Cross-species Microsatellite Markers Research Articles

Population genetic structure of wild Malayan tapirs (Tapirus indicus) in Peninsular Malaysia revealed by nine cross-species microsatellite markers

The Malayan tapir (Tapirus indicus) is an endangered species endemic to Southeast Asia. Previous research have used maternally inherited, mitochondrial DNA genetic markers to investigate the population genetics of the species but not the biparentally inherited, nuclear genetic markers. However, the increasing pressure of habitat fragmentation and roadkill on the Malayan tapirs has called for an urgent need to assess the species’ genetic status. In this study, the genetic diversity, population genetic structure, and sex-biased dispersal patterns of the tapir population in Peninsular Malaysia were investigated using nine cross-species microsatellite markers, using sixty-seven tapir samples (39 wild, 11 captive-born, and 17 of unknown origin) provided by the wildlife department, zoo and conservation centre. Low genetic diversity of the wild population was found based on heterozygosity and allelic richness, and a cryptic pattern of population genetic structure was inferred. One (K = 3) to two (K = 4) genetic clusters in proximity to the south-western part of the major forest complexes seemed to be experiencing restricted gene flows compared to the other more widespread clusters or clines. Effective to census population size was estimated at 0.39–0.46. Sex-biased dispersal was not found in the current dataset. Even though these results may need cautious interpretation due to possible sampling bias, this study is the first study that investigated the genetic diversity distribution for the species using nuclear markers, and therefore will have conservation implications for the implementation of the Central Forest Spine Master Plan in Peninsular Malaysia to connect major forest complexes.

Development of high conserved cross-species microsatellite markers from cucumber genome and their applicability in genetic diversity and comparative mapping

One of the most widely applicable and used markers considered in plant research is simple sequence repeats (SSRs). The Gy14 cucumber genome provided information for the development of SSR marker in this species which might be used in other related species additionally. We, therefore, developed genome-wide SSR markers from cucumber, which were used for genetic diversity analysis in 16 different cucurbits species and comparative mapping with melon and watermelon. A total of 2171 SSR motifs were identified from seven cucumber chromosomes. Out of them, 1749 markers belong to melon while 442 markers to watermelon. We experimentally validated 70 SSR markers to check their cross-species transferability among 16 different cucurbits species belongs to six genera. Maximum% polymorphism has been recorded for Momordica charantia (82%) followed by Momordica cochinchinesis (78.18%) and Luffa harmaphrodita (73.68%) while Benincasa hispida (49.21%) exhibited minimum% polymorphism. The large number of cucumber SSR markers developed in the present study provides a valuable resource for genetic linkage map construction, molecular mapping, and marker-assisted selection (MAS) in cucumber. Furthermore, the cross-species transferable SSR markers could also be useful in various molecular markers related studies in other closely related species in the Cucurbitaceae family in which draft genomes are not yet available.

Development of an SSR-based DNA fingerprinting method for black wattle (Acacia mearnsii De Wild)

Background: The most commonly used method for extracting DNA from plant leaf tissue involves cetyl trimethylammonium bromide but some species, such as Acacia mearnsii, contain high levels of secondary metabolites and polysaccharides that interfere with this process. Various modifications have been proposed for effective removal of these biomolecules but these methods can be time consuming. Therefore, this study was initiated to optimise the cetyl-trimethylammonium bromide protocol for the extraction of high-quality genomic DNA and to develop a fingerprinting tool using cross species transferable simple sequence repeat markers for genetic diversity studies in A. mearnsii.
 Methods: Five CTAB-based modification were examined and 49 cross-species microsatellite markers, developed for several Acacia species, were tested in four multiplex panels of A. mearnsii populations.
 Results: The modified protocol yields high quantity and quality DNA from A. mearnsii leaves using high concentration of NaCl to remove polysaccharides and polyvinylpolypyrrolidone (PVPP) to eliminate polyphenols during DNA purification. In addition, omitting the selective precipitation and NaCl gradient steps in the extraction protocol, enabled us to extract DNA 10–20 min faster than the normal protocol. Of the tested microsatellite loci, 11 were successful in amplifying sharp and high-intensity bands in all the four multiplex panels and were polymorphic. The level of polymorphism ranged from 0.115 to 0.794, with a mean 0.50 and mean number of alleles varied from 2 to 10, with overall mean of 6 alleles per locus. The mean observed and expected heterozygosity ranged from 0.058 to 0.970 and 0.102 to 0.796, respectively. The 11 microsatellite loci that were effectively amplified from A. mearnsii DNA were adequate in detecting genetic variation among the tested populations.
 Conclusions: These PCR-based, multi-allelic, co-dominant microsatellite markers provide a powerful tool for genetic, breeding and conservation studies in A. mearnsii.

Development of 17 Novel Cross-Species Microsatellite Markers for Dabry’s Sturgeon (Acipenser dabryanus) from Chinese Sturgeon (Acipenser sinensis) via Next-Generation Sequencing

Development of 17 Novel Cross-Species Microsatellite Markers for Dabry’s Sturgeon (Acipenser dabryanus) from Chinese Sturgeon (Acipenser sinensis) via Next-Generation Sequencing

Assessing introgressive hybridization between blue wildebeest (Connochaetes taurinus) and black wildebeest (Connochaetes gnou) from South Africa

Introgressive hybridization poses a threat to the genetic integrity of black wildebeest (Connochaetes gnou) and blue wildebeest (Connochaetes taurinus) populations in South Africa. Black wildebeest is endemic to South Africa and was driven to near extinction in the early 1900s due to habitat destruction, hunting pressure and disease outbreaks. Blue wildebeest on the other hand are widely distributed in southern and east Africa. In South Africa the natural distribution ranges of both species overlap, however, extensive translocation of black wildebeest outside of its normal distribution range in South Africa have led to potential hybridization between the two species. The molecular identification of pure and admixed populations is necessary to design viable and sustainable conservation strategies, since phenotypic evidence of hybridization is inconclusive after successive generations of backcrossing. The aim of this study was to assess levels of hybridization in wildebeest using both species-specific and cross-species microsatellite markers. Black wildebeest (157) and blue wildebeest (122) from provincial and national parks and private localities were included as reference material, with 180 putative hybrid animals also screened. A molecular marker panel consisting of 13 cross-species and 11 species-specific microsatellite markers was developed. We used a Bayesian clustering model to confirm the uniqueness of blue- and black wildebeest reference groups, assign individuals to each of the two clusters, and determine levels of admixture. Results indicated a clear partition between black wildebeest and blue wildebeest (the average proportions of membership to black wildebeest and blue wildebeest clusters were QI = 0.994 and QI = 0.955 respectively). From the putative hybrid samples, only five hybrid individuals were confirmed. However, high levels of linkage disequilibrium were observed in the putative hybrid populations which may indicate historical hybridization. Measures of genetic diversity in the black wildebeest populations were found to be lower than that of the blue wildebeest. The observed lower level of genetic diversity was expected due to the demographic history of the specie. This study will make a significant contribution to inform a national conservation strategy to conserve the genetic integrity of both species.

Characterization of six cross-species microsatellite markers suitable for estimating the population parameters of the black-backed jackal (Canis mesomelas) using a non-invasive genetic recovery protocol

AbstractThe black-backed jackal has proven difficult to study in the wild due to its elusive behaviour. As a rabies vector and predator of livestock, understanding dispersal patterns and population structure is of importance when considering the management of this species in South Africa’s rural areas. We have characterised a suite of six cross-species microsatellite markers suitable for this purpose and present a method to isolate suitable quantities of host DNA from faeces in order to undertake endpoint PCR. Allelic dropout and null allele frequencies were shown to be negligible for all loci. No evidence of selection could be detected at any of the loci examined. The genotype frequencies of the total sampled population were tested for concordance with Hardy–Weinberg equilibrium. No significant deficit of heterozygote individuals was detected globally when tested by locus and marker set. An excess of heterozygosity was recorded at three loci within a single study site. The total inbreeding coefficient FS...

Development of 27 novel cross-species microsatellite markers for the endangered Hucho bleekeri using next-generation sequencing technology

In this study, we developed 27 polymorphic microsatellite loci in Hucho bleekeri, a glacial relict and freshwater resident salmonid fish in China. The number of alleles varied from 2 to 8 for each primer set. The observed and expected heterozygosity ranged from 0.250 to 0.906 and 0.508 to 0.845, respectively. The polymorphic information content ranged from 0.371 to 0.808. These microsatellite loci should be useful to study population genetics, paternity identification, speciation and adaptive evolution of this lineage.

Development of novel, cross-species microsatellite markers for Acropora corals using next-generation sequencing technology

The genus Acropora (Scleractinia, Acroporidae) is one of the most widespread coral genera, comprising the largest number of extant species among scleractinian (reef-building) corals. Molecular phylogenetic studies have suggested that A. tenuis belongs to the most basal clade (clade I) while A. digitifera belongs to a derived clade (clade IV). In order to develop microsatellite markers that would be useful for most Acropora species, we sequenced the genomic DNA of A. tenuis, using a next generation sequencer (Illumina MiSeq), and designed primer sets that amplify microsatellite loci. Afterward we selected primer pairs with perfectly matched nucleotide sequences from which at least one primer was uniquely mapped to the A. digitifera genome. Fourteen microsatellite markers showed non-significant departure from Hardy–Weinberg equilibrium (HWE) in both A. tenuis and A. digitifera. Thus these markers could be used for wide range of species and may provide powerful tools for population genetics studies and conservation of Acropora corals.

Novel and cross-species microsatellite markers for parentage analysis in Sanderling Calidris alba

We isolated and tested six novel microsatellite loci in Sanderling (Calidris alba) from Greenland for paternity analyses. In addition, we tested 11 already pub- lished microsatellite markers which were originally developed for the congeneric species, the Pectoral Sand- piper (C. melanotos). All loci were polymorphic, but five of the cross-species loci were not scorable due to suboptimal amplification patterns. The 12 successful loci were tested on 87 individuals, yielding an average of 9.0 (range 4-19) alleles per locus and mean expected heterozygosity of 0.70. Because this dataset contained families, tests for Hardy-Weinberg equilibrium, linkage disequilibrium and probability of identity were done on a subset of the data containing 25 adults caught in the same year. The overall probability of identity was 1.0 9 10 -13 . Only one locus displayed significant homozygote excess and all loci were unlinked. On the basis of female heterozygotes, all loci are assumed to be autosomal.

Population genetic structure of wild and hatchery black rockfish Sebastes inermis in Korea, assessed using cross-species microsatellite markers

The population structure of the black rockfish, Sebastes inermis (Sebastidae), was estimated using 10 microsatellite loci developed for S. schlegeli on samples of 174 individuals collected from three wild and three hatchery populations in Korea. Reduced genetic variation was detected in hatchery strains [overall number of alleles (N(A)) = 8.07; allelic richness (A(R)) = 7.37; observed heterozygosity (H(O)) = 0.641] compared with the wild samples (overall N(A) = 8.43; A(R) = 7.83; H(O) = 0.670), but the difference was not significant. Genetic differentiation among the populations was significant (overall F(ST) = 0.0237, P < 0.05). Pairwise F(ST) tests, neighbor-joining tree, and principal component analyses showed significant genetic heterogeneity among the hatchery strains and between wild and hatchery strains, but not among the wild populations, indicating high levels of gene flow along the southern coast of Korea, even though the black rockfish is a benthic, non-migratory marine species. Genetic differentiation among the hatchery strains could reflect genetic drift due to intensive breeding practices. Thus, in the interests of optimal resource management, genetic variation should be monitored and inbreeding controlled within stocks in commercial breeding programs. Information on genetic population structure based on cross-species microsatellite markers can aid in the proper management of S. inermis populations.

Mining online genomic resources inAnolis carolinensisfacilitates rapid and inexpensive development of cross‐species microsatellite markers for theAnolislizard genus

Online sequence databases can provide valuable resources for the development of cross-species genetic markers. In particular, mining expressed tag sequences (EST) for microsatellites and developing conserved cross-species microsatellite markers can provide a rapid and relatively inexpensive method to develop new markers for a range of species. Here, we adopt this approach to develop cross-species microsatellite markers in Anolis lizards, which is a model genus in evolutionary biology and ecology. Using EST sequences from Anolis carolinensis, we identified 127 microsatellites that satisfied our criteria, and tested 49 of these in five species of Anolis (carolinensis, distichus, apletophallus, porcatus and sagrei). We identified between 8 and 25 new variable genetic markers for five Anolis species. These markers will be a valuable resource for studies of population genetics, comparative mapping, mating systems, behavioural ecology and adaptive radiations in this diverse lineage.

Comparative genetic diversity of wild and released populations of Pacific abalone Haliotis discus discus in Jeju, Korea, based on cross-species microsatellite markers including two novel loci

Pacific abalone Haliotis discus discus is an important fisheries resource in Jeju, Korea. For basic information about its current genetic status in relation to stock enhancement, the level and distribution of genetic variation between wild and released stocks of Pacific abalone in Jeju were examined at nine cross-species microsatellite markers including the use of two novel primers. High levels of polymorphism were observed between the two populations. A total of 146 different alleles were found at all loci, with some alleles being unique. The allelic variability ranged from five to 27 in the wild population and from four to 16 in the released sample. The average observed and expected heterozygosities were estimated to be 0.74 and 0.84 in the wild sample and 0.70 and 0.78 in the released sample, respectively. Although a considerable loss of rare alleles was observed in the released sample, no statistically significant reductions were found in heterozygosity or allelic diversity in the released sample compared to the wild population. Low but significant genetic differentiation was found between the wild and released populations. These results suggest that the intensive breeding practices for stock enhancement may have resulted in a further decrease in genetic diversity, and that the cross-species microsatellite markers used in this study represent a potentially efficient means for further genetic studies, providing beneficial information for the protection and management of H. discus discus.

Comparative Genetic Diversity of Wild and Captive Populations of the Bare-Faced Curassow (Crax fasciolata) Based on Cross-Species Microsatellite Markers: Implications for Conservation and Management

The bare-faced curassow (Crax fasciolata) is a large Neotropical bird that suffers anthropogenic pressure across much of its range. A captive population is maintained for conservation management, although there has been no genetic screening of stocks. Based on the six microsatellite markers developed for Crax globulosa, the genetic variability of C. fasciolata and possible differences between a wild and a captive population were investigated. Only three loci were polymorphic, with a total of 27 alleles. More than half of these alleles were private to the wild (n = 8) or captive (n = 7) populations. Significant deviations from Hardy-Weinberg equilibrium were restricted to the captive population. Despite the number of private alleles, genetic drift has probably promoted differentiation between populations. Our results indicate that wild C. fasciolata populations are genetically impoverished and structured, but species-specific microsatellite markers will be necessary for a more reliable assessment of the species' genetic diversity.

Conservation of Human Microsatellites across 450 Million Years of Evolution

The sequencing and comparison of vertebrate genomes have enabled the identification of widely conserved genomic elements. Chief among these are genes and cis-regulatory regions, which are often under selective constraints that promote their retention in related organisms. The conservation of elements that either lack function or whose functions are yet to be ascribed has been relatively little investigated. In particular, microsatellites, a class of highly polymorphic repetitive sequences considered by most to be neutrally evolving junk DNA that is too labile to be maintained in distant species, have not been comprehensively studied in a comparative genomic framework. Here, we used the UCSC alignment of the human genome against those of 11 mammalian and five nonmammalian vertebrates to identify and examine the extent of conservation of human microsatellites in vertebrate genomes. Out of 696,016 microsatellites found in human sequences, 85.39% were conserved in at least one other species, whereas 28.65% and 5.98% were found in at least one and three nonprimate species, respectively. An exponential decline of microsatellite conservation with increasing evolutionary time, a comparable distribution of conserved versus nonconserved microsatellites in the human genome, and a positive correlation between microsatellite conservation and overall sequence conservation, all suggest that most microsatellites are only maintained in genomes by chance, although exceptionally conserved human microsatellites were also found in distant mammals and other vertebrates. Our findings provide the first comprehensive survey of microsatellite conservation across deep evolutionary timescales, in this case 450 Myr of vertebrate evolution, and provide new tools for the identification of functional conserved microsatellites, the development of cross-species microsatellite markers and the study of microsatellite evolution above the species level.