Development Of Microsatellite Markers Research Articles

Development of Microsatellite Markers from Transcriptome of Eriocheir sinensis and Their Application in Multiplex PCR Panels

Background: The development of genetic markers is crucial for aquaculture and population genetics. Polyallelic microsatellite markers are widely used for breeding and family management of Eriocheir sinensis (E. sinensis), contributing to the sustainable development of the industry. Methods: Through transcriptome analysis, 100 highly polymorphic SSR markers were identified and developed. From these, four multiplex PCR panels, each containing 16 markers, were constructed and applied to parentage assignment in six E. sinensis families. Results: The number of alleles (Na) ranged from 7 to 21, the observed heterozygosity (Ho) varied from 0.356 to 0.951, and the expected heterozygosity (He) varied from 0.656 to 0.909. The polymorphism information content (PIC) ranged from 0.633 to 0.900. The CERVUS analysis showed that using only the two most informative multiplex PCR panels, assignment success rates exceeded 90.34%, with 100% accuracy in assigning offspring to parents when three or four panels were used. Conclusions: This method significantly enhances the efficiency of microsatellite marker analysis, which provides a convenient and cost-effective method for breeding programs and family management of E. sinensis.

Development and Application of Microsatellite Markers for Genetic Diversity Assessment and Construction of a Core Collection of Myrciaria dubia (Kunth) McVaugh Germplasm from the Peruvian Amazon

The Amazonian shrub Myrciaria dubia (camu-camu) produces vitamin C-rich fruits of growing commercial interest. However, sustainable utilization requires assessment and protection of the genetic diversity of the available germplasm. This study aimed to develop and apply microsatellite markers to assess genetic diversity and construct a core collection of M. dubia germplasm from the Peruvian Amazon. Sixteen polymorphic microsatellite loci were developed using an enrichment approach. The evaluation of 336 genotypes from 43 accessions of the germplasm bank, originating from eight river basins, was conducted using these newly developed markers. Genetic diversity parameters, including observed and expected heterozygosity, were calculated. Analysis of molecular variance (AMOVA) was performed to assess the distribution of genetic variation within and among accessions and river basins. Bayesian clustering analysis was employed to infer population structure. A core collection was constructed to maximize allelic richness. High genetic diversity was observed, with heterozygosity values ranging from 0.468 to 0.644 (observed) and 0.684 to 0.817 (expected) at the river basin level. AMOVA indicated significant genetic variation within (73–86%) compared to among (14–27%) accessions and river basins. Bayesian clustering detected ten genetic clusters, with several degrees of admixture among river basins, except for the genetically homogeneous Putumayo River basin. A core collection comprising 84 plant genotypes (25% of the full collection) was established, representing 90.82% of the overall allelic diversity. These results have important implications for M. dubia conservation strategies and breeding programs, in demonstrating a need for genetic connectivity between populations but preserving unique genetic resources in isolated basins. These results validate the expected levels of diversity and population subdivision in a crop and stress the need to secure genetically diverse germplasms, underscoring the importance of thorough genetic characterization for ex situ germplasm management.

The Characteristics of Microsatellites and Development of SSR Markers in the Genome of Periplaneta americana

The research was to analyze the number and pattern of microsatellites in Periplaneta americana’s genome, and also developed tetranucleotide SSR markers. We thoroughly scrutinized and dissected the inherent traits that govern the allocation of microsatellite sequences within the profound domain of P. americana’s genome, software MSDBv2 allowed for the utilization of 2.67 Gb. There were precisely 1,498,458 flawless microsatellite sequences, encompassed approximately 1.57%. The cumulative length of microsatellites was 45,076,707 bp, and the abundance of microsatellites was 16889.577 loci/Mb. Out of all the microsatellite repeat variations, the trinucleotide repeats accounted for 44.83% of the total, with a count of 671,830, which were the most abundant type. The tetranucleotide, mononucleotide, pentanucleotide, dinucleotide, and hexanucleotide repeats accounted for 29.01%, 13.62%, 8.37%, 3.70% and 0.47%, respectively. The numbers of different repeat copy categories in each repeat type were also quite different, such as the A in mononucleotide repeat type, the AT in dinucleotides, the AAT in trinucleotides, and AAAT in tetranucleotide were the most of each categories. 143 primers were designed. After undertaking the arduous task of enhancing the initial PCR conditions to perfection, we successfully determined and analyzed a gargantuan number of 38 different polymorphic tetranucleotide microsatellite markers with utmost precision, employed the assistance of two-color fluorescence markers and ingenious genotyping scaned for their comprehensive characterization. The genetic variation in P. americana population involved analyzing the diversity of microsatellite loci, which exhibited varying numbers of alleles per locus ranging from 4 to 21 among the 32 individuals studied. Among them, there were 24 microsatellite loci whose alleles were greater than 10, accounted for 63.16% of the total number of polymorphic microsatellite. The calculated degrees of genetic diversity varied between 0 and 1, the observed heterozygosities was between 0.219 and 1.0, with a mean of 0.6391. The expected heterozygosities was between 0.312 and 0.942, with a mean of 0.7663. The PIC was between 0.296 and 0.923, with a mean of 0.7294, and there were 36 microsatellite loci whose PIC was greater than 0.5, accounted for 94.74%. This study indicated that new development of microsatellite markers for P. americana was feasible. Furthermore, these new development microsatellite markers will provide adequate and reliable molecular genetics data for carrying out the research of molecular ecology and conservation genetics for P. americana.

A genome sequence resource for the European chestnut (Castanea sativa Mill.) and the development of genic microsatellite markers

A genome sequence resource for the European chestnut (Castanea sativa Mill.) and the development of genic microsatellite markers

Males conditionally inseminate at three female body locations according to female mating history and female maturity status in a squid.

In some squids, such as those in the family Loliginidae, upon copulation, females receive and store male-delivered sperm capsules, spermatangia, at two different body locations: the buccal membrane and the distal end of the oviduct. This insemination site dimorphism is associated with alternative reproductive strategies. However, in Loliolus sumatrensis, a species of Loliginidae, the females possess three insemination sites: buccal membrane (BM), basal left IV arm (ARM) and lateral head behind the left eye (EYE), therefore we studied such the unusual phenomena. We developed microsatellite markers and genotyped the paternity of each spermatangium on three sites. We found multiple paternity at every single site and simultaneous usage of all three sites by a few males. The seasonal dynamics of a population in the Seto Inland Sea revealed a set priority for the initial use of insemination sites as BM, followed by ARM and then EYE, whereas the maximum number of stored spermatangia was greater in EYE > ARM > BM. Female maturity status was correlated with the usage pattern of insemination sites but not with the number of stored spermatangia at any insemination site. These results suggest that a male squid inseminates at different locations according to female mating history and female maturity status.

Microsatellites for butterfly conservation: historical challenges, current relevance, and a guide to implementation

We are nearing the 20-year anniversary of a groundbreaking paper which details how microsatellite marker development in Lepidoptera is “extremely difficult for no apparent reason.” How far have we come in these past 20 years? Microsatellites are still the marker of choice in many population genetics studies for their ease of use, high degrees of polymorphism, species-specificity, and low cost. The rise of next-generation sequencing technologies (e.g. 454, Illumina, PacBio, etc.) has greatly advanced our abilities to generate many microsatellite markers per species. In this paper, we summarize the improvements in marker development using next-generation technology. Using case studies, we review the use and implementation of microsatellite markers in different conservation programs. Lastly, we provide a guide to data interpretation of microsatellite data generated for butterflies, with the goal of supporting student researchers and conservation practitioners in evaluating the meaning in their data.

Development and application of microsatellite markers in Hippophae rhamnoides subsp. sinensis Rousi (Hippophae rhamnoides L.) based on transcriptome sequencing.

Hippophae rhamnoides subsp. sinensis Rousi is a cold- and drought-tolerant pioneer species with significant economic and ecological value. Evaluating its genetic diversity and population structure is of great importance for guiding the development and utilization of resources. In this study, a total of 41,804 SSRs were generated by transcriptome sequencing of Hippophae rhamnoides subsp. sinensis Rousi. Among the different SSR motif types, mononucleotide repeats (26,972) were the most abundant, followed by trinucleotides, tetranucleotides, and pentanucleotides. 200 pairs of SSR primers were selected to detect polymorphisms, of which 15 pairs primers were selected as validated polymorphic SSRs used for genetic diversity and population structure analysis. A total of 63 alleles were identified with 15 pairs primers, with Nei's genetic diversity index ranged from 0.27 to 0.83 (average: 0.54), and the expected heterozygosity ranged from 0.16 to 0.73 (average: 0.46). The polymorphism information content ranged from 0.23 to 0.81 (average: 0.48). Genetic structure analyses showed that the 10 populations could be broadly categorized into two groups. AMOVA denoted that genetic variations primarily originated from within the populations, with minimal differences observed between the groups, accounting for only 7% of the total genetic variation. This implies that mutation in H. rhamnoides subsp. sinensis Rousi mainly occurred within the populations. The results showed that the 10 populations of H. rhamnoides subsp. sinensis Rousi are rich in genetic diversity, with low levels of population differentiation and a high degree of gene exchange, which should be taken into consideration for the future work of germplasm resource preservation and seedling breeding.

Development of novel microsatellite markers for aquaculture important species, milkfish (Chanos chanos Forsskål, 1775) with implications of conservation and management

Development of novel microsatellite markers for aquaculture important species, milkfish (Chanos chanos Forsskål, 1775) with implications of conservation and management

Development of microsatellite markers for the American species Vachellia aroma (Fabac

Background and aims: There are currently no microsatellite markers available for any American species of Vachellia, and particularly, for V. aroma. Then the aims of this study were to develop SSR markers specific for V. aroma, for the first time, and test its amplification in a close related species. M&M: For the development of the SSR in V. aroma, total genomic DNA was extracted and it was sequenced in a one-fourth run on a Roche 454 GS FLX+ platform. The study area included two Argentinean populations: San José and Robles, Santiago del Estero province. Results: We detected 422 sequences containing SSR loci. A set of 39 primer pairs presented amplified products in V. aroma and V. caven, but 12 revealed clear, replicable and polymorphic loci in V. aroma. Conclusions: The results of this work indicate that a new set of SSR markers was developed for V. aroma and their transferability to V. caven was assessed. The analysis of variability, showed that these 12 polymorphic markers are highly informative, and a powerful tool to investigate population genetics parameters in V. aroma and related species.

Development of polymorphic microsatellite markers for genetic stock identification of green chromide, Etroplus suratensis using next generation sequencing technology

Development of polymorphic microsatellite markers for genetic stock identification of green chromide, Etroplus suratensis using next generation sequencing technology

Analysis of seven populations of cultured redclaw crayfish, Cherax quadricarinatus, using newly developed microsatellite markers

Cherax quadricarinatus, also known as the redclaw crayfish, is native to northern Australia and southern Papua New Guinea and is an important freshwater aquaculture species in many countries. Studying the genetic structure of the current cultured population can improve the breeding plan for redclaw crayfish to better manage the hybridization and germplasm bank and promote their rational utilization; however, microsatellite resources for redclaw crayfish remain insufficient. The purpose of this study was to analyze the genetic diversity of different redclaw crayfish populations in China. After screening 18 pairs of microsatellite markers from redclaw crayfish transcriptome data, 240 samples from seven main breeding areas (Changsha, Guangzhou, Haikou, Huzhou, Jiangmen, Wuxi, and Zhanjiang) were evaluated and detected using these microsatellite markers. In total, 97 alleles were detected, of which the minimum number was two and the maximum number was twelve. The observed heterozygosity ranged from 0.258 to 0.807, with an average of 0.559, and the expected heterozygosity ranged from 0.288 to 0.815, with an average value of 0.558. The polymorphic information content values of seven populations ranged from 0.268 to 0.791, with an average of 0.511. Moreover, pairwise genetic differentiation within the population was between 0.007 and 0.031. The UPGMA clustering results showed that the Zhanjiang population stands alone on a branch, but the genetic variation between branches is very small. Meanwhile, STRUCTURE analysis further revealed that the genetic backgrounds of each population are essentially identical, with no noticeable genetic differentiation arising from geographic differences. These results provide a basic assessment of the genetic diversity of the main cultured populations of redclaw crayfish and a molecular foundation for future genetic monitoring and selective breeding research.

Development and Characterization of 12 Microsatellite Markers for an Economically Important Fish, Caranx ignobilis, in the Philippines

The giant trevally (Caranx ignobilis) is an important food fish that faces increasing demand due to human population growth, necessitating genetic monitoring. This study intends to spearhead the development of microsatellite markers to assess the genetic variation of C. ignobilis populations. A total of 150 C. ignobilis specimens from six localities in the Philippines were analyzed. Based on next-generation sequencing data, 12 primer pairs were developed with high amplification rates and polymorphism across different loci. This study introduces the first microsatellite markers exclusively for C. ignobilis, which can be used for monitoring and management purposes.

Genetic diversity assessment of Helichrysum arenarium (Asteraceae) for the genetic restoration of declining populations.

Helichrysum arenarium (L.) Moench (Asteraceae) is a self-compatible, insect-pollinated herb occurring in sand grasslands, and is declining and endangered in many parts of its European distribution range. A recovery plan of H. arenarium has been conducted in southern Belgium, involving plant translocations. We developed multiplex genotyping protocol for nine microsatellite markers previously published for Helichrysum italicum and two newly developed microsatellite markers for H. arenarium. Eleven polymorphic loci were associated (pooled) in two multiplex panels, to assess the genetic status of the only small remaining population in Belgium and of three large German populations used as seed source for propagating transplants. The small Belgian population was characterized by high clonality, with only two, however heterozygous, genets detected. The three large German populations showed high genetic diversity (H e ranging from 0.635 to 0.670) and no significant inbreeding coefficient values, despite expectations of geitonogamous selfing. Management practices (grazing livestock) increasing seed dispersal distances, inbreeding depression at early stages of development, and mechanisms preventing or delaying selfing might be hypothesized to explain the observed patterns. The two Belgian genotypes remained within genetic variation range of German populations so that the high genetic differentiation between Belgian and German populations (F ST values ranging from 0.186 to 0.206) likely resulted from genetic drift effects and small sample size. Transplants obtained from seeds sampled from the three large source populations from Germany constitute a highly diverse, noninbred gene pool, and are thus of high genetic quality for plant translocations.

Genome-wide development of intra- and inter-specific transferable SSR markers and construction of a dynamic web resource for yam molecular breeding: Y2MD.

Microsatellite markers are widely used in population genetics and breeding. Despite the economic significance of yams in developing countries, there is a paucity of microsatellite markers, and as of now, no comprehensive microsatellite marker database exists. In this study, we conducted genome-wide microsatellite marker development across four yam species, identified cross-species transferable markers, and designed an easy-to-use web portal for the yam researchers. The screening of Dioscorea alata, Dioscorea rotundata, Dioscorea dumetorum, and Dioscorea zingiberensis genomes resulted in 318,713, 322,501, 307,040, and 253,856 microsatellites, respectively. Mono-, di-, and tri-nucleotides were the most important types of repeats in the different species, and a total of 864,128 primer pairs were designed. Furthermore, we identified 1170 cross-species transferable microsatellite markers. Among them, 17 out of 18 randomly selected were experimentally validated with good discriminatory power, regardless of the species and ploidy levels. Ultimately, we created and deployed a dynamic Yam Microsatellite Markers Database (Y2MD) available at https://y2md.ucad.sn/. Y2MD is embedded with various useful tools such as JBrowse, Blast, insilicoPCR, and SSR Finder to facilitate the exploitation of microsatellite markers in yams. This study represents the first comprehensive microsatellite marker mining across several yam species and will contribute to advancing yam genetic research and marker-assisted breeding. The released user-friendly database constitutes a valuable platform for yam researchers.

Isolation and characterization of microsatellite markers from Garcinia morella using next generation sequencing technology and cross-species amplification

The fruit-bearing medicinal tree, Garcinia morella, grows in the tropical rain forests of India’s Western Ghats, Indo-Chinese Himalayan regions and Sri Lanka. Its fruit rinds are used as a garnish and in seasoning during food preparation. In order to genetically exploit it and assess diversity, development of microsatellite markers was attempted. We partially sequenced genomic DNA using the Illumina Hiseq 2000 platform and examined sequence data for microsatellite loci. We obtained high-quality 10653 Mbp data and was assembled into 1613263 contigs. A total of 121199 SSRs were discovered, Di nucleotide repeats were predominant (42.5%), followed by mono and tri nucleotide repeats (30.4 and 7.9%, respectively). We were able to design primers for 52901 microsatellites. Genetic analysis of 48 SSR loci, showed PIC values ranging from 0.067 to 0.939 with a mean value of 0.7547. The allele per locus ranged from 2 to 24 with a mean of 13. These microsatellite markers can be employed for genetic diversity analysis, molecular characterization and mapping different traits.

Development and utility of SSR markers based on Brassica sp. whole-genome in triangle of U.

Simple sequence repeats (SSR), also known as microsatellites, are crucial molecular markers in both animals and plants. Despite extensive previous research on SSRs, the development of microsatellite markers in Brassica crops remains limited and inefficient. Krait software was used to identify microsatellites by genome-wide and marker development based on three recently sequenced basic species of Brassica crops in the triangle of U (Brassica rapa, B. nigra and B. oleracea), as well as three allotetraploids (B. juncea, B. napus and B. carinata) using public databases. Subsequently, the primers and the characteristics of microsatellites for most of them were accordingly designed on each chromosome of each of the six Brassica species, and their physical locations were identified,and the cross-transferability of primers have been carried out. In addition, a B-genome specific SSR marker was screened out. A total of 79341, 92089, 125443, 173964, 173604, and 222160 SSR loci have been identified from the whole genome sequences of Brassica crops within the triangle of U crops, B. rapa (AA), B. nigra (BB), B. oleracea (CC), B. napus (AACC), B. juncea (AABB) and B. carinata (BBCC), respectively. Comparing the number distribution of the three allotetraploid SSR loci in the three subgenomes AA, BB and CC, results indicate that the allotetraploid species have significant reduction in the number of SSR loci in the genome compared with their basic diploid counterparts. Moreover, we compared the basic species with their corresponding varieties, and found that the microsatellite characters between the allotetraploids and their corresponding basic species were very similar or almost identical. Subsequently, each of the 40 SSR primers was employed to investigate the polymorphism potential of B. rapa (85.27%), B. nigra (81.33%) and B. oleracea (73.45%), and B. rapa was found to have a higher cross-transfer rate among the basic species in the triangle of U. Meanwhile, a B-genome specific SSR marker, BniSSR23228 possessing the (AAGGA)3 sequence characteristics was obtained, and it located in chromosome B3 with a total length of 97 bp. In this study, results suggest that the pattern of distribution may be highly conserved during the differentiation of basic Brassica species and their allotetraploid counterparts. Our data indicated that the allotetraploidization process resulted in a significant reduction in SSR loci in the three subgenomes AA, BB and CC. The reasons may be partial gene dominated chromosomal homologous recombination and rearrangement during the evolution of basic diploid species into allotetraploids. This study provides a basis for future genomics and genetic research on the relatedness of Brassica species.

Development and validation of genome-wide SSR molecular markers of Tapes dorsatus.

Tapes dorsatus is an economically important benthic animal in the Beibu Gulf of China. However, the deficiency of microsatellite markers has hindered the study of its genetics. The development of microsatellite markers will provide useful tools for genetic improvement, variety identification, phylogenetic analysis and resource conservation. Within the genome sequence, 145,008 simple sequence repeats (SSRs) were identified, and 29,691 primer pairs were designed successfully. A total of 100 primer pairs were randomly synthesized for testing, and 93 primers yielded products. Sixty highly polymorphic primers were used to reveal the genetic diversity of 50 T. dorsatus individuals. The average number of alleles (Na) of the population was 10.40; the average number of effective alleleswas 6.16, the average expected heterozygosity (He) was 0.82, and the average polymorphic information contentwas 0.80. The genetic structure of the population was detected, by which the population could be divided into three subpopulations. We identified 145,008 SSRs in the genome of T. dorsatus and designed 29,691 primer pairs in this study. Of 100 synthesized primers, 60 were highly polymorphic and used to reveal the genetic diversity and structure of the population. The SSR markers identified here will provide useful tools and a foundation for genetic diversity, linkage mapping and molecular marker-aided breeding in T. dorsatus.

Molecular genetic characteristics of Darevskia portschinskii lizard populations based on microsatellite markers analysis

The Caucasian rock lizard species Darevskia portschinskii is one of the bisexual species participating in interspecific hybridisation as the paternal ancestor with the maternal ancestors D. mixta and D. raddei resulting in the successful formation of the parthenogenetic D. dahli and D. rostombekowi, respectively. Populations of D. portschinskii have been previously divided into two subspecies, D. p. portschinskii and D. p. nigrita according to their geographical distribution and the morphological data, but they have not been characterised genetically. Here, we used ten microsatellite markers to determine the genetic structure of the D. portschinskii populations. The utility of the developed microsatellite markers for investigating the genetic variability within and among populations with a heterogeneous spatial distribution was demonstrated. Our results showed that the intra- and interspecific differentiation of the studied populations were consistent with the morphological data on the subspecies status of the D. p. portschinskii and D. p. nigrita populations. A potential applicability of the developed microsatellite markers to study genetic diversity of Darevskia species and subspecies complexes is suggested.

MegaSSR: a web server for large scale microsatellite identification, classification, and marker development.

Next-generation sequencing technologies have opened new avenues for using genomic data to study and develop molecular markers and improve genetic resources. Simple Sequence Repeats (SSRs) as genetic markers are increasingly used in molecular diversity and molecular breeding programs that require bioinformatics pipelines to analyze the large amounts of data. Therefore, there is an ongoing need for online tools that provide computational resources with minimal effort and maximum efficiency, including automated development of SSR markers. These tools should be flexible, customizable, and able to handle the ever-increasing amount of genomic data. Here we introduce MegaSSR (https://bioinformatics.um6p.ma/MegaSSR), a web server and a standalone pipeline that enables the design of SSR markers in any target genome. MegaSSR allows users to design targeted PCR-based primers for their selected SSR repeats and includes multiple tools that initiate computational pipelines for SSR mining, classification, comparisons, PCR primer design, in silico PCR validation, and statistical visualization. MegaSSR results can be accessed, searched, downloaded, and visualized with user-friendly web-based tools. These tools provide graphs and tables showing various aspects of SSR markers and corresponding PCR primers. MegaSSR will accelerate ongoing research in plant species and assist breeding programs in their efforts to improve current genomic resources.

Influence of climate and landscape on genetic differentiation of aardvarks (Orycteropus afer)

AbstractAimAs climate change accelerates, assessing how climate shapes gene flow and neutral and adaptive genetic differentiation on landscapes is increasingly important. Aardvarks (Orycteropus afer) are ecologically important in sub‐Saharan Africa but are sensitive to human pressures and increasing aridity. We used individual, population and landscape genetic approaches to infer the influence of landscape, climate and potential adaptive differences on gene flow.LocationWe surveyed 8 protected and 4 privately owned areas in South Africa, 2 protected areas in Eswatini and one location in Kenya during 2016–2018.MethodsWe developed microsatellite markers and methods for DNA extraction from faeces, collected and genotyped faecal samples from focal areas and estimated genetic structure. We inferred space use from individual redetections, tested for close relatives and estimated genetic neighbourhood distance. We applied individual‐based landscape genetic analyses at multiple scales to test hypotheses about genetic differentiation by landscape resistance and potential adaptive differences.ResultsWe developed 19 variable microsatellite loci and collected 253 faecal samples from 13 focal areas. We genotyped 104 samples successfully at ≥8 loci as needed for individual identification. The genetic structure suggested 3 regional divisions in South Africa. We detected individuals at locations ≤7.3 km distant and closely related individuals at ≤44 km; genetic neighbourhood distance was <55 km. Lower precipitation increased landscape resistance and strongly predicted genetic differentiation at most spatial scales. Temperature differences at sampling sites also influenced structure, suggesting climate‐associated adaptive differences.Main ConclusionsThe genetic structure of aardvarks is strongly shaped by climate, with arid areas limiting gene flow and reflects apparent isolation by adaptation associated with temperature. Dispersal distances likely are <55 km. The markers we developed will facilitate studies of space use, dispersal, population density or survival. Aridification will increase fragmentation and we recommend monitoring aardvark presence as an indicator of ecosystem change associated with aridification.