Development Of Microsatellite Markers Research Articles

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 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.

Development and Application of Potentially Universal Microsatellite Markers for Pheasant Species

Pheasants are widely distributed in the southwest of China, but many of them are endangered due to habitat fragmentation and environmental changes. Genetic diversity is crucial for species to maintain their evolutionary potential, and thus it is important to develop universal genetic markers for facilitating the assessment of genetic diversity and planning effective conservation actions in these endangered species. In this study, 471 microsatellite loci which are common among eight pheasant species were screened based on genome data, and 119 loci were selected to develop microsatellite markers. After PCR amplifications and reaction condition optimizations, and validation of microsatellite loci in 14 species of 11 genera within Phasianidae. Finally, 49 potentially universal microsatellite markers in pheasant species were obtained. These microsatellite markers were successfully applied to assess the genetic diversity of 3 pheasant species. The Sichuan hill partridge (Arborophila rufipectus), blood pheasant (Ithaginis cruentus), buff-throated partridge (Tetraophasis szechenyii) and Sichuan hill partridge had a relatively low genetic diversity level. These 49 microsatellite loci are potentially universal microsatellite loci for pheasants and are of great significance to establish a shared platform in population genetics study of pheasants.

Genome Assembly and Microsatellite Marker Development Using Illumina and PacBio Sequencing in the Carex pumila (Cyperaceae) from Korea.

This study is the first to report the characterization of Carex pumila genomic information. Assembly of the genome generated a draft of C. pumila based on PacBio Sequel II and Illumina paired-end sequencing, which was assembled from 2941 contigs with an estimated genome size of 0.346 Gb. The estimate of repeats in the genome was 31.0%, and heterozygosity ranged from 0.426 to 0.441%. The integrity evaluation of the assembly revealed 1481 complete benchmarked universal single-copy orthologs (BUSCO) (91.76%), indicating the high quality of the draft assembly. A total of 23,402 protein-coding genes were successfully predicted and annotated in the protein database. UpsetR plots showed that 7481 orthogroups were shared by all species. The phylogenetic tree showed that C. pumila is a close but distant relative of Ananas comosus. C. pumila had greater contraction (3154) than expansion (392). Among the extended gene families, aquaporins have been found to be enriched. Primers for microsatellite markers determined 30 polymorphic markers out of 100. The average number of alleles amplified by these 30 polymorphic markers was 4 to 12, with an average polymorphism information content (PIC) value of 0.660. In conclusion, our study provides a useful resource for comparative genomics, phylogeny, and future population studies of C. pumila.

The novel developed microsatellite markers revealed potential hybridization among Cymbidium species and the interspecies sub-division of C. goeringii and C. ensifolium

BackgroundOrchids (Cymbidium spp.) exhibit significant variations in floral morphology, pollinator relations, and ecological habitats. Due to their exceptional economic and ornamental value, Cymbidium spp. have been commercially cultivated for centuries. SSR markers are extensively used genetic tools for biology identification and population genetics analysis.ResultIn this study, nine polymorphic EST-SSR loci were isolated from Cymbidium goeringii using RNA-Seq technology. All nine SSR loci showed transferability in seven other congeneric species, including 51 cultivars. The novel SSR markers detected inter-species gene flow among the Cymbidium species and intra-species sub-division of C. goeringii and C. ensifolium, as revealed by neighborhood-joining and Structure clustering analyses.ConclusionIn this study, we developed nine microsatellites using RNA-Seq technology. These SSR markers aided in detecting potential gene flow among Cymbidium species and identified the intra-species sub-division of C. goeringii and C. ensifolium.

Development of Polymorphic Microsatellite Markers and Identification of Applications for Wild Walnut (Juglans regia L.) in Middle Asia

The common walnut (Juglans regia L.), a species of significant economic and ecological importance, along with its variant, holds developmental value. Simple sequence repeat (SSR) markers are used as an effective method for material identification due to their co-dominant inheritance. In this work, a total of 357629 SSR loci were searched based on previously published walnut genome sequences, with a density of 662.28 (SSRs/Mb). The majority of all these loci were found to be single nucleotide A/T (58.49%), followed by the dinucleotide AT (19.48%). Based on the results of genome-wide SSR site design, 22 effective primer pairs were designed and screened to analyze the genetic diversity of 48 wild walnut samples from three countries. Each locus had an average of 5.17 effective alleles (Ne), with an average polymorphism information content (PIC) of 0.71, average heterozygosity (He) of 0.52, and average genetic differentiation index (Fst) of 0.09. The analysis of 48 wild walnut samples from three countries (Kyrgyzstan, Tajikistan, and China) using the unweighted pairwise clustering method and arithmetic mean (UPGMA) along with principal coordinate analysis revealed the division of all samples into three groups. In summary, these novel SSR markers serve as a reference for the identification of wild walnut germplasm in Central Asia and for the development and utilization of wild walnut seed resources.

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.

Development and characterization of expressed sequence tag-simple sequence repeat markers for Anaphalis margaritacea var. yedoensis (Asteraceae).

RNA-sequencing was used to develop 16 microsatellite markers for the pearly everlasting, Anaphalis margaritacea var. yedoensis (Franch. et Sav.) Ohwi (Asteraceae), which inhabits gravel bars throughout the Japanese archipelago. The mean number of alleles for these 16 markers in two populations in the Hokkaido and Shizuoka Prefectures, was 3.5 and 4.0, respectively, while the mean expected heterozygosity was 0.525 and 0.560, respectively, with a significant genetic differentiation between the two populations. All markers could also be amplified in two conspecific taxa, A. margaritacea var. margaritacea and var. angustifolia, whereas 11 of the 16 markers were amplifiable in two congeneric species, A. sinica and A. alpicola. These newly developed microsatellite markers will support understanding of population genetics and mating systems in A. margaritacea var. yedoensis, and several will potentially be of use in similar studies in other Anaphalis species.