterization of polymorphic microsatellite loci in Candidia barbata (Cyprinidae) using PCR-based isolation of microsatellite arrays (PIMA)

Candidia barbata is an endemic cyprinid fish in Taiwan and suffers habitat destruction caused by human overexploitation. In this study, a total of 14 polymorphic microsatellite loci from C. barbata were isolated and characterized using an optimized protocol to construct a microsatellite-enriched genomic library. The analysis of variability was performed in 30 specimens of Taiwan. The mean number of alleles across loci was 4.92 ± 1.44. The levels of expected and observed heterozygosities varied from 0.1266 to 0.5079, and from 0.0667 to 0.9667, respectively. Frequencies of null alleles of the 14 loci are not significantly greater than zero. No linkage disequilibrium was detected between loci in either population.     

Isolation and characterization of polymorphic microsatellite loci for the dace complex: Leuciscus leuciscus (Teleostei: Cyprinidae)

Ten novel polymorphic microsatellites were isolated from the dace complex (Leuciscus leuciscus), which is a European cyprinid species. Four multiplex polymerase chain reaction sets were optimized in order to genotype 26 polymorphic loci in all, including 16 previously published cyprinid-specific loci. The level of genetic diversity was assessed in 142 dace individuals. We also successfully applied 26 of the microsatellites to 10 related species. These primers thus will be useful to assess population structure of the dace and other cyprinid species, with application for conservation issues and phylogeographical approaches.     

Ten new polymorphic microsatellite loci for North American river otters (Lontra canadensis) and their utility in related mustelids

We describe 10 novel North American river otter (Lontra canadensis) polymorphic microsatellite loci. Individuals from two river drainages in New York were sampled, and the number of alleles per locus ranged from two to 12 (Drainage 1) and two to 10 (Drainage 2). Observed heterozygosities ranged from 0.21 to 0.83 (Drainage 1) and 0.20 to 0.80 (Drainage 2). Preliminary screening revealed that loci amplified in five other mustelids [Martes pennanti (n = 6), Martes fiona (n = 8), Mustela frenata (n = 8), Mustela erminea (n = 8) and Mustela vison (n = 5)].     

Development of eighteen tetranucleotide microsatellite markers in Tibetan Macaque (Macaca thibetana) and genetic diversity analysis of captive population

In this study, eighteen tetranucleotide microsatellite loci were isolated from AAAG-enriched and GATA-enriched libraries of the Tibetan macaque, Macaca thibetana. These loci were tested on 53 individuals of M. thibetana, and most loci were proved to be highly polymorphic. A total of 109 alleles were detected with an average of 6.06 alleles per locus. The PIC values of these loci ranged from 0.192 to 0.879, with an average of 0.624. The observed and expected heterozygosities ranged from 0.170 to 0.800 and from 0.217 to 0.898, with an average of 0.583 and 0.675, respectively. 5 loci significantly deviated from Hardy–Weinberg equilibrium (HWE). Significant linkage disequilibrium (LD) was found between 9 pairs of loci. The newly identified polymorphic markers would facilitate the study of M. thibetana on the population structure and genetic diversity.