Set Of Microsatellite Markers Research Articles

Introduced Herbivores Threaten the Conservation Genetics of Two Critically Endangered Single-Island Endemics, Crambe sventenii and Pleudia herbanica.

Crambe sventenii Pett. ex Bramwell & Sunding and Pleudia herbanica (A.Santos & M.Fernández) M.Will, N.Schmalz & Class.-Bockh. are two single-island endemic species from Fuerteventura (Canary Islands), inhabiting the same areas and similar habitats. They are under the "Critically Endangered" category due to historical herbivore pressure, mainly goats, leading to habitat fragmentation and poor population recruitment. The main aim of our study was to provide insights into the conservation genetics and habitat suitability of these two species. For this purpose, we sampled all known populations on the island and developed two new sets of microsatellite markers. Moreover, to assist restoration plans, we performed species distribution models to determine the most suitable areas for reintroduction. While Crambe sventenii is highly fragmented, with low genetic diversity indices in some populations, Pleudia herbanica's genetic structure is quite homogeneous, grouped in three main regions, with signs of inbreeding and an overall low genetic diversity. Both species could present moderate to high levels of autogamy. Our findings can provide guidance to local governments regarding conservation actions to be implemented in the field, like the identification of propagule sources and new suitable areas for restoration.

Population genetic structure of the endemic sponge Lubomirskia baikalensis in Lake Baikal in the light of mass disease and mortality events

Abstract The study of the state of Baikal endemic sponge populations is of great interest because of the occurrence of mass mortalities and disease in recent decades. To identify possible signs of species vulnerability to extinction, it is crucial to develop appropriate genetic markers that help developing measures for conservation. In this paper, we describe the population genetic structure of the Baikal endemic sponge Lubomirskia baikalensis examined with the set of microsatellite markers we developed. We analysed 251 samples from eight locations that cover all three basins of Lake Baikal. A genetic subdivision into three clusters was revealed. Such a structure can be explained mainly by the low ability of larvae to disperse. Despite the presence of dead and diseased individuals in all studied locations, all populations were in Hardy–Weinberg equilibrium and no bottleneck effect was found at all. This is the first time that a genetic connectivity study has been performed for L. baikalensis, a species endemic of Lake Baikal. Reconstruction of the changes in the effective population size agrees with the results obtained during drill sample analysis and it demonstrates that the effective population size was 55.5 times lower about 24,000 years ago, which indicates that apparently there is no threat of extinction of the Baikal endemic sponges at present.

The Development of a Fluorescent Microsatellite Marker Assay for the Pitaya Canker Pathogen (Neoscytalidium dimidiatum).

Pitaya canker, caused by Neoscytalidium dimidiatum, is a destructive disease that significantly threatens the safety of the pitaya industry. The authors of previous studies have mainly focused on its biological characteristics and chemical control. However, there are no molecular markers available thus far that can be used for the population genetics study of this pathogen. In the present study, a draft genome of N. dimidiatum with a total length of 41.46 MB was assembled in which 9863 coding genes were predicted and annotated. In particular, the microsatellite sequences in the draft genome were investigated. To improve the successful screening rate of potentially polymorphic microsatellite makers, another five N. dimidiatum isolates were resequenced and assembled. A total of eight pairs of polymorphic microsatellite primers were screened out based on the polymorphic microsatellite loci after investigating the sequencing and resequencing assemblies of the six isolates. A total of thirteen representative isolates sampled from different pitaya plantations were genotyped in order to validate the polymorphism of the resulting eight markers. The results indicated that these markers were able to distinguish the isolates well. Lastly, a neighbor-joining tree of 35 isolates, sampled from different pitaya plantations located in different regions, was constructed according to the genotypes of the eight molecular markers. The developed tree indicated that these molecular markers had sufficient genotyping capabilities for our test panel of isolates. In summary, we developed a set of polymorphic microsatellite markers in the following study that can effectively genotype and distinguish N. dimidiatum isolates and be utilized in the population genetics study of N. dimidiatum.

Environmental effects on faecal genotyping success in mesocarnivores

Non-invasive genetic sampling can be used in research, monitoring, and conservation of wild animals to, for example, provide insights into diets, identify individuals and estimate population sizes. Non-invasive genetic sampling may be especially useful to monitor elusive species because DNA can be derived from materials such as hairs and faeces without handling individuals. However, the reliability of the results derived from this technique is dependent on the quality of DNA obtained from samples, which can deteriorate from exposure to environmental conditions and sample age. While freshness of the sample is an important factor, the combined effect of different field conditions on the genotyping success is not fully understood. To address this gap, we systematically investigated the effects of sample age and environmental conditions on genotyping success of faeces, in an experimental setting of four treatments that combined rain and temperature conditions typical of central European climates. We compared the performance of a microsatellite marker set and a SNP panel for red fox (Vulpes vulpes), as well as a microsatellite marker set for pine marten (Martes martes) from faeces resampled over 21 days. We found that genotyping success decreased significantly with sample age, however environmental treatments did not impact the success. Furthermore, the SNPs we used amplified more successfully over time than the respective microsatellites. Therefore, the use of SNPs instead of conventional microsatellites, when using faecal samples for analysis relying on correct amplifications, could be advantageous. We recommend to prioritise the collection of fresh faeces regardless of environmental conditions.

Unveiling the genetic structure of pig population in a Himalayan state Uttarakhand through microsatellite and mitochondrial DNA analyses.

This study traced the maternal lineage of the domestic swine populations using mitochondrial DNA control region markers and genetic diversity using microsatellite markers in Uttarakhand, an Indian state situated at the foothills of the world's youngest (geo-dynamically sensitive) mountain system, "the Himalayas". Analysis of 68 maternally unrelated individuals revealed 20 haplotypes. The maternal signature of the Pacific, Southeast Asian, European, and ubiquitously distributed Chinese haplotypes was present in Uttarakhand's domestic pig population. The D3 haplotype reported in wild pigs from North India was also identified in 47 domestic samples. A unique gene pool, UKD (Uttarakhand Domestic), as another lineage specific to this region has been proposed. Genotypes were analyzed, using 13 sets of microsatellite markers. The observed (Ho) and expected (He) heterozygosities were 0.83 ± 0.02 and 0.84 ± 0.01, respectively. The average polymorphic information content value of 0.83 ± 0.01 indicated the high informativeness of the marker. The overall mean FIS value for all the microsatellite markers was low (F = 0.04, P < 0.01). Seven loci deviated from Hardy-Weinberg equilibrium (HWE) at a significant level (p < 0.05). Two clusters were identified, indicating overlapping populations. These results suggested that though belonging to different maternal lineages, the traditional management practices in Uttarakhand have allowed for genetic mixing and the sharing of genetic material among pig populations. It could contribute to increased genetic diversity but might also result in the loss of distinct genetic characteristics or breed purity of the local breeds if not carefully managed.

Identification of microsatellite markers for the invasive freshwater mussel Sinanodonta lauta (E. von Martens, 1877) (Bivalvia: Unionida)

The Far Eastern freshwater mussel Sinanodonta lauta has recently been recorded in European Russia outside of its native range. As an invasive species affecting native ecosystems, this mussel is still poorly investigated in many aspects, including population genetics. In this study, we describe for the first time eight microsatellite loci that were developed based on a previously published set of microsatellite markers of the Chinese Pond Mussel (Sinanodonta woodiana).

Parentage Determination of Chinese Hooksnout Carp (Opsariichthys bidens) Based on Microsatellite DNA Markers

In this study, a panel of 14 microsatellite markers was screened to determine the parentage of 144 progenies in 12 Chinese hooksnout carp (Opsariichthys bidens) full-sib families. The combined exclusion probabilities for 14 loci were 97.6% and 99.9%, respectively, when there was no available parent information or only one parent information was provided. Simulation analysis demonstrated that the power of five loci to exclude false parents exceeded 99.0%, while that of eight loci reached 99.9% based on allele frequency data obtained from full-sib families (168 individuals). Moreover, the cumulative assignment success rate reached 100% with known parental and filial information when utilizing 13 or more loci, even in cases where no parent information was available. The results showed that this set of microsatellite markers proved to be a reliable and efficient tool for parentage determination of O. bidens.

The Poultry Red Mite, Dermanyssus gallinae, travels far but not frequently, and takes up permanent residence on farms

Management of Dermanyssus gallinae, a cosmopolitan hematophagous mite responsible for damage in layer poultry farming, is hampered by a lack of knowledge of its spatio-temporal population dynamics. Previous studies have shown that the circulation of this pest between farms is of strictly anthropogenic origin, that a mitochondrial haplogroup has been expanding on European farms since the beginning of the 21st century and that its local population growth may be particularly rapid. To refine our understanding of how D. gallinae spreads within and among farms, we characterized the genetic structure of mite populations at different spatial scales and sought to identify the main factors interrupting gene flow between poultry houses and between mitochondrial haplogroups. To this end, we selected and validated the first set of nuclear microsatellite markers for D. gallinae and sequenced a region of the CO1-encoding mitochondrial gene in a subsample of microsatellite-genotyped mites. We also tested certain conditions required for effective contamination of a poultry house through field experimentation, and conducted a survey of practices during poultry transfers. Our results confirm the role of poultry transport in the dissemination of mite populations, but the frequency of effective contamination after the introduction of contaminated material into poultry houses seems lower than expected. The high persistence of mites on farms, even during periods when poultry houses are empty and cleaned, and the very large number of nodes in the logistic network (large number of companies supplying pullets or transporting animals) undoubtedly explain the very high prevalence on farms. Substantial genetic diversity was measured in farm populations, probably as a result of the mite's known haplodiploid mode of sexual reproduction, coupled with the dense logistic network. The possibility of the occasional occurrence of asexual reproduction in this sexually reproducing mite was also revealed in our analyses, which could explain the extreme aggressiveness of its demographic dynamics under certain conditions.

Identification and Characterization of Relict Olive Varieties (Olea europaea L.) in the Northwest of the Iberian Peninsula

Olives (Olea europaea L.) are an important crop in the Mediterranean Basin, but it is not well-known that they have also been grown in other areas, such as Galicia in northwestern Spain. Although commercial production ended long ago in this peripheral growing region, it remains home to olive resources that are well-adapted to the prevailing environmental conditions, providing a valuable but largely undocumented source of genetic variation. Following a survey of Galicia to locate examples of centuries-old olive trees, those detected were subjected to molecular characterization using a set of microsatellite markers, as well as full botanical characterization using the features established by the International Union for the Protection of New Varieties of Plants, along with others proposed by the present authors. These procedures allowed 11 undescribed varieties to be identified, which are new genetic resources that might be of use in olive improvement programs or studies of how the species adapts to different climates. The trees also underwent preliminary health checks, allowing disease-free specimens of each variety to be propagated. The addition of this material to the Community Plant Variety Office’s register of commercial varieties is underway.

Far from home: tracing the origin of non-native water frogs (genus Pelophylax) in Malta by molecular markers

AbstractOne of the most frequently translocated species outside their native range in Europe are water frogs of the genus Pelophylax. Recently, water frogs belonging to the same genus have also been recorded on the island of Gozo in Malta. To trace their origin, we genetically examined 17 individuals from three Gozitan localities where water frogs have been recorded recently. We analysed one mitochondrial (NADH dehydrogenase 2, ND2) and one nuclear (serum albumin intron 1, SAI-1) fragment to identify the geographic origin of the frogs and a set of microsatellite markers to determine their population-genetic structure and the predicted number of source populations. Based on the ND2 and SAI-1 markers, the water frogs on the island of Gozo originate from southern Anatolia, Turkey. According to sequence variation in ND2, they were assigned to a caralitanus mtDNA clade, which is endemic to southern Anatolia and taxonomically represents either an evolutionary lineage within P. cf. bedriagae or a separate species P. caralitanus. All Gozo water frogs had only one haplotype in the ND2 and one allele in the SAI-1 fragment, indicating a recent and single introduction event. These results are supported by microsatellite analysis, which revealed low genetic variability and the absence of any population-genetic structure, suggesting that Gozo water frogs originate from only one source population.

Creation of new rice genotypes tolerant to long-term water flooding using DNA markers

The aim of this work is to search for informative molecular markers for the identification of the Sub1A gene in rice breeding material. Studies have been carried out to identify specialized DNA marker systems that provide a clear control of the inheritance of the target locus on the basis of tolerance to prolonged flooding with water, as a factor in the fight against weeds. Of the studied set of microsatellite markers (13 SSRs), two SSR markers (RM 7481, PrC3) showed high efficiency in detecting polymorphism between donor varieties and varieties of domestic selection. Based on them, the DNA of the obtained hybrid rice plants of the F2 segregating population was analyzed for this trait and a laboratory express method was performed to evaluate the phenotype. During the statistical analysis, it was found that both SSR markers are co-heritable with the trait of tolerance to prolonged flooding under water, and on their basis, plants were selected that have the target gene in the genotype in the homozygous state, which were transferred to the breeding process for study by morphometric characteristics and economically valuable traits.

Development of polymorphic microsatellite markers for Fagus pashanica (Fagaceae) using next-generation sequencing.

Fagus pashanica is an endangered and endemic tree species in China. To understand its genetic diversity and structure for effective conservation, we used next-generation sequencing data to develop a set of microsatellite markers. Twenty-three of the 68 designed loci were successfully amplified. Fifteen polymorphic loci with clear peaks were selected for further analyses in three F. pashanica populations sampled from Nanjiang, Wangcang and Pingwu counties in Sichuan Province, China. The number of alleles per locus ranged from two to 11. The levels of observed and expected heterozygosity ranged from 0.033-0.852 and 0.033-0.787, respectively. All 23 loci were also successfully amplified in F. longipetiolata and F. lucida, and 19 were successfully amplified in F. engleriana. These microsatellite markers will be useful for population genetic studies of F. pashanica and other Fagus species.

Assessing the recovery of Y chromosome microsatellites with population genomic data using Papio and Theropithecus genomes

Y chromosome markers can shed light on male-specific population dynamics but for many species no such markers have been discovered and are available yet, despite the potential for recovering Y-linked loci from available genome sequences. Here, we investigated how effective available bioinformatic tools are in recovering informative Y chromosome microsatellites from whole genome sequence data. In order to do so, we initially explored a large dataset of whole genome sequences comprising individuals at various coverages belonging to different species of baboons (genus: Papio) using Y chromosome references belonging to the same genus and more distantly related species (Macaca mulatta). We then further tested this approach by recovering Y-STRs from available Theropithecus gelada genomes using Papio and Macaca Y chromosome as reference sequences. Identified loci were validated in silico by a) comparing within-species relationships of Y chromosome lineages and b) genotyping male individuals in available pedigrees. Each STR was selected not to extend in its variable region beyond 100 base pairs, so that loci can be developed for PCR-based genotyping of non-invasive DNA samples. In addition to assembling a first set of Papio and Theropithecus Y-specific microsatellite markers, we released TYpeSTeR, an easy-to-use script to identify and genotype Y chromosome STRs using population genomic data which can be modulated according to available male reference genomes and genomic data, making it widely applicable across taxa.

A Procedure for Modeling Genetic Diversity Distortions in Populations of Organisms with Mixed Reproductive Strategies

We propose an approach for modeling the pattern of the genetic diversity of microsatellite markers in a population with a mixed breeding strategy. Part of the population is reproduced sexually, and part is produced asexually. The method of the proposed simulation is different from others in that it produces a set of microsatellite markers as the outcome of a computer simulation of processes in a fixed-size population. These markers can be utilized with the assistance of available software to calculate various metrics of genetic diversity. Our approach is implemented in Python 3.10 and is accompanied by additional scripts that ensure result compatibility with programs that calculate different population characteristics.

Development of microsatellite markers for the vulnerable Depressed River Mussel Pseudanodonta complanata (Rossmässler, 1835) (Bivalvia: Unionida)

The Depressed River Mussel (Pseudanodonta complanata) is a highly threatened bivalve species. Distributed across Europe, this species is still poorly investigated in terms of population genetics. In this study, we describe for the first time eight microsatellite loci that were developed based on a previously published set of microsatellite markers of the Duck Mussel (Anadonta anatina).

Genome-Wide Development of Polymorphic Microsatellite Markers and Genetic Diversity Analysis for the Halophyte Suaeda aralocaspica (Amaranthaceae).

Suaeda aralocaspica, which is an annual halophyte, grows in saline deserts in Central Asia with potential use in saline soil reclamation and salt tolerance breeding. Studying its genetic diversity is critical for effective conservation and breeding programs. In this study, we aimed to develop a set of polymorphic microsatellite markers to analyze the genetic diversity of S. aralocaspica. We identified 177,805 SSRs from the S. aralocaspica genome, with an average length of 19.49 bp, which were present at a density of 393.37 SSR/Mb. Trinucleotide repeats dominated (75.74%) different types of motifs, and the main motif was CAA/TTG (44.25%). We successfully developed 38 SSR markers that exhibited substantial polymorphism, displaying an average of 6.18 alleles with accompanying average polymorphism information content (PIC) value of 0.516. The markers were used to evaluate the genetic diversity of 52 individuals collected from three populations of S. aralocaspica in Xinjiang, China. The results showed that the genetic diversity was moderate to high, with a mean expected heterozygosity (He) of 0.614, a mean Shannon's information index (I) of 1.23, and a mean genetic differentiation index (Fst) of 0.263. The SSR markers developed in this study provide a valuable resource for future genetic studies and breeding programs of S. aralocaspica, and even other species in Suaeda.

Development and characterization of twenty microsatellite markers for Phelsuma inexpectata (Squamata: Gekkonidae), a critically endangered gecko endemic to Reunion Island

The Manapany day gecko (Phelsuma inexpectata) is endemic to the south of Reunion Island. Threatened by habitat fragmentation and loss, human activities and invasive species, P. inexpectata is considered as critically endangered. Conservation measures are required but data on the species are missing, notably on its genetic diversity and population structure for which no specific markers are available to date. Here, we aimed to develop molecular markers to allow genetic studies of P. inexpectata. We developed and characterized 20 polymorphic microsatellite markers based on 23 P. inexpectata individuals sampled from 10 sites. Then, the markers were tested on a total of 101 individuals, 30 from a natural site and 71 from an anthropized site. The mean values of Na, Ho and He were 2.3 (± 0.2), 0.353 (± 0.053) and 0.345 (± 0.046) in the natural site and 2.8 (± 0.3), 0.345 (± 0.051) and 0.338 (± 0.048) in the anthropized site, respectively. Based on the combined loci, the probability of identity (PID) for unrelated specimens were 2.7 × 10-7 and 2.6 × 10-7 in the natural and anthropized site, respectively. This work provides the first set of microsatellite markers for P. inexpectata, constituting a valuable tool to conduct classical genetic studies on the species, such as estimating genetic diversity, population structure and kinship relationships among individuals. Such studies will provide relevant information on P. inexpectata and will therefore be helpful in the implementation of conservation measures for this threatened species.

Development and testing of microsatellite loci for the study of population genetics of Ixodes ricinus Linnaeus, 1758 and Ixodes inopinatus Estrada-Peña, Nava and Petney, 2014 (Acari: Ixodidae) in the western Mediterranean region

Ixodes ricinus is an important vector of several human and veterinary infectious agents. Its wide geographical distribution and permissive feeding behaviour have prompted earlier studies on its population genetics. Results were, nevertheless, not conclusive. Furthermore, no research has fully focused on the south-western distribution range of I. ricinus, where exchanges between European and North African populations are more likely to occur. The presence of an additional species, Ixodes inopinatus, in the area further confuses the topic, as the two species are hard to differentiate morphologically. The present work describes the testing of microsatellite markers previously described for I. ricinus using Portuguese and Tunisian tick populations of both species. In addition, new microsatellite loci were developed to complement the available marker toolbox. Loci showed different amplification successes across subpopulations, with Tunisian DNA less readily amplified. Altogether, 15 loci were considered suitable for genetic analyses of Portuguese subpopulations, 10 for Tunisian samples, and seven, common to both populations, were considered to be informative at the inter-continental level. A preliminary analysis of both datasets revealed two isolated populations, which can correspond to two different species. Furthermore, Tunisian specimens identified by sequencing of 16S rDNA as having I. ricinus or I. inopinatus sequence profiles all clustered together in one single population using the proposed microsatellites. This confirms that taxonomic decisions based only on 16S rRNA gene sequencing can be misleading. The application of the proposed set of microsatellite markers to a larger sample, representative of the south-western Ixodes’ distribution range, will be crucial to clarify the distribution of both species.

Characterization of 35 new microsatellite markers for the blacktip reef shark (Carcharhinus melanopterus) and cross-species amplification in eight other shark species.

Shark species are overfished at a global scale, as they are poached for the finning industry or are caught as bycatch. Efficient conservation measures require fine-scale spatial and temporal studies to characterize shark habitat use, infer migratory habits, analyze relatedness, and detect population genetic differentiation. Gathering these types of data is costly and time-consuming, especially when it requires collection of shark tissue samples. Genetic tools, such as microsatellite markers, are the most economical sampling method for collecting genetic data, as they enable the estimation of genetic diversity, population structure and parentage relationships and are thus an efficient way to inform conservation strategies. Here, a set of 45 microsatellite loci was tested on three blacktip reef shark (Carcharhinus melanopterus) populations from three Polynesian islands: Moorea, Morane and Tenararo. The set was composed of 10 previously published microsatellite markers and 35 microsatellite markers that were developed specifically for C. melanopterus as part of the present study. The 35 novel and 10 existing loci were cross-amplified on eight additional shark species (Carcharhinus amblyrhynchos, C. longimanus, C. sorrah, Galeocerdo cuvier, Negaprion acutidens, Prionacea glauca, Rhincodon typus and Sphyrna lewini). These species had an average of 69% of successful amplification, considered if at least 50% of the individual samples being successfully amplified per species and per locus. This novel microsatellite marker set will help address numerous knowledge gaps that remain, concerning genetic stock identification, shark behavior and reproduction via parentage analysis.

DNA markers reveal genetic association between the sea-side Lithuanian and Bavarian Scots pine populations

Due to complex history of European forests, natural populations may not necessarily represent autochthonous genepools for forest trees. Eastern Prussian forests were famous for using non-local sources for afforestation. We studied efficiency of a set of nuclear microsatellite markers (nSSR) for genetic association and diversity studies of 194 adult trees from 5 populations genotyped at 11 nSSR loci. The Bayesian and UPGMA clustering revealed two genetically distinct groups: (a) the Baltic group, and (b) the Bavarian one and an over 200-years-old sea-side Lithuanian population of Juodkrante from the sea-side Curonian spit of Neringa. We interpret this result as putative introduction of Bavarian Scots pine back in the 18th century, when reforestation efforts were made to sustain moving sands in the dunes of Neringa. The genomic SSRs were more variable than the EST SSRs. However, the association between the variability of the nuclear microsatellite loci and their efficiency in detecting population differentiation was not strong. Keywords: nSSR, Pinus sylvestris (L.), population structure, provenance transfer, FRM transfer, molecular markers