Microsatellite-enriched Library Research Articles

Characterization of microsatellite loci for three species of Tomoplagia (Diptera: Tephritidae) and absence of cross-species amplification

Tomoplagia (Diptera: Tephritidae) are endophagous herbivores on flowerheads of Asteraceae, with varying levels of host specialization. We isolated, characterized, and tested the heterologous amplification for sets of microsatellites of three species from this genus associated with native hosts in the Espinhaco mountain range, Brazil. We used two protocols to build microsatellite-enriched libraries and characterized 12 polymorphic loci for each fruit-fly species. The samples of T. grandis, a monophagous species, presented a lower number of alleles than the samples of the oligophagous species T. incompleta and T. bicolor. The average observed heterozygosity did not vary among species and the high values of Polymorphism Information Content suggest that the characterized microsatellite loci are quite informative. The primers developed for each Tomoplagia species failed to amplify the microsatellite loci for the other two species, which indicates a low conservation of the flanking nucleotide sequences in the three evaluated species. The sets of polymorphic microsatellites characterized for these species of Tomoplagia may be used in future ecological and evolutionary studies that aim to detect patterns of diversity on a fine scale.

Isolation and characterization of 15 new microsatellite markers for the globally endangered Lear's macaw Anodorhynchus leari.

A set of 16 microsatellite markers was characterized for Lear's macaw (Anodorhynchus leari) using DNA samples from captive individuals. Extending this molecular toolkit, including the use of samples from wild individuals, is expected to provide the required power of resolution for pedigree inference of both wild and captive individuals, and could support research on the genetic structure of wild populations.We characterize a set of 15 microsatellite markers optimized for the Lear's macaw, developed from a microsatellite-enriched library in a three-step procedure. Primer pairs were initially designed for 62 microsatellites with > 7 tandem repetitions. After amplification of DNA of five wild individuals from different localities, 22 loci seemed to be polymorphic and were further tested on 12 wild nestling samples. Fifteen unlinked loci showed unambiguous peaks and low to moderate polymorphism levels. The combination of the four most polymorphic markers allowed individual identification even of putative sibs.These markers complement previously described microsatellites developed for A. leari and constitute a fundamental genetic toolkit for the investigation of the genetics of both wild and captive populations, thus assisting integrated management plans for the conservation of this globally endangered species.

Microsatellites for the Neotropical Ant, Odontomachus chelifer (Hymenoptera: Formicidae)

Odontomachus chelifer (Latreille) (Ponerinae) is a ground-dwelling, predominantly carnivorous ant whose colonies may contain multiple egg-laying queens and are potentially susceptible to border effects in the Brazilian savanna known as Cerrado. The ecology and natural history of O. chelifer is well studied, but very little is known about the genetic diversity of O. chelifer colonies. In this study, we developed microsatellite markers for the study of genetic variation in O. chelifer. We created a microsatellite-enriched library that resulted in the development and characterization of 22 markers, of which 18 were found to be polymorphic in the population studied. The mean expected heterozygosity was 0.59, whereas the mean rarified allelic richness was determined as 4.27 alleles per locus. The polymorphism level detected was similar to genetic diversity estimates found in other poneromorph ant species. The microsatellites developed here are likely to be useful for the investigation of colony structure, functional polygyny, breeding system, and population genetics in O. chelifer. Moreover, the description of O. chelifer’s genetic diversity is crucial for its conservation and maintenance of its ecological role in the Cerrado savanna.

Development of novel g-SSR markers in guava (Psidium guajava L.) cv. Allahabad Safeda and their application in genetic diversity, population structure and cross species transferability studies

Dearth of genomic resources particularly, microsatellite markers in nutritionally and commercially important fruit crop, guava necessitate the development of the novel genomic SSR markers through the library enrichment techniques. Three types of 3' -biotinylated oligonucleotide probes [(CT)14, (GT)12, and (AAC)8] were used to develop microsatellite enriched libraries. A total of 153 transformed colonies were screened of which 111 positive colonies were subjected for Sanger sequencing. The clones having more than five motif repeats were selected for primer designing and a total of 38 novel genomic simple sequence repeats could be identified. The g-SSRs had the motif groups ranging from monomer to pentamer out of which dimer group occurred the most (89.47%). Out of 38 g-SSRs markers developed, 26 were found polymorphic, which showed substantial genetic diversity among the guava genotypes including wild species. The average number of alleles per locus, major allele frequency, gene diversity, expected heterozygosity and polymorphic information content of 26 SSRs were 3.46, 0.56, 0.53, 0.29 and 0.46, respectively. The rate of cross-species transferability of the developed g-SSR loci varied from 38.46 to 80.77% among the studied wild Psidium species. Generation of N-J tree based on 26 SSRs grouped the 40 guava genotypes into six clades with two out-groups, the wild guava species showed genetic distinctness from cultivated genotypes. Furthermore, population structure analysis grouped the guava genotypes into three genetic groups, which were partly supported by PCoA and N-J tree. Further, AMOVA and PCoA deciphered high genetic diversity among the present set of guava genotypes including wild species. Thus, the developed novel g-SSRs were found efficient and informative for diversity and population structure analyses of the guava genotypes. These developed novel g-SSR loci would add to the new genomic resource in guava, which may be utilized in genomic-assisted guava breeding.

Novel 28 microsatellite loci using high-throughput sequencing for an endangered species on Metasequoia glyptostroboides (Cupressaceae).

Metasequoia glyptostroboides is a living fossil and an endangered species listed in the International Union for Conservation of Nature (IUCN). Distinguishing the genotypes of all wild individuals of M. glyptostroboides is important to delimit management units and key germplasm resources. We characterized 28 novel polymorphic microsatellite loci using a streptavidin-biotin microsatellite-enriched library and Illumina high-throughput sequencing. Characteristics of each locus were tested using 140 individuals collected from five natural populations of M. glyptostroboides. The number of alleles per locus ranged from 3 to 20, with a mean number of about 8 alleles. The observed and expected heterozygosities in each population ranged from 0.0000 to 1.0000 and from 0.0000 to 0.8958, respectively. Four to nine loci were cross-amplified successfully in seven species of Cupressaceae. The novel SSR markers will provide a toolkit for DNA identification of all of the extant wild individuals guiding further conservation efforts of M. glyptostroboides.

Microsatellite markers in Spanish lime (Melicoccus bijugatus Jacq., Sapindaceae), a neglected Neotropical fruit crop

Spanish lime (Melicoccus bijugatus Jacq.) is a Neotropical fruit tree cultivated, mainly, in orchards for self-consumption or local sale. The genus Melicoccus includes other nine species with edible fruits, some of these species are at risk of extinction. Like for the vast majority of tropical fruit trees, there is no information on the genetic diversity of Spanish lime and its related species, and this is mostly due to the lack of molecular markers. The objectives of this study were to present the first microsatellite markers developed for Spanish lime, testing its usefulness on a sample of cultivated accessions, as well as its transferability to Huaya India (M. oliviformis). To do this, we performed high-throughput sequencing of microsatellite-enriched libraries of Spanish lime using Roche 454, assembled 9567 DNA contig sequences and identified 10,117 microsatellites. After screening 384 of those microsatellites on four DNA samples, 31 polymorphic markers were used to screen 25 accessions of Spanish lime and five of Huaya India collected in Yucatan, Mexico. Genetic diversity was low in Spanish lime (A = 20.61, HE = 0.38) and similar for both sexes of this species. Neighbor-Joining and PCoA analyses clearly discriminated between the two Melicoccus species studied. Nine of the markers showed unique alleles for Huaya India. The set of microsatellite markers developed has a great potential to generate information in relation to conservation genetics, improvement of elite cultivars and breeding programs for Spanish lime and related species.

Isolation and characterization of 21 polymorphic microsatellite loci for the rockpool shrimp Palaemon elegans using Illumina MiSeq sequencing

The rockpool shrimp Palaemon elegans is considered an important crustacean species within the European coastline fauna. This species is experiencing an ongoing geographical expansion beyond its native distribution range due to unintentional human introductions. A better knowledge of the genetic diversity, geographic structure and connectivity of its populations is necessary. In the present study, microsatellite loci were isolated using the Illumina MiSeq platform. The microsatellite-enriched library sequencing produced 3.9 million raw reads. Reads were processed and primer pairs were designed for microsatellite sequences amplification. Ninety-six microsatellite loci were preliminary screened in individuals from Atlantic and Mediterranean localities. From them, 21 loci exhibited reliable polymorphism and were thoroughly characterized in 30 individuals from a Cantabrian locality (Spain). No linkage disequilibrium between pairs of loci was detected. Number of alleles per locus ranged from 2 to 12. Observed and expected heterozygosities ranged from 0.033 to 0.833 and from 0.033 to 0.869 respectively. No significant departure from the Hardy-Weinberg equilibrium was detected in most of loci. This is the first time that microsatellite markers have been developed for P. elegans. This characterized microsatellite suite provides new suitable tools for further analyses, facilitating the understanding of population genetics both in natural and introduced populations.

Development and application of SSR markers derived from Bauhinia Strychnifolia a semi-endemic plant in Thailand

ABSTRACTBauhinia strychnifolia is a rare and semi-endemic plant in Thailand. Microsatellite-enriched libraries were constructed using (CT)15/(CAG)15 and (GT)15/(AAG)15 probes. Fourteen polymorphic loci produced a total of 104 alleles with a mean of 7.4 alleles per locus. The average expected and observed heterozygosity were 0.727 and 0.497, respectively. The polymorphism information content value ranged from 0.335 to 0.910. Nine microsatellite loci were successfully transferred across species to B. galpiniii, B. Siamensis, and B. tomentosa. The information obtained in this study will be useful in establishing a conservation strategy for B. strychnifolia in Thailand and for genetic studies of other Bauhinia sp.

Fifteen microsatellite markers for Herbertia zebrina (Iridaceae): An endangered species from South American grasslands.

Premise of the study:Polymorphic microsatellite loci were developed and used to genotype individuals of Herbertia zebrina (Iridaceae) as a first step for assessment of intraspecific genetic diversity.Methods and Results:Primer pairs for 47 markers were developed: 20 from a microsatellite-enriched library and 27 from a next-generation sequencing run using the Illumina MiSeq platform. Of those, 15 loci were considered successful, of which 12 were polymorphic and three were monomorphic. The primers were tested in 50 individuals from three populations of H. zebrina. Two to 14 alleles per locus were identified, and observed and expected heterozygosity were 0.00–0.95 and 0.18–0.89, respectively. Tests of cross-amplification to evaluate the applicability of these markers showed positive results in one congeneric species, H. darwinii, and in a phylogenetically closely related species, Calydorea crocoides.Conclusions:These microsatellite markers can be used for studies of genetic variation and genetic population structure, as well as to support conservation efforts.

Identification and characterization of simple sequence repeats (SSRs) for population studies of Puccinia novopanici

Switchgrass (Panicum virgatum L.) can be severely affected by rust disease. Recently switchgrass rust caused by P. emaculata (now confirmed to be Puccinia novopanici) has received most of the attention by the research community because this pathogen is responsible for reducing the biomass production and biofuel feedstock quality of switchgrass. Microsatellite markers found in the literature were either not informative (no allele frequency) or showed few polymorphisms in the target populations, therefore additional markers are needed for future studies of the genetic variation and population structure of P. novopanici. This study reports the development and characterization of novel simple sequence repeat (SSR) markers from a Puccinia emaculata s.l. microsatellite-enriched library and expressed sequence tags (ESTs). Microsatellites were evaluated for polymorphisms on P. emaculata s.l. urediniospores collected in Iowa (IA), Mississippi (MS), Oklahoma (OK), South Dakota (SD) and Virginia (VA). Puccinia novopanici single spore whole genome amplifications were used as templates to validate the SSR reactions protocol and to assess a preliminary population genetics statistics of the pathogen. Eighteen microsatellite markers were polymorphic (average PIC=0.72) on individual urediniospores, with an average of 8.3 alleles per locus (range 3 to 17). Of the 49 SSRs loci initially identified in P. emaculata s.l., 18 were transferable to P. striiformis f. sp. tritici, 23 to P. triticina, 20 to P. sorghi and 31 to P. andropogonis. Thus, these markers could be useful for DNA fingerprinting and population structure analysis for population genetics, epidemiology and ecological studies of P. novopanici and potentially other related Puccinia species.

Effective DNA fragmentation technique for simple sequence repeat detection with a microsatellite-enriched library and high-throughput sequencing.

Two different techniques for genomic DNA fragmentation before microsatellite-enriched library construction-restriction enzyme (NlaIII and MseI) digestion and sonication-were compared to examine their effects on simple sequence repeat (SSR) detection using high-throughput sequencing. Tens of thousands of SSR regions from 5 species of the plant family Myrtaceae were detected when the output of individual samples was >1 million paired-end reads. Comparison of the two DNA fragmentation techniques showed that restriction enzyme digestion was superior to sonication for identification of heterozygous genotypes, whereas sonication was superior for detection of various SSR flanking regions with both species-specific and common characteristics. Therefore, choosing the most suitable DNA fragmentation method depends on the type of analysis that is planned.

Rapid and effective isolation of candidate sequences for development of microsatellite markers in 30 fish species by using kit-based target capture and multiplexed parallel sequencing

Recent advances in next-generation sequencing (NGS) technology have accelerated the development of microsatellite markers for wildlife conservation genetics. Although the discovery of microsatellite-containing sequences based on NGS is more efficient with sequencing of a microsatellite-enriched library than with whole-genome shotgun sequencing, the process of constructing a microsatellite-enriched library is somewhat complicated. Therefore, many researchers prefer to use external services for the microsatellite-enrichment, which requires more time. To facilitate the rapid and effective development of novel microsatellite markers, we attempted to simplify the process of constructing a microsatellite-enriched library for multiplexed parallel sequencing. To capture microsatellite-containing sequences, we applied an easy-to-use commercially available kit for the hybridization and wash steps. After preparing shotgun libraries of 30 fish species for NGS, we captured microsatellite-containing DNA fragments directly from the shotgun libraries by using the commercially available kit. Next, three runs of multiplexed parallel sequencing were conducted on the 454 GS Junior platform. The resulting sequences for each species included high proportions of microsatellite-containing sequences (from 46 to 79%). Thus, sufficient numbers of primer sets, ranging from 1029 to 6606, were effectively designed for each species. Microsatellite capture and sequencing were completed in about a week, so the time required was substantially reduced. To validate the effectiveness of our strategy, we screened 44 potential primer sets designed for ayu (Plecoglossus altivelis). The results of polymorphisms revealed that allelic variability at 23 markers will be useful for studying population structure. These results prove the effectiveness of our improved approach for microsatellite marker development.

Development and characterization of novel microsatellite loci for the amphipod Cymadusa filosa

Microsatellites are molecular tools for answering a wide variety of questions in population genetics. Here, we developed a novel set of microsatellite loci for the amphipod Cymadusa filosa using a microsatellite-enriched library. In total, 22 loci were described, and ten polymorphic markers were characterized for 30 individuals from southeastern Brazil. The number of alleles ranged from two to 26 and the observed heterozygosity from 0.033 to 0.933. No significant deviation from Hardy-Weinberg equilibrium was found; there was no evidence for linkage disequilibrium, and null alleles were detected only in three loci at low frequencies. These markers will be useful tools for ecological and evolutionary studies involving C. filosa.

Isolation and Characterization of Microsatellite Markers for Stenotaphrum Trin. Using 454 Sequencing Technology

St. augustinegrass (Stenotaphrum sp.) is a warm-season perennial turfgrass that grows widely in tropical regions around the world. St. augustinegrass is valued for both its turf performance and high levels of resistance to biotic and abiotic stresses. The current study was aimed at developing nuclear microsatellite markers for st. augustinegrass. Pyrosequencing of an enriched microsatellite library on the Roche FLX platform using a 454 Titanium kit produced 57,306 sequence reads; 2614 of which contained short tandem repeats. One hundred primer pairs were tested with 18 accessions from the U.S. Department of Agriculture National Plant Germplasm System st. augustinegrass collection grown in Griffin, GA. This collection contains both Stenotaphrum dimidiatum and Stenotaphrum secundatum accessions. Among revealed 100 primer pairs, 33 were polymorphic. A total of 175 alleles were amplified. The number of observed alleles per primer pair ranged from two to 10, with an average of 5.3. Shannon’s information index and Nei’s genetic diversity values were 0.4403 and 0.2873, respectively. This set of microsatellite markers is useful for assessment of genetic diversity and construction of molecular genetic linkage maps in st. augustinegrass.

Characterization of 16 microsatellite markers for the Oreinotinus clade of Viburnum (Adoxaceae).

Premise of the study:Microsatellite loci were isolated from four species of Viburnum (Adoxaceae) to study population structure and assess species boundaries among morphologically similar South American Viburnum species of the Oreinotinus clade.Methods and Results:Using a microsatellite-enriched library and mining next-generation sequence data, 16 microsatellites were developed. Each locus was tested on two populations of V. triphyllum and one population of V. pichinchense. For nuclear loci, one to 13 alleles were recovered, expected heterozygosity ranged from 0 to 0.8975, Simpson diversity index ranged from 0.0167 to 1.000, and Shannon diversity index ranged from 0 to 2.3670 in a given population. For the mitochondrial locus, three to six alleles were recovered and unbiased haploid diversity values ranged from 0.756 to 0.853 in a given population.Conclusions:The 16 microsatellite loci developed for the Oreinotinus clade (Viburnum, Adoxaceae) will inform investigations of population structure and species boundaries within this group.

Development of 14 microsatellite markers in Odontites vernus s.l. (Orobanchaceae) and cross-amplification in related taxa.

Premise of the study:Microsatellite primers were developed for the first time in the root hemiparasite herb Odontites vernus (Orobanchaceae). These markers will be useful to investigate the role of polyploidization in the evolution of this diploid-tetraploid complex, as well as the extent of gene flow between different ploidy levels.Methods and Results:Fourteen polymorphic and reproducible loci were identified and optimized from O. vernus using a microsatellite-enriched library and 454 Junior sequencing. The set of primers amplified di- to pentanucleotide repeats and showed two to 13 alleles per locus. Transferability was tested in 30 taxa (19 belonging to Odontites and 11 from eight other genera of Orobanchaceae tribe Rhinantheae).Conclusions:The results indicate the utility of the newly developed microsatellites in O. vernus and several other species, which will be useful for taxon delimitation and conservation genetics studies.

Development of genomic simple sequence repeats (g-SSR) markers in Tinospora cordifolia and their application in diversity analyses

Tinospora cordifolia, a medicinal plant of economic importance, has very rich diversity and distributed across India growing in wild. However, for its judicious exploitation through targeted breeding program there is complete lack of genomic resources which is necessary for its desired utilization. In the present study, we have developed novel genomic-SSRs (g-SSR) markers, which due to their wider distribution across the genome find greater usefulness in assessing genetic diversity. For development of g-SSR markers, four microsatellite enriched libraries [(CT)14, (AC)10, (GT)12 and (AAC)8] were constructed from genomic DNA of T. cordifolia accession (IC-281965) by using a streptavidin-biotin based enrichment approach. The 536 random recombinant clones from the four SSR enriched libraries were further verified by colony PCR for the presence of insert. From such screening, 356 clones were found positive and were subjected to sequencing using Sanger sequencing method. The sequence analyses revealed that 114 clones (32%) were having SSR repeats. Further analysis, based on microsatellite library enrichment showed that 60% SSR repeats were found in clones from GT enriched libraries followed by 38%, 23% and 19% in CT, AAC and AC enriched libraries respectively. Altogether 90 SSR sequences were submitted to the National Center for Biotechnology Information (NCBI) with accession numbers from KT384079 to KT384168. For diversity study 42 primer pairs were designed and all primers reproducibly amplified across all accessions of T. cordifolia. However, when such markers were used in analysis for Tinospora rumphii and Tinospora sinensis, only 83.3% and 85.7% of the markers were transferred successfully. The alleles generated across 28 accessions of three Tinospora species were assessed for genetic relatedness and it grouped accessions into three major clusters suggesting sufficient diversity in Tinospora. The g-SSR markers generated in the present study are a valuable genomic resource for effective utilization in crop improvement of Tinospora germplasm.

Microsatellite markers for Senna spectabilis var. excelsa (Caesalpinioideae, Fabaceae).

Premise of the study:Senna spectabilis var. excelsa (Fabaceae) is a South and Central American tree of great ecological importance and one of the most common species in several sites of seasonally dry forests. Our goal was to develop microsatellite markers to assess the genetic diversity and structure of this species.Methods and Results:We designed and assessed 53 loci obtained from a microsatellite-enriched library and an intersimple sequence repeat library. Fourteen loci were polymorphic, and they presented a total of 39 alleles in a sample of 61 individuals from six populations. The mean values of observed and expected heterozygosities were 0.355 and 0.479, respectively. Polymorphism information content was 0.390 and the Shannon index was 0.778.Conclusions:Polymorphism information content and Shannon index indicate that at least nine of the 14 microsatellite loci developed are moderate to highly informative, and potentially useful for population genetic studies in this species.

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 Eucommia ulmoides (Eucommiaceae), an endangered tree, using next-generation sequencing.

Eucommia ulmoides Oliver, a single extant species of Eucommiaceae, is an endemic dioecious tree in China. The natural resources of E. ulmoides have rapidly declined in recent years because of the over-collection of its cortex. To design a suitable protection strategy, it is necessary to develop a set of molecular markers to investigate genetic diversity and population structure of E. ulmoides. Pyrosequencing of an enriched microsatellite library by Roche 454 FLX+ platform was used to isolate simple sequence repeats (SSRs) for E. ulmoides. A total of 1568 SSRs that contained enough flanking sequences for primer pair design were identified from 45,236 raw sequence reads. One hundred SSRs were randomly selected to design primer pairs and polymerase chain reaction was performed. Among these 100 tested primer pairs, 16 were polymorphic across 18 individuals from three E. ulmoides populations. The number of alleles ranged from 3 to 8, with an average of 5.1. The expected heterozygosity ranged from 0.110 to 0.830, with an average of 0.648, and the observed heterozygosity ranged from 0.111 to 0.833, with an average of 0.524. The inbreeding coefficient ranged from -0.349 to 0.547. This set of microsatellite markers could be valuable for landscape genetic structure assessment and molecular marker-assisted breeding in E. ulmoides.