Polymorphic Microsatellite Loci Research Articles

Development and Characterization of Fifteen Polymorphic Microsatellite Loci for Rare and Endangered Species within Luciobarbus Heckel, 1843 Genus in the Aral Basin and Their Conservation Application

Biodiversity conservation entails not only the preservation of specific taxa but also genetic diversity. Despite the crucial role of molecular data in freshwater fish conservation management, there is a scarcity of information regarding the genetic diversity of Luciobarbus Heckel, 1843 (Actinopterygii, Cyprinidae) populations in the Aral system. Therefore, the primary aim of this study was to provide genetic information on two native species of the Luciobarbus genus found in the Aral system: L. conocephalus (Kessler, 1872) and L. brachycephalus (Kessler, 1872). These species, like many others in the Aral system, confront the imminent threat of extinction due to system alterations. However, genetic studies on these species at the nuclear level are challenging because Luciobarbus is an allotetraploid genus. Consequently, genetic investigations thus far have focused mainly on sequencing mitochondrial genes due to their haploid nature. This study has successfully developed fifteen new polymorphic microsatellite loci, which can prove to be valuable for population genetics, conservation, and other pertinent research on these species.

Polyandry and sperm competition in two traumatically inseminating species of Strepsiptera (Insecta)

Polyandry, the practice of females mating with multiple males, is a strategy found in many insect groups. Whether it increases the likelihood of receiving beneficial genes from male partners and other potential benefits for females is controversial. Strepsiptera are generally considered monandrous, but in a few species females have been observed copulating serially with multiple males. Here we show that the offspring of a single female can have multiple fathers in two Strepsiptera species: Stylops ovinae (Stylopidae) and Xenos vesparum (Xenidae). We studied female polyandry in natural populations of these two species by analysis of polymorphic microsatellite loci. Our results showed that several fathers can be involved in both species, in some cases up to four. Mating experiments with S. ovinae have shown that the first male to mates with a given female contributes to a higher percentage of the offspring than subsequent males. In X. vesparum, however, we found no significant correlation between mating duration and offspring contribution. The prolonged copulation observed in S. ovinae may have the advantage of reducing competition with sperm from other males. Our results show that monandry may not be the general pattern of reproduction in the insect order Strepsiptera.

Same landscape, different connectivity: contrasting patterns of gene flow in two sympatric ungulates in a mountain area

Comparative landscape genetics studies provide insights on the impact of landscape elements on gene flow patterns of different species inhabiting the same geographic area. We investigated the population genetic structure of two sympatric ungulates, roe deer Capreolus capreolus and Northern chamois Rupicapra rupicapra, in a mountain area of the central Italian Alps (Trentino, northern Italy). A total of 122 chamois and 72 roe deer samples were genotyped by two species-specific panels of 11 polymorphic microsatellite loci and analyzed by aspatial and spatially explicit analyses. While the roe deer population resulted unstructured, a clear population structure was detected in chamois, with two main groups, one inhabiting the eastern and the other spread in the western part of the study area. Landscape genetics analysis confirmed these scenarios and revealed a different effect of landscape on gene flow. An IBD (Isolation-By-Distance) model best explained genetic variation in roe deer, while IBR (Isolation-By-Resistance) was found as the process underlying genetic variation patterns in chamois, suggesting arable lands, coniferous forests, watercourses, and main roads as potential barriers. Species distribution and landscape use might explain these results: roe deer mostly occupy valley floors relatively connected to each other, and their spatial behavior may promote gene flow across areas. On the other hand, chamois prefer higher elevations and their movements may be hindered by valleys, rivers, and road networks. This study highlights the different impacts of natural and anthropic landscape elements on gene flow in two sympatric species, resulting from their different ecological requirements.

Development and characterization of nuclear microsatellite markers for the African walnut Coula edulis Baill (Coulaceae).

Coula edulis Baill (Coulaceae) is a common tree species in the Guineo-Congolian forests producing an edible fruit known as African walnut, which is an important food and income resource for rural populations. However, the species suffers from a deficit of natural regeneration. We developed here nuclear microsatellite markers for C. edulis to be able to study the genetic structure of its natural populations and gene flow. A genomic library was obtained using the Illumina platform, and 21 polymorphic microsatellite loci were developed. The polymorphic microsatellites displayed eight to 22 alleles per locus (average: 14.2), with a mean expected heterozygosity ranging from 0.33 to 0.72 in five populations from Central and West Africa. The high polymorphism of the nuclear microsatellite markers developed makes them useful to investigate gene flow and the organization of genetic diversity in C. edulis, and to assess whether particular genetic resources require conservation efforts.

Dig-up Primers: A Pipeline for Identification of Polymorphic Microsatellites Loci within Assemblies of Related Species.

Simple sequence repeats (SSRs) have become one of the most popular molecular markers and are used in numerous fields, including conservation genetics, population genetic studies, and genetic mapping. Advances in next-generation sequencing technology and the growing amount of genomic data are driving the development of bioinformatics tools for SSR marker design. These tools work with different combinations of input data, which can be raw reads or assemblies, and with one or more input datasets. We present here a new strategy and implementation of a simple standalone pipeline that utilizes more than one assembly for the in silico design of PCR primers for microsatellite loci in more than one species. Primers are tested in silico to determine if they are polymorphic, eliminating the need to test time-consuming cross-species amplification in the laboratory. The end result is a set of markers that are in silico polymorphic in all analyzed species and have great potential for the identification of interspecies hybrids. The efficiency of the tool is demonstrated using two examples at different taxonomic levels and with different numbers of input assemblies to generate promising, high-quality SSR markers.

Genetic characterization of juvenile chum salmon with otolith marks of Japanese salmon hatcheries in the catches of trawl surveys 2016–2021

Chum salmon sample from the catches of autumn trawl surveys in the Sea of Okhotsk (2016–2021), including individuals with otolith marks of Japanese salmon hatcheries (SHs), was analyzed based on microsatellite loci polymorphism. Reference database of eight microsatellite loci frequencies, created before and included 26 samples of chum salmon from major reproductive regions in the Sea of Okhotsk basin, has been updated with data on the microsatellite allele variety of chum salmon from Hokkaido and Honshu. Result s of the research indicate of strong similarity between chum salmon of the Kuril Islands and individuals released from Japanese SHs.

Polymorphism of Microsatellite Loci in Populations of Caucasian Rock Lizards and Its Use for Assessing the Genetic Diversity of Darevskia raddei

Polymorphism of Microsatellite Loci in Populations of Caucasian Rock Lizards and Its Use for Assessing the Genetic Diversity of Darevskia raddei

Emerging patterns of genetic diversity in the critically endangered Malayan tiger (Panthera tigris jacksoni)

Southeast Asia experiences some of the highest deforestation in the world. Loss of tropical forest typically leads to widespread habitat fragmentation, with detrimental effects on dispersal ability and gene flow—particularly for large carnivores. We conducted mtDNA and microsatellite analysis to assess—for the first time—contemporary patterns of genetic diversity in the Malayan tiger. We collected 295 suspected carnivore samples in Peninsular Malaysia, from which we identified 26 as originating from tiger using 16 polymorphic microsatellite loci, comprising 22 individual tigers. Despite limitations of the study, our findings suggest tiger subpopulations in the north of the peninsula maintain some genetic connectivity and migration between two putative geographic subpopulations in the Main Range and Greater Taman Negara, with negligible population segregation due to dispersal barriers such as road infrastructure. We identified consistently lower levels of genetic diversity in tigers in the Greater Taman Negara region compared to tigers in the Main Range and small but emerging differences in nuclear and mitochondrial genetic diversity. Our mtDNA haplotype and nuclear DNA analyses suggest the levels of genetic diversity in Malayan tigers may be amongst some of the lowest of the surviving tiger subspecies, though the study is limited both in scale and genomic loci. Our findings are consistent with an expected lag between the rapid decline of tigers in Peninsular Malaysia by over 95% in the last 70 years and observed differences in their levels of genetic diversity.

Establishment of a Microfluidic Chip Based Rapid Detection Method for <i>Wasmannia auropunctata</i>

Invasive alien ants are one of the most aggressive, competitive, and widespread invasive alien species around the world. <i>Wasmannia auropunctata, </i>native to the neotropical zoogeographic area, has been inadvertently introduced all over the world. The first disclosed record of field establishment of <i>W. auropunctata</i> in Chinese mainland was reported in 2022. In this paper, we aim to establish a rapid detection method targeting the <i>W. auropunctata </i>polymorphic microsatellite locus Waur-275, based on the microfluidic chip technology, to contribute to an active response to the crisis caused by this invasive pest. Eight primer sets were generated using the PrimerExplorer v5 online service, and the fifth primer set selected had the lowest Ct in the volume ratio of 8 (FIP/BIP:F3/B3). Method validity with six <i>W. auropunctata </i>samples confirmed the efficiency of this assay. High specificity was demonstrated by the positive result for <i>W. auropunctata</i> out of 6 related insect samples tested. Sensitivity analysis showed that this microfluidic chip method could achieve the detection limit of 1.00×10<sup>1</sup> copies/μL. A rapid screening method is essential for the whole process of interception and control of <i>W. auropunctata </i>in China. It can also help determine the origin of invasion, clarify the path of introduction to ensure biosecurity, and also uncover other possible undetected establishments in the wild.

Isolation and characterization of polymorphic microsatellite loci for the three Iberian vipers, Vipera aspis, V. latastei and V. seoanei by Illumina MiSeq sequencing

BackgroundEuropean vipers (genus Vipera) are a well-studied taxonomic group, but the low resolution of nuclear sanger-sequenced regions has precluded thorough studies at systematic, ecological, evolutionary and conservation levels. In this study, we developed novel microsatellite markers for the three Iberian vipers, Vipera aspis, V. latastei and V. seoanei, and assessed their polymorphism in north-central Iberian populations.Methods and resultsGenomic libraries were developed for each species using an Illumina Miseq sequencing approach. From the 70 primer pairs initially tested, 48 amplified reliably and were polymorphic within species. Cross-species transferability was achieved for 31 microsatellites loci in the three target species and four additional loci that were transferable to one species only. The 48 loci amplified in average seven alleles, and detected average expected and observed heterozygosities of 0.7 and 0.55, in the three genotyped populations/species (26 V. aspis, 20 V. latastei and 10 V. seoanei).ConclusionsOur study provides a selection of 48 polymorphic microsatellite markers that will contribute significantly to current knowledge on genetic diversity, gene flow, population structure, demographic dynamics, systematics, reproduction and heritability in these species, and potentially in other congeneric taxa.

Isolation and characterisation of 17 microsatellite DNA loci from RAD reduced-representation genomes for Asian warty newts, genus Paramesotriton (Caudata: Salamandridae).

Asian warty newts, genus Paramesotriton, are endemic to southern China and northern Vietnam. Despite the achievements in biodiversity, molecular systematics and biogeography of species in this genus, population genetic diversity studies are lacking due to the lack of economical and available genetic markers. In this study, we developed 17 highly polymorphic microsatellite loci from RAD simplified genomic data for the Asian warty newts, genus Paramesotriton and successfully completed cross-species amplification tests on 20 samples of four species of Paramesotriton. These microsatellite markers can be used as important tools to study population genetic structure, levels of gene flow, population differentiation, mating systems and landscape genetics within the genus Paramesotriton and, thus, to make scientific conservation decisions and actions for the conservation of these rare and endangered amphibians.

Forest fragmentation causes an isolated population of the golden takin (Budorcas taxicolor bedfordi Thomas, 1911) (Artiodactyla: Bovidae) in the Qinling Mountains (China)

Budorcas taxicolor bedfordi is a rare animal uniquely distributed in the Qinling Mountains (China). Human disturbance and habitat fragmentation have directly affected the survival of B. t. bedfordi. It is urgent to clarify the genetic diversity and genetic structure of the B. t. bedfordi population and implement effective conservation measures. In this study, 20 new polymorphic microsatellite loci were isolated by Illumina sequencing. The genetic diversity and population structure of 124 B. t. bedfordi individuals from three populations (Niubeliang population, Zhouzhi population, and Foping population) were analysed according to these 20 microsatellite loci. Our results indicated that B. t. bedfordi had a low level of genetic variability and that there was inbreeding in the three populations. The population genetic structure analyses showed that the Niubeliang population had a trend of differentiation from other populations. National roads can affect population dispersal, while ecological corridors can promote population gene exchange. None of the three B. t. bedfordi populations experienced bottleneck effects. For conservation management plans, the Zhouzhi population and Foping population should be considered one management unit, and the Niubeliang population should be considered another management unit. We suggest building an ecological corridor to keep the habitat connected and formulating tourism management measures to reduce the influence of human disturbance on B. t. bedfordi.

Genetic structure of California's elk: a legacy of extirpations, reintroductions, population expansions, and admixture

AbstractCalifornia, USA, is home to 3 subspecies of North American elk (Cervus canadensis): Roosevelt (C. c. roosevelti), Rocky Mountain (C. c. nelsoni), and tule (C. c. nannodes). Effective management requires a baseline understanding of each subspecies' range, admixture zones, and geographic patterns of genetic diversity. To address these questions, we genotyped 1,271 individual elk from California (n = 1,204) and reference populations of Rocky Mountain and Roosevelt elk from Nevada (n = 32) and Oregon (n = 35), USA. Using 19 polymorphic microsatellite loci, we detected admixture between Roosevelt and Rocky Mountain elk at a contact zone in northern California, and between Roosevelt and tule elk in north‐coastal California and central‐coastal California. We identified a genetically distinct population of Roosevelt elk in northwestern California, likely reflecting the remnant population that survived a large demographic decline from overhunting during the 1800s. Tule elk exhibited lower levels of heterozygosity (0.44 ± 0.03 [SD]) and allelic richness (2.9 ± 0.2) than Rocky Mountain (0.58 ± 0.05, 4.9 ± 0.4, respectively) and Roosevelt (0.50 ± 0.06, 4.4 ± 0.6, respectively) elk. Among tule elk populations, heterozygosity varied, with the lowest heterozygosity (0.23 ± 0.05) corresponding to the oldest enclosed herd used over the past century as a source of translocations. Among tule elk populations, genetic structure revealed several cases of successful and unsuccessful reintroduction or augmentation attempts. Results provide an essential baseline for future monitoring and decisions about harvest management, translocations to preserve genetic diversity, and landscape‐level conservation planning to maintain, enhance, or obstruct connectivity of elk populations. Genome‐wide sequencing and analyses are needed to quantify inbreeding absolutely and assess genetic load and the age of admixture where subspecies currently exchange genes.

Genetic Variability of Mass-Selected and Wild Populations of Yellow Drum (Nibea albiflora) Revealed Using Microsatellites

In this study, twelve polymorphic microsatellite loci were screened to evaluate the genetic diversity of five yellow drum (Nibea albiflora) populations in the Zhoushan Sea region of the East China Sea, including one wild population (WP), one artificially propagated population (common population, CP), and three breeding populations (parent population, F4; all-female population, AF; and neo-male population, NeoG). The results of genetic diversity analyses showed that all five yellow drum populations had relatively high genetic diversity, with the highest in WP and the lowest in NeoG. Genetic structure analyses showed that the level of genetic differentiation among populations was low, with that between CP and F4 being the largest, whereas that between CP and WP was the smallest. Mutation–drift equilibrium analysis showed that the five populations likely did not experience a recent bottleneck. Our results suggest the CP population was the most suitable for large-scale release for stock enhancement, and precautionary measures shall be taken for the AF population before it is used for cage culture to avoid potential genetic concerns of the wild population. Nevertheless, further genetic diversity monitoring is needed to evaluate genetic effects and avoid the negative impact of excessive genetic differentiation between breeding and wild populations.

Microsatellites reveal divergence in population genetic diversity, and structure of osyris lanceolata (santalaceae) in Uganda and Kenya

BackgroundOsyris lanceolata (Hochst. & Steud.) (Santalaceae) is a multipurpose plant highly valued culturally and economically in Africa. However, O. lanceolata populations have rapidly dwindled in East Africa due to overexploitation and this is believed to cause further consequences on the species’ genetic diversity and structure within the region. Information regarding a species’ genetic diversity and structure is necessary for conservation but this is currently lacking for O. lanceolata in Uganda and Kenya. Lack of adequate scientific data hinders conservation efforts hence threatening the species survival and livelihoods. This study investigated patterns in genetic diversity and structure of O. lanceolata in Uganda and Kenya. Ten polymorphic microsatellite loci were used to genotype 210 individuals: 96 from Ugandan and 114 from Kenyan populations.ResultsAll populations were highly polymorphic (80–100% polymorphism). A genetic differentiation was found between Kenyan and Ugandan populations. The highest genetic differentiation was among individuals and the least among populations. The Kenyan populations showed higher genetic diversity than Ugandan populations. The Ugandan populations showed more marker deviations from Hardy-Weinberg equilibrium and inbreeding coefficient. Two populations showed evidence of going through a recent bottleneck. There was significant genetic differentiation and structuring at higher K values into larger clusters and observed admixture between populations. The populations were significantly isolated by altitude as opposed to distance and climatic variables. Main barriers were associated with altitude differences. The data supports the idea of long-distance gene-flow between high altitude populations in both countries.ConclusionThe divergence in genetic structure suggests unrecognised taxonomic units within O. lanceolata which are characteristic to lower altitudes and higher altitudes including most Kenyan populations with divergent evolutionary patterns. Geographical barriers and environmental gradients could have influenced this genetic divergence, and such patterns may escalate the species microevolutionary processes into full allopatric speciation. Further investigations into the species’ genetic admixture and emerging taxonomic units are necessary to guide conservation strategies in the region.

A Multiplex PCR System of Novel Microsatellite Loci for Population Genetic Application in Walnuts.

Multiplex polymerase chain reaction (PCR) of microsatellite loci allows for simultaneous amplification of two or more pairs of primers in a single PCR reaction; hence, it is cost and time effective. However, very few attempts have been reported in non-model species. In this study, by combining a genome-based de novo development and cross-species application approach, a multiplex PCR system comprising 5 PCR reactions of 33 microsatellites consisting of 26 novel genomic and 7 literature-sourced loci was tested for polymorphisms, cross-species transferability, and the ability to assess genetic diversity and population structure of three walnut species (Juglans spp.). We found that the genome-based approach is more efficient than other methods. An allelic ladder was developed for each locus to enhance consistent genotyping among laboratories. The population genetic analysis results showed that all 33 loci were successfully transferred across the three species, showing high polymorphism and a strong genetic structure. Hence, the multiplex PCR system is highly applicable in walnut species. Furthermore, we propose an efficient pipeline to characterize and genotype polymorphic microsatellite loci. The novel toolbox developed here will aid future ecology and evolution studies in walnut and could serve as a model for other plant species.

Weak Population Genetic Structure of the Eurasian Crane <i>Grus grus</i> L.

The article presents an extended (as compared with our previous studies) analysis of the population genetic structure of a migratory wide-range bird species with a high abundance, the Eurasian Crane Grus grus L. Using seven highly polymorphic microsatellite loci, high and very similar values of genetic diversity parameters were obtained in the samples of both the western (G. g. grus) and eastern (G. g. lilfordi) subspecies. Coefficients of genetic differentiation between these subspecies (FST = 0.008, GST = 0.002) were also found to be low. According to AMOVA, 99% of genetic variation of G. grus is attributed to individual levels. Using the Bayesian clustering algorithm implemented in STRUCTURE software, no clear population-genetic structuring of the species was revealed. However, visualization of spatial patterns of genetic variability in Geneland showed the presence of a cluster of “pure” subspecies G. g. grus and G. g. lilfordi surrounding a cluster of individuals from the zone of intergradation of these subspecies. This result, along with the observed very low FST values, may indicate subtle genetic differences between cranes from the studied area, possibly having a mutational nature. The lower allelic richness and lack of private alleles in the eastern subspecies suggest that G. g. lilfordi is relatively evolutionary young and suggest its recent origin from some eastern marginal populations of the nominative western subspecies.

Development of a novel molecular tool to study molecular ecology of Ornithomya (Hippoboscidae) avian louse flies

Louse flies (Diptera: Hippoboscidae) are obligatory hematophagous ectoparasites of birds and mammals. These widely distributed parasitic flies may have a significant impact on wild and farm animals by feeding on their blood and transmitting bloodborne pathogens. However, despite their ecological importance, louse flies are clearly underrepresented in host-parasite research and implementation of genetic approaches in this group is generally hampered by lacking molecular tools. In addition, louse flies that parasitize long-distance migrants can travel long distances with their avian hosts, facilitating the large-scale spread of pathogens across landscapes and geographic regions. Given the wide diversity of louse flies that parasitize a variety of avian hosts, their direct negative impact on host survival, and their high potential to transmit bloodborne pathogens even along avian migration routes, it is surprising that our knowledge of louse fly ecology is rather modest and incomplete. Here, we aimed to develop a novel molecular tool for polyxenous avian louse flies from the genus Ornithomya, which are among the most common and widely distributed representatives of Hippoboscidae family, to improve research of their genetic population structure and molecular ecology.Using the Illumina Mi-seq sequencing, we conducted a genome-wide scan in Ornithomya avicularia to identify putative microsatellite markers. A panel of 26 markers was selected to develop amplification protocols and assess polymorphism in the Central European population of O. avicularia, as well as to test for cross-amplification in a congeneric species (O. chloropus).A genome-scan in O. avicularia identified over 12 thousand putative microsatellite markers. Among 26 markers selected for a population-wide screening; one did not amplify successfully and three were monomorphic. 22 markers were polymorphic with at least two alleles detected. Two markers showed presence of null alleles. A cross-amplification of microsatellite markers in O. chloropus revealed allelic polymorphism at 14 loci, with the mean allelic richness of 3.78 alleles per locus (range: 2–8).Our genome-wide scan in O. avicularia provides a novel and powerful tool for molecular research in Ornithomya louse flies. Our panel of polymorphic microsatellite loci should allow genotyping of louse flies from geographically distinct populations and from a wide spectrum of avian hosts, enhancing population genetic and phylogeographic research in Ornithomya.

Temporal population genetic structure of Phormia regina (Diptera: Calliphoridae).

The genetic structure of forensically important blow fly (Brauer & Bergenstamm) (Diptera: Calliphoridae) populations has remained elusive despite high relatedness within wild-caught samples. This research aimed to determine if the implementation of a high-resolution spatiotemporal sampling design would reveal latent genetic structure among blow fly populations and to elucidate any environmental impacts on observed patterns of genetic structure. Adult females of the black blow fly, Phormia regina (Meigen) (Diptera: Calliphoridae), were collected from 9 urban parks in Indiana, USA over 3 yr and genotyped at 6 polymorphic microsatellite loci. The data analysis involved 3 clustering methods: principal coordinate analysis (PCoA), discriminant analysis of principal components (DAPC), and STRUCTURE. While the PCoA did not uncover any discernible clustering patterns, the DAPC and STRUCTURE analyses yielded significant results, with 9 and 4 genetic clusters, respectively. Visualization of the STRUCTURE bar plot revealed N = 11 temporal demarcations indicating barriers to gene flow. An analysis of molecular variance of these STRUCTURE-inferred populations supported strong temporally driven genetic differentiation (FST = 0.048, F'ST = 0.664) relative to geographic differentiation (FST = 0.009, F'ST = 0.241). Integrated Nested Laplace Approximation and Boosted Regression Tree analyses revealed that collection timepoint and 4 main abiotic factors (temperature, humidity, precipitation, and wind speed) were associated with the genetic subdivisions observed for P. regina. A complex interplay between environmental conditions, the unique reproductive strategies of the blow fly, and the extensive dispersal abilities of these organisms likely drives the strong genetic structure of P. regina in the Midwestern US.

Genetic evidence for variability in the social mating system of the Midday Gerbil (Meriones meridianus)

Abstract Social mating systems (e.g., monogamy, polygamy, or polyandry) are relatively stable behavioral strategies developed by environmental in animals, but the genetic imprint of a particular mating system is often incongruent with the social mating system due to extrapair matings. However, the genetics of mating systems remain little understood in rodents. In this study, we investigated the genetic signature of the mating system of 141 (63 females, 78 males) field-captured midday gerbils (Meriones meridianus)—a rodent species commonly found in the Minqin Desert in China—through microsatellite site analyses of genetic structure and relatedness. Seven pairs of highly polymorphic microsatellite loci were selected and were highly polymorphic, with the combined exclusion probability greater than 0.99. The parent pair paternity test by Cervus 3.0 software show that 11 mother–offspring and nine father–offspring relationships were identified in 2018, involving 26 individuals from 10 families. Similarly, 19 mother–offspring and 19 father–offspring relationships were identified in 2019, involving 48 individuals from 18 families. All three types of genetic mating structure were identified—monogamy (19 families), polyandry (4 families), and polygyny (5 families), providing evidence that the genetics underlying mating systems in this species are variable, can be incongruent with behavioral evidence for social mating systems, and could vary based on environmental cues, including degree of perceived or actual predation.