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.     

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.     

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 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.     

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.     

Application of bovine microsatellite markers for genetic diversity analysis of Swiss yak (Poephagus grunniens)

In order to assess the applicability of bovine microsatellite markers for population genetic studies in Swiss yak, 131 bovine microsatellite markers were tested on a panel of 10 animals. Efficient amplification was observed for 124 markers (94.6%) with a total of 476 alleles, of which 117 markers (94.3%) were polymorphic. The number of alleles per locus among the polymorphic markers ranged from two to nine. Seven loci (ILSTS005, BMS424B, BMS1825, BMS672, BM1314, ETH123 and BM6017) failed to amplify yak genomic DNA. Two cattle Y-chromosome specific microsatellite markers (INRA126 and BM861) amplified genomic DNA from both male and female yaks. However, two additional markers on cattle Y-chromosome (INRA124 and INRA189) amplified DNA from only males. Of the polymorphic markers, 24 microsatellites proposed by CaDBase for within- and cross-species comparisons and two additional highly polymorphic markers (MHCII and TGLA73) were used to investigate the genetic variability and the population structure of a Swiss yak herd that included 51 additional animals. The polymorphic information content ranged from 0.355 to 0.752, while observed heterozygosity (HO) ranged from 0.348 to 0.823. Furthermore, a set of 13 markers, organized into three multiplex polymerase chain reactions, was evaluated for routine parentage testing. This set provided an exclusion probability in a family of four yaks (both parents and two offspring) of 0.995. These microsatellites serve as useful tools for genetic characterization of the yak, which continues to be an important domestic livestock species.     

Development of microsatellite markers in Fragaria, their use in genetic diversity analysis, and their potential for genetic linkage mapping.

We have developed 21 new microsatellites in the model diploid perennial species Fragaria vesca from an enriched genomic library developed using F. vesca 'Ruegen'. The transferability of the primer pairs to other Fragaria species was high; all 31 primer pairs produced amplicons in 3 accessions of the octoploid strawberry Fragaria x ananassa, whereas 24 (77%) amplified a product in 7 other diploid Fragaria species. We analysed the allelic variation among 15 F. vesca accessions using the 21 microsatellites reported here and 10 F. vesca microsatellites described previously. The level of polymorphism detected at these microsatellite loci was high; five loci were monomorphic. Only two microsatellites were required to unambiguously discriminate among the 15 F. vesca accessions. A preliminary survey of segregation in an F2 progeny indicates that 20 of the 26 polymorphic loci (77%) could be mapped.     

Ingestion of bacteria in suspension Indian major carps (Catla catla,Labeo rohita) and Chinese carps (Hypophthalmichthys molitrix,Aristichthys nobilis)

Single-species groups of fry of the planktivorous, filter-feeding carps (silver carp Hypophthalmichthys molitrix, bighead carp Aristichthys nobilis, rohu Labeo rohita and catla Catla catla) were exposed to the bacterium Chromobacterium violaceum to assess if they could detect or ingest unattached bacteria. The results indicate that planktivorous carp fry are able to ingest unattached bacteria and that the numbers ingested increase with time and with bacterial concentration in the media, although probably by passive means. It is also suggested that unattached bacteria are of little nutritional significance to carps.     

Evaluation of the genetic variability of 13 microsatellite markers in native Indian pigs

We analysed polymorphism of 13 microsatellites in two Indian domesticated pig types (North Indian and Northeast Indian). Heterozygosity, polymorphism information content, and probability of identity of two random individuals were calculated for all microsatellites in both types. The number of alleles observed at a locus varied between five and 12. The evaluated microsatellites exhibited a very high heterozygosity and polymorphism information content. The probability of identity of two random individuals from different populations taking into account all the 13 microsatellites was as low as 3.51 × 10^-19. On the basis of these results, we propose that these microsatellite markers may be used with reliability for studying the genetic diversity and for identification of individuals in Indian pig types.     

Polymorphic microsatellite loci for the genetic analysis of Lycoris radiata (Amaryllidaceae) and cross-amplification in other congeneric species

Lycoris radiata is a perennial herb that has been used in traditional Chinese medicine for a long time and has two main medicinal components in its bulb, lycorine and galanthamine. However, the original microsatellite loci have not been developed for any species of Lycoris. Total genomic DNA was extracted from fresh bulbs using a modified CTAB protocol. We isolated 10 microsatellite loci from 21 L. radiata individuals of a natural population from Yellow Mountain in Anhui Province, China. The number of alleles ranged from two to nine. The observed and expected heterozygosities ranged from 0.238 to 0.952 and from 0.455 to 0.784, respectively. One locus significantly deviated from Hardy-Weinberg equilibrium and no significant linkage disequilibrium was found between pairs of loci. Cross-species amplification of these microsatellite loci was characterized in additional five species (L. sprengeri, L. anhuiensis, L. albiflora, L. longituba, and L. chinensis) of Lycoris. The results suggest that these microsatellite markers would contribute to the population genetic studies of L. radiata and other related species.     

Development and characterisation of 20 microsatellite loci isolated from the large bent-wing bat, Miniopterus schreibersii (Chiroptera: Miniopteridae) and their cross-taxa utility in the family Miniopteridae

The large bent-wing bat, Miniopterus schreibersii (Kuhl 1819), has a long history of taxonomic uncertainty and many populations are known to be in a state of decline. Microsatellite loci were developed for the taxonomic and population genetic assessment of the Australian complex of this species. Of the 33 primer sets designed for this research, seven (21%) were deemed suitably polymorphic for population-level analyses of the Australian taxa, with five (71%) of these loci revealing moderate to high levels of polymorphism (PIC = 0.56 to 0.91). The cross-taxa utility of the M. schreibersii microsatellite markers was assessed in the microbat (Chiroptera) family Miniopteridae. Sub-species and species covering the Miniopteridae's global distribution (with the exception of the Middle East) were selected, numbering 25 taxa in total. Amplification was successful for 26 loci, of which 20 (77%) were polymorphic. High cross-taxa utility of markers was observed with amplification achieved for all taxa for between four (20%) and 20 (100%) loci, and polymorphism was considered moderate to high (PIC = 0.47-0.91) for 12 (60%) of these loci. The high cross-taxa utility of the microsatellites reported herein reveal versatile and cost-effective molecular markers, contributing an important genetic resource for the research and conservation of Miniopteridae species worldwide.     

Allelic and population variation of microsatellite loci in aspen (Populus tremuloides)

To develop a robust basis for inferences about population genetics and evolution, this work assayed 192 aspens (Populus tremuloides) from 11 sites in Wisconsin, USA, for allelic and population variation at 16 microsatellite loci distributed across the Populus genome. Frequency distributions of fluorochrome-labeled alleles resolved by capillary electrophoresis were analyzed for relationships to repeat size and number. Population-level statistics were compared with those of other studies, especially in Populus. All loci were polymorphic, varying widely in the number of alleles per locus (mean = 8.25, range 2-20). Expected and observed heterozygosities were high (0.45 and 0.41, respectively), with little differentiation among populations (F(ST) = 0.006-0.045) and a moderate level of inbreeding (F(IS) = 0.09), intermediate among levels reported in studies based on isozymes. Contrary to several other reports, allele frequencies clustered tightly around the modal frequency, and the genetic diversity (measured as alleles per locus or as expected heterozygosity) was not related to either the repeat unit size or to the number of repeats.