First Set Of Microsatellite Markers Research Articles

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 validation of microsatellite markers for kikuyu grass using next generation sequencing technology

Context The kikuyu grass (Cenchrus clandestinus) is native to Africa and is one of the most widely used grasses for forage feeding in dairy regions around the world. Aims To obtain the first set of microsatellite markers for the population genetics study of kikuyu grass, using nextgeneration sequencing technology (Illumina MiSeq). Methods Sixty loci were evaluated, in which a subset of 12 loci were selected to be used for a complete population analysis in 108 samples of kikuyu, and were grouped in to three zones of Colombia. The three approaches with which the genetic structure was evaluated. Key results Obtained same tendency of grouping reflects a low genetic differentiation, specifically evidencing differences between the northern zone of Antioquia and the zones that comprise the other territories of Colombia. Conclusions These reads of microsatellite loci help to complement the information on the genetic structure of the populations of the kikuyu, and will be useful for the characterisation and evaluation of the diversity of germplasm in other parts of the world. Implications The set of microsatellite markers developed has a species-specific reproducibility and could be used for studies in other Cenchrus individuals and particularly in future investigations with Kikuyu grass. Likewise, this research presents findings in a broad context and relates them to other pasture species.

First set of microsatellite markers for immortelle (Helichrysum italicum (Roth) G. Don): A step towards the selection of the most promising genotypes for cultivation

Immortelle (Helichrysum italicum (Roth) G. Don), also known as curry plant, is a herbaceous perennial plant with documented biological properties that contribute to its attractiveness and use in the cosmetic, pharmaceutical, and food industry. Availability of DNA markers can significantly improve the knowledge of complex genetics and biochemistry of this aromatic plant. To improve in-depth genetic studies, high-throughput sequencing of a genomic DNA has been performed and a set of 24 SSR markers was developed. In total, 17,025,076 high quality reads were assembled in contigs with the cumulative length of 258.6 Mbp. Genome-wide microsatellite sequences were identified in 8.19% (36,882) contigs, among which dinucleotide (71.2%) and trinucleotide (24.4%) repeats were the most abundant. Twenty-four finally selected microsatellite loci (including di- and tri-nucleotides) which exhibited polymorphism with unambiguous and reproducible amplification products, were used for detailed genetic diversity analysis on a set of 28 H. italicum samples. The Unweighted Pair Group Method with Arithmetic Mean (UPGMA) method based on the Jaccard index differentiated 27 genotypes into two main clusters according to their geographical origin (Northern and Southern Adriatic region), whereas the commercially available plant did not cluster with other H. italicum individuals and morphological evaluation confirmed that it belongs to Helichrysum angustifolium (Lam.) DC. The great diversity of H. italicum samples from the Adriatic observed by new microsatellites will serve for selection of most promising genotypes for propagation and their implementation in agricultural production. The first set of microsatellites developed in the present study provides a valuable resource of DNA markers for further breeding programs, germplasm characterization, and cross-species transferability studies.

Development of Microsatellite Markers for the Hermatypic Coral <i>Porites lutea</i>

By mining the EST and genomic microsatellite sequence resources of Porites species in GenBank, and using interspecific amplification and resequencing, we developed 20 microsatellite markers for the stony coral Porites lutea , meanwhile, validated and excluded 43 microsatellite markers of its symbiotic zooxanthella. These markers of P. lutea were genotyped in one wild population (n=16) from the southwest of Hainan Island fringing reefs. Nineteen of the 20 markers showed polymorphism. The number of alleles was 2~7 (mean 3.5±1.8), and the observed heterozygosity ( H o ) and expected heterozygosity ( H e ) were 0.06~0.94 (mean 0.34±0.29) and 0.18~0.83 (mean 0.57±0.20), respectively. Four markers remained deviated from the Hardy-Weinberg Equilibrium (HWE) after multiple comparison correction for Bonferroin ( P HWE < 0.05). This is the first set of microsatellite markers of P. lutea . They should be useful for the research on the genetic diversity and connectivity of this species and the conservation of endangered coral reef ecosystems and reef-building coral communities in the Indo-Pacific region.

Development of novel EST-SSR markers for Ephedra sinica (Ephedraceae) by transcriptome database mining.

Premise of the Study Ephedra sinica (Ephedraceae) is a gymnosperm shrub with a wide distribution across Central and Eastern Asia. It is widely cultivated as a medicinal plant, but its wild populations are monitored to determine whether protection is needed.Methods and ResultsThirty‐six microsatellite markers, including 11 polymorphic markers, were developed from E. distachya RNA‐Seq data deposited in the National Center for Biotechology Information dbEST database. Among 100 genotyped E. sinica individuals originating from five different population groups, the allele number ranged from three to 22 per locus. Levels of observed and expected heterozygosity ranged from 0 to 0.866 (average 0.176) and 0 to 0.876 (average 0.491), respectively. Allelic polymorphism information content ranged from 0.000 to 0.847 (average 0.333). Cross‐species amplifications were successfully conducted with two related Ephedra species for all 11 di‐ or trinucleotide simple sequence repeats.ConclusionsThis study provides the first set of microsatellite markers for genetic monitoring and surveying of this medicinal plant.

Genome survey and development of 15 microsatellite molecular markers in Syagrus coronata (Martius) Beccari (Arecaceae) by next-generation sequencing

Syagrus coronata (Martius) Beccari belongs to the Arecaceae family, an economically and ecologically important species of palm tree. It grows in northeastern Brazil and has a widespread geographic distribution in the “Caatinga” (a seasonally dry tropical forest biome) and Atlantic forest domains (a rain forest biome). The aim of this study was to perform a genome survey and develop SSR markers using high-throughput sequencing. We obtained 78,231,881 million Illumina single-end and paired-end reads, and these reads were used to obtain contigs with a minimum of 500 nucleotides using Ray software. Mining of the SSRs was performed with Phobos software, while the primers were designed by Primer3. Thirty loci were analyzed, three of which were not amplified, 12 were monomorphic, and 15 were polymorphic. Five to 11 alleles per locus were observed, with the observed and expected heterozygosity at 0.06–0.63 and 0.73–0.88, respectively. Here, we report the first set of microsatellite markers developed for S. coronata, and their polymorphism levels indicate they can be used as powerful tools for evaluating genetic diversity and population structure, and thus contribute to conservation studies.

Genome-wide discovery and characterization of microsatellite markers from Melipona fasciculata (Hymenoptera: Apidae), cross-amplification and a snapshot assessment of the genetic diversity in two stingless bee populations

Brazilian native meliponines are currently threatened by increased human impacts. The assessment of their genetic variation by microsatellite DNA markers can assist in the conservation of populations and help in the planning and establishment of efficient management strategies. The purpose of this study was to develop the first set of microsatellite markers for Melipona fasciculata, selected from partial genome assembly of Illumina paired-end reads. Primer pairs were designed for each detected locus at their flanking regions. Bee samples were genotyped from two different populations of Northeastern Brazil for marker characterization and validation. A total of 17 microsatellite loci displayed polymorphism. Mean HE and HO heterozygosities were 0.453 and 0.536, respectively. PIC across all loci ranged from 0.108 to 0.714. A genetic diversity analysis revealed high values for population differentiation estimates (FST = 0.194, RST = 0.230, and Dest = 0.162) within the investigated region. PCoA and Bayesian clustering showed a separation of the species into two distinct clusters. These microsatellite markers have demonstrated strong potential for population-level genetic studies. Moreover, the preliminary analysis of the genetic diversity in M. fasciculata provides provisional evidence of significant population differentiation between the two studied populations.

Isolation and characterization of novel microsatellites for Abies koreana and A. nephrolepis (Pinaceae).

Abies koreana is an endemic and rare species from Korea and is classified as endangered by the International Union for Conservation of Nature. Although the genetic diversity assessment for current population of A. koreana needs to be performed urgently, no microsatellite markers have been developed for this species. In the present study, we developed 22 novel polymorphic microsatellite loci and the characteristics of these loci were determined in A. koreana as well as in Abies nephrolepis, the most closely related species, and these loci were compared with previously reported microsatellite markers developed for the Abies genus. Genomic sequence (161 Mbp; 325,776 reads) was obtained from one individual of A. koreana using Roche 454 GS-FLX Titanium sequencing and 19,258 repeat motifs were identified from it. A total of 288 primer pairs with high copy numbers of di-repeat motifs were evaluated for amplification in A. koreana and A. nephrolepis. A total of 71 primer pairs successfully amplified fragments, of which 22 showed polymorphisms in A. koreana and A. nephrolepis. The average expected diversity was 0.767 and 0.717 in A. koreana and A. nephrolepis, respectively; these heterozygosity levels were moderate compared to the previously reported microsatellite loci from Abies species. This is the first set of microsatellite markers developed for A. koreana as well as A. nephrolepis and further population genetic studies of both species and genetic delimitation can be carried out for the species conservation and management.

Isolation and characterisation of 20 microsatellite loci from the Chinese green tree viper Trimeresurus stejnegeri stejnegeri

The Chinese green tree viper (Trimeresurus stejnegeri stejnegeri), one of the most common snakes in Southeast Asia, can be a good model species for evolutionary and behavioral research. However, there is no high polymorphic co-dominant marker that can be used for individual-based genetic analyses available for this species. Therefore, we developed 20 polymorphic microsatellite loci for T. s. stejnegeri in Taiwan by screening a microsatellite-enriched DNA library. The allele numbers of these loci ranged from 3 to 22, and the observed heterozygosity were 0.042-1.000. The probability of false parent non-exclusion was 2 × 10−5 for those loci that conformed to the Hardy–Weinberg equilibrium. These highly polymorphic microsatellite markers are useful for both individual-based and population-level analysis. Furthermore, this is the first set of microsatellite markers developed for Trimeresurus, and could be applied to closely related species to address various research questions on evolution and animal behavior.

Isolation and characterization of microsatellite markers for Jasminum sambac (Oleaceae) using Illumina shotgun sequencing

Premise of the study:Microsatellite markers of Jasminum sambac (Oleaceae) were isolated to investigate wild germplasm resources and provide markers for breeding.Methods and Results:Illumina sequencing was used to isolate microsatellite markers from the transcriptome of J. sambac. A total of 1322 microsatellites were identified from 49,772 assembled unigenes. One hundred primer pairs were randomly selected to verify primer amplification efficiency. Out of these tested primer pairs, 31 were successfully amplified: 18 primer pairs yielded a single allele, seven exhibited fixed heterozygosity with two alleles, and only six displayed polymorphisms.Conclusions:This study obtained the first set of microsatellite markers for J. sambac, which will be helpful for the assessment of wild germplasm resources and the development of molecular marker–assisted breeding.

Development and transferability of microsatellite markers in Habenaria nuda and H. repens (Orchidaceae)

We report here the first set of microsatellite markers developed for species of Habenaria. A set of microsatellite markers for Habenaria nuda Lindl. and H. repens Nutt. were isolated, characterized, and tested for cross-amplification between these congeneric species. These species constitute two of the many existing complexes of the genus Habenaria. A total of nine markers were isolated from both species and characterized in two populations of each, with some of the markers being successfully cross-amplified. The numbers of alleles per locus ranged from four to 12 in H. nuda, and the mean numbers of alleles per locus were 6.0 and 6.14 in each of the populations analyzed. The observed (Ho) and expected (He) heterozygosities within the two populations varied from 0.294 to 0.850 and from 0.517 to 0.851, respectively. The numbers of alleles per locus ranged from four to seven in H. repens, and the mean numbers of alleles per locus were 2.8 and 4.0 in each of the populations analyzed. The Ho ranged from 0.000 to 0.563, and the He ranged from 0.000 to 0.666. The polymorphism levels found indicate these markers as powerful tools for future population genetic analyses in these and related species and could be used to address ecological, evolutionary, and taxonomic questions.

Isolation and characterization of microsatellite markers for Axonopus compressus (Sw.) Beauv. (Poaceae) using 454 sequencing technology.

Axonopus compressus (Sw.) Beauv. is a perennial herb widely used as a garden lawn grass. In this study, we used Roche 454 pyrosequencing, combined with the magnetic bead enrichment method FIASCO, to isolate simple sequence repeat markers from the A. compressus genome. A total of 1942 microsatellite loci were identified, with 53,193 raw sequencing reads. One hundred micro-satellite loci were selected to test the primer amplification efficiency in 24 individuals; 14 primer pairs yielded polymorphic amplification products. The number of observed alleles ranged from two to six, with an average of 3.5. Shannon's Information index values ranged from 0.169 to 0.650, with an average of 0.393. Nei's genetic diversity values ranged from 0.108 to 0.457, with an average of 0.271. This first set of microsatellite markers developed for Axonopus will assist in the development of molecular marker-assisted breeding and the assessment of genetic diversity in A. compressus.

Development and characterization of nine polymorphic microsatellite markers for the blue sheep (Pseudois nayaur)

Pseudois nayaur is a major component of Caprini and a keystone species in Qinghai-Tibet Plateau ecosystems. Due to the excessive hunting and habitat loss, its numbers are being reduced drastically. We developed and characterized the first set of microsatellite markers in this species to investigate its population genetic diversity and structure. Nine polymorphic loci displayed an average of 5.73 alleles (range between 2 and 14) and the levels of expected heterozygosity ranged from 0.422 to 0.816. Cross-amplification within dwarf blue sheep was successful for the nine loci. The loci characterized here will be useful for detecting fine-scale spatial structuring and resolving the taxonomic status of divergent Pseudois populations.

Novel polymorphic EST-derived microsatellite markers for the red-listed five needle pine, Pinus dabeshanensis

Pinus dabeshanensis is an endangered five needle pine endemic to mid-South of China. This pine finds its very limited natural occurrence in the Dabie mountains area with altitudes ranging 900–1400 m. We identified 3,516 SSR loci from the transcriptome assembly of the congeneric species P. lambertiana, and evaluated 671 loci we found. Finally, 28 novel polymorphic microsatellite markers were developed for P. dabeshanensis. Markers were characterized using 36 individuals, representing all existing populations from across the species geographic range. Species level analysis of these 28 polymorphic markers revealed the mean number per locus was 2.786 (range 2–6) and mean observed and expected heterozygosities were 0.332 (range 0.000–1.000) and 0.316 (range 0.033–0.691), respectively. This study provides the first set of microsatellite markers for the conservation genetics which in turn will facilitate conservation management for this pine.

Microsatellite markers for the Arctic copepod Calanus glacialis and cross-amplification with C. finmarchicus

Calanus glacialis is a major component of Arctic zooplankton and a keystone species in Arctic marine ecosystems. Due to the observed climate warming, its numbers are being reduced to the advantage of a sibling Atlantic species Calanus finmarchicus. We developed and characterized the first set of microsatellite markers in this species to investigate its population genetic structure and dispersal capabilities. Nine polymorphic loci displayed an average of 7.3 alleles (range between 2 and 13) and the levels of expected heterozygosity ranged from 0.039 to 0.806. These provide a valuable tool to understand present connectivity patterns across Arctic regions, look for signatures of past climate effects and predict the response to future climate-driven environmental changes. Additionally, due to the cross-amplification with C. finmarchicus, the markers can be used to discriminate between these sibling species.

Development of novel polymorphic microsatellite markers by technology of next generation sequencing in western white pine

The invasive fungal pathogen Cronartium ribicola has seriously damaged five-needle pines in North America. Genetic diversity assessment using DNA markers among natural populations will benefit conservation and restoration programs. This report presents characterization of the first set of microsatellite markers developed by analyzing a reference transcriptome de novo assembled from RNA-seq data and expressed sequence tags in western white pine (Pinus monticola). A total of 1,948 microsatellite loci were detected in 1,876 contigs. Forty-six loci were verified to be polymorphic, with allele number per locus ranging from 2 to 12. The mean observed and expected heterozygosity per locus was measured as 0.671 and 0.485, respectively. These microsatellite markers will be useful for characterizing genetic diversity and gene-flow among natural stands and population structure among seed families for conservation of this conifer species across western North America.

Development and validation of microsatellite markers for Brachiaria ruziziensisobtained by partial genome assembly of Illumina single-end reads

BackgroundBrachiaria ruziziensis is one of the most important forage species planted in the tropics. The application of genomic tools to aid the selection of superior genotypes can provide support to B. ruziziensis breeding programs. However, there is a complete lack of information about the B. ruziziensis genome. Also, the availability of genomic tools, such as molecular markers, to support B. ruziziensis breeding programs is rather limited. Recently, next-generation sequencing technologies have been applied to generate sequence data for the identification of microsatellite regions and primer design. In this study, we present a first validated set of SSR markers for Brachiaria ruziziensis, selected from a de novo partial genome assembly of single-end Illumina reads.ResultsA total of 85,567 perfect microsatellite loci were detected in contigs with a minimum 10X coverage. We selected a set of 500 microsatellite loci identified in contigs with minimum 100X coverage for primer design and synthesis, and tested a subset of 269 primer pairs, 198 of which were polymorphic on 11 representative B. ruziziensis accessions. Descriptive statistics for these primer pairs are presented, as well as estimates of marker transferability to other relevant brachiaria species. Finally, a set of 11 multiplex panels containing the 30 most informative markers was validated and proposed for B. ruziziensis genetic analysis.ConclusionsWe show that the detection and development of microsatellite markers from genome assembled Illumina single-end DNA sequences is highly efficient. The developed markers are readily suitable for genetic analysis and marker assisted selection of Brachiaria ruziziensis. The use of this approach for microsatellite marker development is promising for species with limited genomic information, whose breeding programs would benefit from the use of genomic tools. To our knowledge, this is the first set of microsatellite markers developed for this important species.

Development of microsatellite markers for Pimenta pseudocaryophyllus (Myrtaceae), a wild South American species

We developed the first set of microsatellite markers for Pimenta pseudocaryophyllus to support further studies on genetic diversity and to inform conservation strategies. The microsatellite-enriched library approach was used to isolate and characterize 12 new molecular markers. It was possible to detect 11 polymorphic microsatellite loci and one monomorphic locus. The polymorphism information content ranged from 0.317 to 0.869. These molecular markers will be valuable tools to aid in understanding the biology of P. pseudocaryophyllus and to detect ongoing consequences of its exploitation, in the context of conservation genetics.

Development and characterization of microsatellite markers for the wild South American Passiflora cincinnata (Passifloraceae)

We developed the first set of microsatellite markers for the passion fruit, Passiflora cincinnata, to provide tools for further study of its genetic diversity and to support current conservation and genetic studies. We used a microsatellite-enriched library approach to isolate and characterize 25 new molecular markers. Seven primer pairs successfully amplified polymorphic microsatellite regions, with an average of five alleles per locus. The mean values of expected and observed levels of heterozygosity were 0.516 and 0.525, respectively. The microsatellite markers identified in this study may be valuable tools for population genetic studies, and this set of markers also may be useful in the design of a genetic conservation strategy, mating system, and hybridization studies.

The first set of microsatellite markers developed for the ancient Parabathynellidae (Syncarida, Malacostraca) and their utility for other groundwater fauna

Aquifers harbor a unique and highly specialized fauna (stygofauna). The stygofauna often comprise short-range endemics, making them particularly vulnerable to extinction. In Australia, the demand for groundwater is ever-increasing, resulting in a conflict of interest concerning the management and conservation of these ecosystems. Knowledge of the levels of connectivity for groundwater taxa is needed to develop appropriate management strategies. We used next-generation sequencing to develop a panel of 10 polymorphic microsatellite markers for the Parabathynnelidae, an ancient group of groundwater invertebrates. These loci were amplified by PCR in 32 individuals from Wellington, New South Wales Australia. The number of alleles per locus ranged from 2 to 7, and observed and expected heterozygosities from 0.32 to 0.90 and 0.31 to 0.84, respectively. Five loci were successfully amplified in stygofauna taxa other than parabathynellids. These molecular markers will be a valuable resource for the conservation management of groundwater invertebrate populations.