Microsatellite-containing Sequences Research Articles

Development of microsatellite markers for the invasive mosquito Aedes koreicus

BackgroundAedes koreicus is a mosquito species native to East Asia which has recently invaded several countries in Europe. In Italy, this mosquito was first detected in the North-East in 2011 and is now widely distributed in the entire northern part of the country. The development of specific genetic markers, such as microsatellites, is necessary to uncover the dispersal routes of this mosquito from its native areas and, eventually, to plan future control interventions.MethodsAvailable raw sequences of genomic DNA of Ae. koreicus were screened in silico using BLASTn to identify possible microsatellite-containing sequences. Specific primer pairs were then designed, and their efficiency was determined through polymerase chain reaction (PCR) on 32 individuals of Ae. koreicus collected in Italy. PCR conditions were optimised in three multiplex reactions. Genotyping of individual mosquitoes was performed on both single and multiplex PCR reactions. Finally, analysis of intra-population variation was performed to assess the level of polymorphism of the markers.ResultsMosquito genotyping provided consistent results in both single and multiplex reactions. Out of the 31 microsatellite markers identified in the Ae. koreicus genome raw sequences, 11 were polymorphic in the examined mosquito samples.ConclusionsThe results show that the 11 microsatellite markers developed here hold potential for investigating the genetic structure of Ae. koreicus populations. These markers could thus represent a novel and useful tool to infer the routes of invasion of this mosquito species into Europe and other non-native areas.Graphical

Comparative genomics approach to build a genome-wide database of high-quality, informative microsatellite markers: application on Phytophthora sojae, a soybean pathogen

Microsatellites are a tract of repetitive, short DNA motifs (usually 1 to 6 bp) abundant in eukaryotic genomes. They are robust molecular markers in many areas of studies. Development of microsatellite markers usually involves three steps: (1) obtaining microsatellite-containing sequences, (2) primer design, and (3) screening microsatellite loci for polymorphism. The first and third steps require considerable resources. Next generation sequencing technologies have greatly alleviated the constraint of the first step. In this study, we leveraged the availability of genome assemblies of multiple individuals in many species and designed a comparative genomics approach to bioinformatically identify polymorphic loci. Our approach can eliminate or greatly reduce the need of experimental screening for polymorphism and ensure that the flanking regions do not have length difference that would confound interpretation of genotyping results using microsatellite markers. We applied this approach on Phytophthora sojae, a soybean pathogen, and identified 157 high-quality, informative microsatellite markers in this oomycete. Experimental validation of 20 loci supported bioinformatics predictions. Our approach can be readily applied to other organisms of which the genomes of multiple individuals have been sequenced.

Development of nuclear microsatellite loci for Pinus albicaulis Engelm. (Pinaceae), a conifer of conservation concern.

Pinus albicaulis (whitebark pine) is a widely-distributed but rapidly declining high elevation western North American tree and a candidate for listing under the U.S. Endangered Species Act. Our objectives were to develop reliable nuclear microsatellite markers that can be used to assess within-population genetic diversity as well as seed and pollen migration dynamics, and to validate markers using two geographically proximal P. albicaulis populations. We identified 1,667 microsatellite-containing sequences from shotgun DNA libraries of P. albicaulis. Primer pairs were designed for 308 unique microsatellite-containing loci, and these were evaluated for PCR amplification success and segregation in a panel of diploid needle tissue. DNA was extracted with an SDS protocol, and primers were screened through gel electrophoresis. Microsatellites were genotyped through fluorescent primer fragment analysis. Ten novel and 13 transferred loci were found to be reproducible in analyses based on 20 foliage samples from each of two locations: Henderson Mountain, Custer Gallatin National Forest, Montana, and Mt. Washburn, Yellowstone National Park, Wyoming (USA). Transferred loci had higher numbers of alleles and expected heterozygosities than novel loci, but also revealed evidence for a higher frequency of null alleles. Eight of the 13 transferred loci deviated significantly from Hardy-Weinberg Equilibrium, and showed large positive FIS values that were likely inflated by null alleles. Mantel’s tests of transferred and novel markers showed no correlation between genetic and geographic distances within or among the two sampled populations. AMOVA suggests that 91% of genetic variability occurs within populations and 9% between the two populations. Studies assessing genetic diversity using these microsatellite loci can help guide future management and restoration activities for P. albicaulis.

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.

Population genetic structure and identification of loci under selection in the invasive tunicate, Botryllus schlosseri, using newly developed EST-SSRs

Botryllus schlosseri, also known as golden star tunicate, is generally considered of European origin and has successfully invaded coastal waters of all continents except Antarctica. Owing to its significantly negative ecological impacts, it is crucial to understand its dispersal dynamics and mechanisms of invasion success. Here, we identified 1020 microsatellite-containing sequences from 98,626 expressed sequence tags (ESTs), and developed and characterized 17 polymorphic microsatellites (i.e. EST-SSRs) based on populations both from native (French coast) and invasive ranges (Canadian coast). The number of alleles, observed heterozygosity, and expected heterozygosity ranged from 2 to 12, 0.200 to 0.783, and 0.523 to 0.888 for the French population, and from 2 to 10, 0 to 0.524 and 0.043 to 0.827 for the Canadian population, respectively. We found significant population genetic differentiation between the native and invasive populations (pairwise FST = 0.1712). Moreover, principal coordinates analysis and Bayesian clustering test suggest long-distance dispersal between distant populations. When all loci were subjected for selection analyses, two loci (BS3244 and BS5339) were under selection based on the LOSITAN test. The results obtained in this study can help understand how ecological and evolutionary processes shape population genetic structure, and further how these processes contribute to invasion success.

Characterization of microsatellites and repetitive flanking sequences (ReFS) from the topmouth culter (Culter alburnus Basilewsky)

The topmouth culter (Culter alburnus) is an economically important freshwater fish in China. We obtained 159 microsatellite containing sequences (MCSs) from genomic DNA in this species enriched by (CAA)8 and (GAA) 8 probes. Careful examination of these sequences revealed the existence of cryptic repeated elements on presumed unique flanking regions. These cryptic elements can be grouped into three families, with the MCSs of the each family sharing regions of similarity ranging between 40 and 130 bp in length, with 96% sequence similarity. Repbase scans revealed that a large proportion of the cryptic repetitive DNA was identified as transposable elements (TEs). Complex patterns were apparent among these sequences. In most (89.2%), a single TE was identified in an MCS, in three instances, the same TE was observed twice in the same MCS. Some MCS have two or even four different TEs. We isolated nine polymorphic microsatellite loci from sequences with no matches to TEs. In a sample of 30 cultured C. alburnus, we found that the average allele number was 8.1 per locus (range = 4–17), with polymorphism informative content ranging from 0.364 to 0.898. These microsatellites can be used to study the population genetic diversity of this species.

Exploiting EST databases for the development and characterization of EST-SSR markers in blueberry (Vaccinium) and their cross-species transferability in Vaccinium spp

Expressed sequence tags (ESTs) databases are an increasingly valuable resource for developing SSR markers. However, to date, only a limited number of SSR markers have been developed from EST databases for blueberry. In this study, 17,267 ESTs in blueberry (Vaccinium spp.) were screened for SSRs with motif lengths varying from 2 to 6 base pairs (bp). In total, 2187 SSRs (12.67% of total screened ESTs) were identified from 1876 microsatellite-containing sequences. Dinucleotide repeats follow by trinucleotide repeats were the predominant motif. A total of 54 EST-SSR markers were characterized from 120 primer pairs designed by assaying 12 samples of the Vaccinium genus section Cyanococcus. The primer pairs exhibited a high degree of polymorphism with a mean PIC of 0.772, indicating high level informativeness within these EST-SSRs. The 54 new EST-SSR primer pairs comprising 540 alleles were used to investigate cross-species transferability among 26 accessions, which represented 8 species (three exotic cultivated species and five natural wild species from China) from 6 sections of Vaccinium. The results indicated that cross-species transferability ranged from 68.5% to 88.9%. Cluster analysis and PCoA based on 54 EST-SSR primer pairs were largely consistent with the traditional taxonomy and geographic locations among the materials tested, and both indicated that the EST-SSR primers were effective for the phylogenetic analysis in Vaccinium. Our results showed that those EST-SSR markers displayed considerable polymorphism and high transferability in both cultivated and wild species of Vaccinium, and will contribute to the studies of genetic diversity, linkage mapping, QTL and molecular assisted breeding of Vaccinium crops.

Microsatellite primers for the Pacific Northwest conifer Callitropsis nootkatensis (Cupressaceae).

• Premise of the study: Microsatellite primers were developed for Nootka cypress (Callitropsis nootkatensis) to provide quantitative measures for gene conservation that can assist in guiding management decisions for a species experiencing climate-induced decline.• Methods and Results: Using multiplexed massively parallel sequencing, we identified 136,785 microsatellite-containing sequences from 489,625 Illumina paired-end 80-bp reads. After stringent filtering, we selected 144 primer pairs and screened variation at these loci in five populations of C. nootkatensis. Loci show between three and 36 dinucleotide repeats per locus, with an average of 13. Screening of these markers in the Pacific Northwest relative Chamaecyparis lawsoniana demonstrated no marker transferability. This finding highlights the narrow taxonomic utility of microsatellite markers in Callitropsis.• Conclusions: These microsatellites show high polymorphism and can be used for routine screening of natural variation in Callitropsis nootkatensis, and will be particularly helpful in identifying clones and inbred relatives at the stand-level.

Development and characterization of microsatellite primers in Geranium carolinianum (Geraniaceae) with 454 sequencing.

• Premise of the study: Microsatellite primers were developed for Geranium carolinanum, a North American winter annual herb, for use in population genetic analyses.• Methods and Results: Genomic DNA enriched for repeat-containing fragments was sequenced on the Roche 454 Titanium platform, resulting in 470 primer pairs developed from 1115 microsatellite-containing sequences. A subset of 37 primer pairs was screened for polymorphism across three native and three invasive populations. We identified four monomorphic and eight polymorphic loci. Polymorphic loci contained between two and seven alleles per locus, and mean within-population expected heterozygosity ranged from 0.100 to 0.290. Within populations, observed heterozygosity for individual loci ranged from zero to 0.857, and expected heterozygosity ranged from 0.046 to 0.559.• Conclusions: These microsatellite markers will be useful for future studies of genetic diversity, structure, and mating systems across the geographic range of G. carolinianum, and may be transferable to other closely related species.

Characterization of microsatellite loci for a threatened species, the King Rail, Rallus elegans, using a next-generation sequencing protocol

The King Rail Rallus elegans (Audubon) has experienced population declines of 4.6 % per year on average since the 1960s. Wetland loss, most severely affecting inland marshes, has significantly reduced this species’ distribution to the coastal margins of its historic range. Polymorphic microsatellite markers were generated by 454 pyrosequencing of genomic DNA from King Rails, and Clapper Rails R. longirostris from Louisiana after AFLP enrichment and barcoding of restriction fragment cut sites across individuals. Of 1,419 microsatellite-containing sequences, 20 hypervariable microsatellite loci with up to 20 different alleles were identified at the alignment stage. We characterized nine loci, tested variability in 45 Atlantic coast King Rail samples, and detected 4–19 alleles per locus. Cross-species amplification revealed variability in the Virginia Rail, R. limicola, and Sora, Porzana carolina. These loci will be useful for studying secretive marsh rails, many of which are threatened or endangered.

Development and characterization of EST-SSR markers in the Chinese medicinal plant Callerya speciosa (Fabaceae).

• Premise of the study: The first microsatellite primers were developed for Callerya speciosa, an important traditional medicinal plant with island-mainland distributions in China, to further investigate its genetic variability and population structure.• Methods and Results: The microsatellite-containing sequences were selected from a cDNA library of C. speciosa. In total, 58 primer pairs were designed, and 25 of the corresponding loci showed clear amplification. Polymorphisms were assessed in two different natural populations. The mean number of alleles per locus ranged from two to nine. Observed and expected heterozygosity per loci ranged from 0.067 to 0.938 and 0.064 to 0.836, respectively. One out of 25 loci showed departure from Hardy–Weinberg equilibrium expectations in both populations, and three pairs of loci showed significant linkage disequilibrium after Bonferroni correction.• Conclusions: These microsatellite markers will be useful tools for genetic and conservation studies and to understand the evolutionary processes in Callerya species.

Isolation and validation of microsatellite markers from a depleted South African sciaenid species, the dusky kob (Argyrosomus japonicus), by means of the FIASCO/454 approach

The dusky kob (Argyrosomus japonicus) is a large, estuarine-dependent sciaenid fish that has been severely depleted in South African waters and that, in recent years, has received considerable attention from the local fish farming industry. Discovery and application of appropriate molecular markers is necessary to improve the understanding of wild population structure, assist the effectiveness of broodstock and breeding programmes, and ensure monitoring of potential interactions between wild and farmed fish. The present study uses a recently tested approach that combines the FIASCO enrichment protocol with 454 GS-FLX Next Generation Sequencing, to identify large numbers of microsatellite-containing sequences at a low cost and high discovery rate from the dusky kob genome. Following the FIASCO enrichment (targeting specifically tetranucleotide repeats), 2,355 potential tetranucleotide microsatellites (perfect repeat motifs including eight or more repeat units flanked by regions for primer design) were identified from 1/5th of a single 454 lane. From these sequences, a test panel of 60 potential markers was selected for validation. A total of eight (13 %) markers were successfully amplified from a test sample of wild dusky kob individuals and showed high levels of polymorphism (observed heterozygosity per locus ranging between 0.375 and 0.905). Cross-species amplification of seven of these markers was also successfully carried out in another closely related and commercially important South African sciaenid species, the silver kob (A. inodorus). The microsatellite markers developed in the present study are readily available tools suitable to address genetic variability of Argyrosomus species of southern Africa.

Microsatellite primers for the Pacific Northwest endemic coniferChamaecyparis lawsoniana(Cupressaceae)

Microsatellite primers were developed for the Pacific Northwest conifer, Chamaecyparis lawsoniana (Cupressaceae), to enhance efficiencies in disease-resistance breeding and germplasm screening for gene conservation of this rare species. Using multiplexed massively parallel Illumina sequencing, we identified over 300000 microsatellite-containing sequences from 2 million paired-end microreads. After stringent filtering and primer evaluation, we selected 11 primer pairs and used these to screen variation in four populations of C. lawsoniana. Loci show between three and 10 repeats per locus, with an average of eight. Screening of these markers in the North American relative Callitropsis nootkatensis demonstrated limited marker transferability, but these markers could have utility in Asian species of Chamaecyparis. These microsatellite primers show high polymorphism and should provide a high level of individual discrimination for paternity analysis in defined pedigrees, and routine screening of wild variation in Chamaecyparis lawsoniana.

Partial sequencing of the bottle gourd genome reveals markers useful for phylogenetic analysis and breeding

BackgroundBottle gourd [Lagenaria siceraria (Mol.) Standl.] is an important cucurbit crop worldwide. Archaeological research indicates that bottle gourd was domesticated more than 10,000 years ago, making it one of the earliest plants cultivated by man. In spite of its widespread importance and long history of cultivation almost nothing has been known about the genome of this species thus far.ResultsWe report here the partial sequencing of bottle gourd genome using the 454 GS-FLX Titanium sequencing platform. A total of 150,253 sequence reads, which were assembled into 3,994 contigs and 82,522 singletons were generated. The total length of the non-redundant singletons/assemblies is 32 Mb, theoretically covering ~ 10% of the bottle gourd genome. Functional annotation of the sequences revealed a broad range of functional types, covering all the three top-level ontologies. Comparison of the gene sequences between bottle gourd and the model cucurbit cucumber (Cucumis sativus) revealed a 90% sequence similarity on average. Using the sequence information, 4395 microsatellite-containing sequences were identified and 400 SSR markers were developed, of which 94% amplified bands of anticipated sizes. Transferability of these markers to four other cucurbit species showed obvious decline with increasing phylogenetic distance. From analyzing polymorphisms of a subset of 14 SSR markers assayed on 44 representative China bottle gourd varieties/landraces, a principal coordinates (PCo) analysis output and a UPGMA-based dendrogram were constructed. Bottle gourd accessions tended to group by fruit shape rather than geographic origin, although in certain subclades the lines from the same or close origin did tend to cluster.ConclusionsThis work provides an initial basis for genome characterization, gene isolation and comparative genomics analysis in bottle gourd. The SSR markers developed would facilitate marker assisted breeding schemes for efficient introduction of desired traits.

잣나무( Pinus koraiensis)의 cDNA library 제작 및 EST 분석

In this study, we report the generation and analysis of a total of 1,211 expressed sequence tags (ESTs) from Pinus koraiensis. A cDNA library was generated from the young leaf tissue and a total of 1,211 cDNA were partially sequenced. EST and unigene sequence quality were determined by computational filtering, manual review, and BLAST analyses. In all, 857 ESTs were acquired after the removal of the vector sequence and filtering over a minimum length 50 nucleotides. A total of 411 unigene, consisting of 89 contigs and 322 singletons, was identified after assembling. Also, we identified 77 new microsatellite-containing sequences from the unigenes and classified the structure according to their repeat unit. According to homology search with BLASTX against the NCBI database, 63.1% of ESTs were homologous with known function and 22.2% of ESTs were matched with putative or unknown function. The remaining 14.6% of ESTs showed no significant similarity to any protein sequences found in the public database. Gene ontology (GO) classification showed that the most abundant GO terms were transport, nucleotide binding, plastid, in terms biological process, molecular function and cellular component, respectively. The sequence data will be used to characterize potential roles of new genes in Pinus and provided for the useful tools as a genetic resource.

Isolation and characterization of polymorphic microsatellite markers from flax (Linum usitatissimum L.)

Flax ( Linum usitatissimum L.) is the third largest natural fiber crop and one of the five major oil crops in the world. Flax can be used as model plant for the bast fiber plants because of its small size and self pollination characters. Development of highly polymorphic co-dominant markers will be important for the molecular and genomic research in flax. Two microsatellite-enriched flax genomic libraries were constructed for trinucleotide TTC and ATC motifs. A total of 206 new microsatellite-containing sequences were identified and classified. Thirty eight polymorphic microsatellite markers were characterized and evaluated in eight cultivars from different countries and regions. These loci produced 2 to 12 alleles per locus with an average of 3.395. The observed and expected heterozygosities ranged from 0.000 to 1.000 (mean 0.257) and from 0.125 to 0.950 (mean 0.516), respectively. The mean polymorphic information content (PIC) value over 38 loci was 0.429, with 13 loci having PIC greater than 0.5. These novel polymorphic microsatellite loci will be useful in genetic linkage map construction, germplasm classification and identification, gene identification and QTL mapping, and marker-assisted selection in breeding of L. usitatissimum .

De novo sequencing and development of EST‐SSR for Gevuina avellana (Proteaceae), a nut tree from South America

• The aim of this study was to develop molecular tools to investigate the genetic structure and diversity of natural populations of Gevuina avellana (Molina, Proteaceae), the Chilean hazelnut. Simple sequence repeats (SSRs) derived from expressed sequence tags (EST-SSRs) were developed, optimized, and characterized. • The microsatellite-containing sequences were selected from a cDNA library developed from the nut. The eight marker loci showed two to seven alleles in 60 unrelated trees, from two different natural populations. The observed heterozygosity (H(o)) was 0.44, ranging from 0.07 to 0.92 for different loci. When the multilocus genotype of the eight EST-SSRs was considered, all trees could be differentiated. • The newly identified EST-SSRs in G. avellana and the multi-pooling strategy involving eight markers will facilitate future studies of clone identification, genetic structure, and diversity.

Recombinant microsatellite amplification: a rapid method for developing simple sequence repeat markers

Recombinant microsatellite (simple sequence repeat; SSR) amplification is a technique by which DNA sequences flanking microsatellites can be isolated rapidly on a large scale. The approach selectively amplifies microsatellite-containing sequences and recombines the amplicons by redigestion and ligation, in order to increase the yield of microsatellite flanks per clone two-fold and to further increase selectivity of amplification. Since this method does not require prior knowledge of the genomic sequence, it is especially useful for species for which abundant genomic sequences are not available. The feasibility of this approach was demonstrated by developing SSR markers in cultivated oats.

Comparative genomic analysis reveals species-dependent complexities that explain difficulties with microsatellite marker development in molluscs

Reliable population DNA molecular markers are difficult to develop for molluscs, the reasons for which are largely unknown. Identical protocols for microsatellite marker development were implemented in three gastropods. Success rates were lower for Gibbula cineraria compared to Littorina littorea and L. saxatilis. Comparative genomic analysis of 47.2 kb of microsatellite containing sequences (MCS) revealed a high incidence of cryptic repetitive DNA in their flanking regions. The majority of these were novel, and could be grouped into DNA families based upon sequence similarities. Significant inter-specific variation in abundance of cryptic repetitive DNA and DNA families was observed. Repbase scans show that a large proportion of cryptic repetitive DNA was identified as transposable elements (TEs). We argue that a large number of TEs and their transpositional activity may be linked to differential rates of DNA multiplication and recombination. This is likely to be an important factor explaining inter-specific variation in genome stability and hence microsatellite marker development success rates. Gastropods also differed significantly in the type of TEs classes (autonomous vs non-autonomous) observed. We propose that dissimilar transpositional mechanisms differentiate the TE classes in terms of their propensity for transposition, fixation and/or silencing. Consequently, the phylogenetic conservation of non-autonomous TEs, such as CvA, suggests that dispersal of these elements may have behaved as microsatellite-inducing elements. Results seem to indicate that, compared to autonomous, non-autonomous TEs maybe have a more active role in genome rearrangement processes. The implications of the findings for genomic rearrangement, stability and marker development are discussed.

Identification of host-defense genes and development of microsatellite markers from ESTs of hard clam Meretrix meretrix

The hard clam Meretrix meretrix is an economically important shellfish in China. However, genomic research on this species is still at early stage, and few genomic resources are available. The objective of the present study was to generate expressed sequence tags (ESTs), and identify host-defense genes and microsatellite markers for M. meretrix. Three cDNA libraries for intestine, mantle and hepatopancreas were constructed using highly efficient SMART (Switching Mechanism At 5' end of the RNA Transcript) method. A total of 3224 random clones were single-pass sequenced from 5'-ends, resulting in 3129 high-quality (>100 bp) ESTs averaging 734 bp. All the ESTs were assembled by software Cap 3, producing 1796 unigenes-1490 singletons and 306 contigs. All the unigenes were compared to the public protein database using tblastx, and 696 (38.8%) were homologues to known genes while the remaining 1100 (61.2%) appeared to be novel sequences. A total of 31 EST clusters were related to immune and defense functions. They included immune recognition receptors, proteases and protease inhibitors, and other immune-related genes. The screening of 1796 unigenes identified 55 (3.1%) microsatellite-containing sequences, with 20 having sufficient flanking sequences for primer design. Polymerase chain reaction amplification was successful for 12 primer pairs and 7 of them showed polymorphic. The EST collection and microsatellite markers obtained in this study provide a useful resource for further gene discovery and population genetic analysis in M. meretrix.