Microsatellite Discovery Research Articles

Pipeline for identification of genome-wide microsatellite markers and its application in assessing the genetic diversity and structure of the tropical sea cucumber Holothuria leucospilota

Microsatellite DNA (SSR) is extensively used as molecular markers in diverse fields, including molecular ecology, conservation biology, and molecular breeding. Nevertheless, the efficient and easy acquisition of high-quality SSR molecular markers for economically cultured marine animals remains a challenge. Holothuria leucospilota is an economically and ecologically important tropical sea cucumber. Recently, artificial breeding and cultivation of this species have been reported. However, there is still a lack of highly polymorphic SSR molecular markers for geographical population differentiation and genetic breeding. This study aims to develop and utilize SSR markers from genomic data to assess the genetic variation and population structure of H. leucospilota in the South China Sea. A comprehensive pipeline was created, integrating microsatellite discovery, primer design, and assessment into a single platform. Through this, 15 highly polymorphic microsatellite loci were identified and validated. The results revealed high genetic diversity and low genetic differentiation among H. leucospilota populations. Larval dispersal and ocean currents in the South China Sea were identified as the primary factors influencing genetic connectivity between populations. In addition, the artificial culture and releasing of H. leucospilota have influenced the genetic structure of their wild populations in the South China Sea. This study provides a pipeline for identifying SSR markers within genome data, and applies it to assess the genetic diversity and structure of H. leucospilota. The outcomes of this research are expected to be beneficial and applicable to the future aquaculture of tropical sea cucumbers.

A first look at the ‘repeatome’ of Benedenia humboldti, a major pathogen in yellowtail aquaculture: Repetitive element characterization, nuclear rRNA operon assembly, and microsatellite discovery

The monogenean Benedenia humboldti is a pathogen of the yellowtail Seriola lalandi in the South-Eastern Pacific ocean. Using low-coverage short Illumina 150bp pair-end reads sequencing, this study examines, for the first time, the 'repeatome' (= repetitive genomic elements), including the 45S ribosomal RNA DNA operon and microsatellites, in B. humboldti. Repetitive elements comprised a large fraction of the nuclear genome and a considerable proportion of them could not be assigned to known repeat element families. Taking into account only annotated repetitive elements, the most frequent belonged to the 45S ribosomal RNA operon or were classified as satellite DNA and Class I - Long Interspersed Nuclear Elements (LINEs) which were considerably more abundant than Class I - LTR elements. The ribosomal RNA gene operon in B. humboldti is comprised of, in the following order, a 5' ETS (length=233bp), ssrDNA (2082bp), ITS1 (346bp), 5.8S rDNA (150bp), ITS2 (572bp), lsrDNA (3887bp), and a 3' ETS (1097bp). A total of 15 SSRs were identified. These newly developed genomic resources will contribute to the better understanding of meta-population connectivity in this species, cryptic species in the genus, and will advance pest management strategies.

Developing an ultra-efficient microsatellite discoverer to find structural differences between SARS-CoV-1 and Covid-19.

MotivationRecently, the outbreak of Coronavirus-Covid-19 has forced the World Health Organization to declare a pandemic status. A genome sequence is the core of this virus which interferes with the normal activities of its counterparts within humans. Analysis of its genome may provide clues toward the proper treatment of patients and the design of new drugs and vaccines. Microsatellites are composed of short genome subsequences which are successively repeated many times in the same direction. They are highly variable in terms of their building blocks, number of repeats, and their locations in the genome sequences. This mutability property has been the source of many diseases. Usually the host genome is analyzed to diagnose possible diseases in the victim. In this research, the focus is concentrated on the attacker's genome for discovery of its malicious properties.ResultsThe focus of this research is the microsatellites of both SARS and Covid-19. An accurate and highly efficient computer method for identifying all microsatellites in the genome sequences is discovered and implemented, and it is used to find all microsatellites in the Coronavirus-Covid-19 and SARS2003. The Microsatellite discovery is based on an efficient indexing technique called K-Mer Hash Indexing. The method is called Fast Microsatellite Discovery (FMSD) and it is used for both SARS and Covid-19. A table composed of all microsatellites is reported. There are many differences between SARS and Covid-19, but there is an outstanding difference which requires further investigation.AvailabilityFMSD is freely available at https://gitlab.com/FUM_HPCLab/fmsd_project, implemented in C on Linux-Ubuntu system. Software related contact: hossein_savari@mail.um.ac.ir.

Optimizing depth and type of high-throughput sequencing data for microsatellite discovery.

PremiseSimple sequence repeat (SSR) markers (microsatellites) are a mainstay of many labs, especially when working on a limited budget, carrying out preliminary analyses, and in teaching. Whether SSRs mined from plant genomes or transcriptomes are preferred for certain applications, and the depth of sequencing needed to allow efficient SSR discovery, has not been tested.MethodsI used genome and transcriptome high‐throughput sequencing data at a range of sequencing depths to compare efficacy of SSR identification. I then tested primers from tomato for amplification, polymorphism, and transferability to related species.ResultsSmall assemblies (two million read pairs) identified ca. 200–2000 potential markers from the genome assemblies and ca. 600–3650 from the transcriptome assemblies. Genome‐derived contigs were often short, potentially precluding primer design. Genomic SSR primers were less transferable across species but exhibited greater variation (partially explained by being composed of more repeat units) than transcriptome‐derived primers.DiscussionSmall high‐throughput sequencing resources may be sufficient for identification of hundreds of SSRs. Genomic data may be preferable in species with low polymorphism, but transcriptome data may result in longer loci (more amenable to primer design) and primers may be more transferable to related species.

Multi-individual microsatellite identification: A multiple genome approach to microsatellite design (MiMi).

Bespoke microsatellite marker panels are increasingly affordable and tractable to researchers and conservationists. The rate of microsatellite discovery is very high within a shotgun genomic data set, but extensive laboratory testing of markers is required for confirmation of amplification and polymorphism. By incorporating shotgun next‐generation sequencing data sets from multiple individuals of the same species, we have developed a new method for the optimal design of microsatellite markers. This new tool allows us to increase the rate at which suitable candidate markers are selected by 58% in direct comparisons and facilitate an estimated 16% reduction in costs associated with producing a novel microsatellite panel. Our method enables the visualisation of each microsatellite locus in a multiple sequence alignment allowing several important quality checks to be made. Polymorphic loci can be identified and prioritised. Loci containing fragment‐length‐altering mutations in the flanking regions, which may invalidate assumptions regarding the model of evolution underlying variation at the microsatellite, can be avoided. Priming regions containing point mutations can be detected and avoided, helping to reduce sample‐site‐marker specificity arising from genetic isolation, and the likelihood of null alleles occurring. We demonstrate the utility of this new approach in two species: an echinoderm and a bird. Our method makes a valuable contribution towards minimising genotyping errors and reducing costs associated with developing a novel marker panel. The Python script to perform our method of multi‐individual microsatellite identification (MiMi) is freely available from GitHub (https://github.com/graemefox/mimi).

A rapid and cost-effective approach for the development of polymorphic microsatellites in non-model species using paired-end RAD sequencing.

As one of the most informative and versatile DNA-based markers, microsatellites have been widely used in population and conservation genetic studies. However, the development of microsatellites has traditionally been laborious, time-consuming, and expensive. In the present study, a rapid and cost-effective "RAD-seq-Assembly-Microsatellite" approach was developed to identify abundant microsatellite markers in non-model species using the roughskin sculpin Trachidermus fasciatus as a representative. Overlapping paired-end Illumina reads generated by restriction-site-associated DNA sequencing (RAD-seq) were clustered based on the similarity of reads containing the restriction enzyme recognition site and then assembled into contigs, which were used for microsatellite discovery and primer design. A total of 121,750 RAD contigs were generated with a mean length of 522bp, and 19,782 contigs contained microsatellite motifs. A total of 156,150 primer pairs were successfully designed based on 16,497 contigs containing priming sites. Experimental validation of 52 randomly selected microsatellite loci demonstrated that 45 (86.54%) loci were successfully amplified and polymorphic in two geographically isolated populations of T. fasciatus. Compared with traditional approaches based on DNA cloning and other approaches based on next-generation sequencing, our newly developed approach could yield thousands of microsatellite loci with much higher successful amplification rate and lower costs, especially for non-model species with shallow background of genomic information. The "RAD-seq-Assembly-Microsatellite" approach holds great promise for microsatellite development in future ecological and evolutionary studies of non-model species.

Microsatellite markers for the dinoflagellate Gambierdiscus caribaeus from high-throughput sequencing data

Members of the benthic dinoflagellate genus Gambierdiscus are the causative agents of ciguatera fish poisoning worldwide. Ciguatera outbreaks appear to be more common in recent years and new incidences are reported from unprecedented regions. To investigate Gambierdicus population dynamics, connectivity, and dispersal routes, we developed microsatellite markers for Gambierdiscus caribaeus, a globally distributed species that is common at our study site at St. Thomas in the US Virgin Islands. We used high-throughput partial genome sequencing along with an existing transcriptome for microsatellite discovery. Screening of contigs with less than three times coverage resulted in 558 (partial genome) and 33 (transcriptome) candidate microsatellites. Four primer pairs from the partial genome and three from the transcriptome successfully amplified polymorphic microsatellites in multiplexed PCR reactions. The seven markers were tested on 150 G. caribaeus strains isolated monthly from August 2013 to July 2015 at St. Thomas, USVI. The numbers of alleles per locus varied between 3 and 14, and the allele diversity ranged from 0.214 to 0.899 in this dataset. These newly developed microsatellites will enable studies of population structure, connectivity, and dispersal in G. caribaeus and can give new insights into the expansion of ciguatera outbreaks worldwide.

Restriction site associated DNA (RAD) for de novo sequencing and marker discovery in sugarcane borer, Diatraea saccharalis Fab. (Lepidoptera: Crambidae).

We present the development of a genomic library using RADseq (restriction site associated DNA sequencing) protocol for marker discovery that can be applied on evolutionary studies of the sugarcane borer Diatraea saccharalis, an important South American insect pest. A RADtag protocol combined with Illumina paired-end sequencing allowed de novo discovery of 12811 SNPs and a high-quality assembly of 122.8M paired-end reads from six individuals, representing 40Gb of sequencing data. Approximately 1.7Mb of the sugarcane borer genome distributed over 5289 minicontigs were obtained upon assembly of second reads from first reads RADtag loci where at least one SNP was discovered and genotyped. Minicontig lengths ranged from 200 to 611bp and were used for functional annotation and microsatellite discovery. These markers will be used in future studies to understand gene flow and adaptation to host plants and control tactics.

Microsatellite discovery in an insular amphibian (Grandisonia alternans) with comments on cross-species utility and the accuracy of locus identification from unassembled Illumina data

The Seychelles archipelago is unique among isolated oceanic islands because it features an endemic radiation of caecilian amphibians (Gymnophiona). In order to develop population genetics resources for this system, we identified microsatellite loci using unassembled Illumina MiSeq data generated from a genomic library of Grandisonia alternans, a species that occurs on multiple islands in the archipelago. Applying a recently described method (PALFINDER) we identified 8001 microsatellite loci that were potentially informative for population genetics analyses. Of these markers, we screened 60 loci using five individuals, directly sequenced several amplicons to confirm their identity, and then used eight loci to score allele sizes in 64 G. alternans individuals originating from five islands. A number of these individuals were sampled using non-lethal methods, demonstrating the efficacy of non-destructive molecular sampling in amphibian research. Although two loci satisfied our criteria as diploid, neutrally evolving loci with the statistical power to detect population structure, our success in identifying reliable loci was very low. Additionally, we discovered some issues with primer redundancy and differences between Illumina and Sanger sequences that suggest some Illumina-inferred loci are invalid. We investigated cross-species utility for eight loci and found most could be successfully amplified, sequenced and aligned across other species and genera of caecilians from the Seychelles. Thus, our study in part supported the validity of using PALFINDER with unassembled reads for microsatellite discovery within and across species, but importantly identified major limitations to applying this approach to small datasets (ca. 1 million reads) and loci with small tandem repeat sizes.

Whole genome shotgun sequences for microsatellite discovery and application in cultivated and wild Macadamia (Proteaceae).

• Premise of the study: Next-generation sequencing (NGS) data are widely used for single-nucleotide polymorphism discovery and genetic marker development in species with limited available genome information. We developed microsatellite primers for the Proteaceae nut crop species Macadamia integrifolia and assessed cross-species transferability in all congeners to investigate genetic identification of cultivars and gene flow.• Methods and Results: Primers were designed from both raw and assembled Illumina NGS paired-end reads. The final 12 microsatellite markers selected were polymorphic among wild individuals of all four Macadamia species—M. integrifolia, M. tetraphylla, M. ternifolia, and M. jansenii—and in commercial macadamia cultivars including hybrids.• Conclusions: We demonstrate the utility of raw and assembled Illumina NGS reads from total genomic DNA for the rapid development of microsatellites in Macadamia. These primers will facilitate future studies of population structure, hybridization, parentage, and cultivar identification in cultivated and wild Macadamia populations.

The effects of read length, quality and quantity on microsatellite discovery and primer development: from Illumina to PacBio

The advent of next-generation sequencing (NGS) technologies has transformed the way microsatellites are isolated for ecological and evolutionary investigations. Recent attempts to employ NGS for microsatellite discovery have used the 454, Illumina, and Ion Torrent platforms, but other methods including single-molecule real-time DNA sequencing (Pacific Biosciences or PacBio) remain viable alternatives. We outline a workflow from sequence quality control to microsatellite marker validation in three plant species using PacBio circular consensus sequencing (CCS). We then evaluate the performance of PacBio CCS in comparison with other NGS platforms for microsatellite isolation, through simulations that focus on variations in read length, read quantity and sequencing error rate. Although quality control of CCS reads reduced microsatellite yield by around 50%, hundreds of microsatellite loci that are expected to have improved conversion efficiency to functional markers were retrieved for each species. The simulations quantitatively validate the advantages of long reads and emphasize the detrimental effects of sequencing errors on NGS-enabled microsatellite development. In view of the continuing improvement in read length on NGS platforms, sequence quality and the corresponding strategies of quality control will become the primary factors to consider for effective microsatellite isolation. Among current options, PacBio CCS may be optimal for rapid, small-scale microsatellite development due to its flexibility in scaling sequencing effort, while platforms such as Illumina MiSeq will provide cost-efficient solutions for multispecies microsatellite projects.

Microsatellite markers from the Ion Torrent: a multi‐species contrast to 454 shotgun sequencing

The development and screening of microsatellite markers have been accelerated by next-generation sequencing (NGS) technology and in particular GS-FLX pyro-sequencing (454). More recent platforms such as the PGM semiconductor sequencer (Ion Torrent) offer potential benefits such as dramatic reductions in cost, but to date have not been well utilized. Here, we critically compare the advantages and disadvantages of microsatellite development using PGM semiconductor sequencing and GS-FLX pyro-sequencing for two gymnosperm (a conifer and a cycad) and one angiosperm species. We show that these NGS platforms differ in the quantity of returned sequence data, unique microsatellite data and primer design opportunities, mostly consistent with the differences in read length. The strength of the PGM lies in the large amount of data generated at a comparatively lower cost and time. The strength of GS-FLX lies in the return of longer average length sequences and therefore greater flexibility in producing markers with variable product length, due to longer flanking regions, which is ideal for capillary multiplexing. These differences need to be considered when choosing a NGS method for microsatellite discovery. However, the ongoing improvement in read lengths of the NGS platforms will reduce the disadvantage of the current short read lengths, particularly for the PGM platform, allowing greater flexibility in primer design coupled with the power of a larger number of sequences.

Genome-Wide Microsatellite Identification in the Fungus Anisogramma anomala Using Illumina Sequencing and Genome Assembly

High-throughput sequencing has been dramatically accelerating the discovery of microsatellite markers (also known as Simple Sequence Repeats). Both 454 and Illumina reads have been used directly in microsatellite discovery and primer design (the “Seq-to-SSR” approach). However, constraints of this approach include: 1) many microsatellite-containing reads do not have sufficient flanking sequences to allow primer design, and 2) difficulties in removing microsatellite loci residing in longer, repetitive regions. In the current study, we applied the novel “Seq-Assembly-SSR” approach to overcome these constraints in Anisogramma anomala. In our approach, Illumina reads were first assembled into a draft genome, and the latter was then used in microsatellite discovery. A. anomala is an obligate biotrophic ascomycete that causes eastern filbert blight disease of commercial European hazelnut. Little is known about its population structure or diversity. Approximately 26 M 146 bp Illumina reads were generated from a paired-end library of a fungal strain from Oregon. The reads were assembled into a draft genome of 333 Mb (excluding gaps), with contig N50 of 10,384 bp and scaffold N50 of 32,987 bp. A bioinformatics pipeline identified 46,677 microsatellite motifs at 44,247 loci, including 2,430 compound loci. Primers were successfully designed for 42,923 loci (97%). After removing 2,886 loci close to assembly gaps and 676 loci in repetitive regions, a genome-wide microsatellite database of 39,361 loci was generated for the fungus. In experimental screening of 236 loci using four geographically representative strains, 228 (96.6%) were successfully amplified and 214 (90.7%) produced single PCR products. Twenty-three (9.7%) were found to be perfect polymorphic loci. A small-scale population study using 11 polymorphic loci revealed considerable gene diversity. Clustering analysis grouped isolates of this fungus into two clades in accordance with their geographic origins. Thus, the “Seq-Assembly-SSR” approach has proven to be a successful one for microsatellite discovery.

Microsatellite Marker Discovery using Single Molecule Real-Time Circular Consensus Sequencing on the Pacific Biosciences RS

Microsatellite sequences are important markers for population genetics studies. In the past, the development of adequate microsatellite primers has been cumbersome. However with the advent of next-generation sequencing technologies, marker identification in genomes of non-model species has been greatly simplified. Here we describe microsatellite discovery on a Pacific Biosciences single molecule real-time sequencer. For the Greater White-fronted Goose (Anser albifrons), we identified 316 microsatellite loci in a single genome shotgun sequencing experiment. We found that the capability of handling large insert sizes and high quality circular consensus sequences provides an advantage over short read technologies for primer design. Combined with a straightforward amplification-free library preparation, PacBio sequencing is an economically viable alternative for microsatellite discovery and subsequent PCR primer design.

Characterisation of thirteen polymorphic microsatellite markers for cowslip (Primula veris L.) developed using a 454 sequencing approach

The cowslip (Primula veris L., Primulaceae) is a distylous perennial plant with a self-incompatibility system making it an obligate outbreeder. It is found in threatened grassland habitats that are becoming increasingly frag- mented. Microsatellite discovery was performed using next generation sequence technologies. We present 13 novel microsatellite markers that have been optimised for P. veris. The loci show clear polymorphic banding patterns, exhib- iting between 2 and 12 alleles, with observed heterozygosity values ranging from 0.296 to 0.889. These markers will facilitate the assessment of genetic diversity and population structure of P. veris populations.

Rapid, economical single‐nucleotide polymorphism and microsatellite discovery based on de novo assembly of a reduced representation genome in a non‐model organism: a case study of Atlantic cod Gadus morhua

By combining next-generation sequencing technology (454) and reduced representation library (RRL) construction, the rapid and economical isolation of over 25 000 potential single-nucleotide polymorphisms (SNP) and >6000 putative microsatellite loci from c. 2% of the genome of the non-model teleost, Atlantic cod Gadus morhua from the Celtic Sea, south of Ireland, was demonstrated. A small-scale validation of markers indicated that 80% (11 of 14) of SNP loci and 40% (6 of 15) of the microsatellite loci could be amplified and showed variability. The results clearly show that small-scale next-generation sequencing of RRL genomes is an economical and rapid approach for simultaneous SNP and microsatellite discovery that is applicable to any species. The low cost and relatively small investment in time allows for positive exploitation of ascertainment bias to design markers applicable to specific populations and study questions.

Development and characterization of new microsatellites for Eugenia dysenterica DC (Myrtaceae)

Microsatellite markers were developed for population genetic analyses of the Neotropical tree Eugenia dysenterica DC (Myrtaceae), after construction of a shotgun genomic library for microsatellite discovery. Nine primers were designed, of which 5 yielded amplified product. These primers were polymorphic for 97 individuals collected in 3 distinct localities. The number of alleles per locus (primer) ranged from 3 to 11 and expected heterozygosities varied from 0.309 to 0.884. The probability of locus identity was ~1.88 x 10(-4) and the probability of paternity exclusion was ~0.9367. The 5 microsatellite primer pairs may be suitable for population genetic studies such as parentage and fine-scale genetic analyses of this species.

Using next-generation DNA sequencing for rapid microsatellite discovery in Mexican leaf-toed geckos (Phyllodactylus tuberculosus)

We use a combination of 454 shotgun pyrose- quencing and traditional molecular cloning to isolate microsatellite loci for the Mexican yellow-bellied leaf-toed gecko (Phyllodactulus tuberculosus). A pyrosequencing run on a 454 GS Junior yields 62 Mbp of data composed of 150 k fragments with an average length of 412 bp. Among the fragments, 18 144 (12 %) contain a suitable microsat- ellite. Dinucleotides and tetranucleotides are most fre- quently encountered. We then genotype 30 individuals of P. tuberculosus using 12 polymorphic loci. We find mod- erate allelic diversity and heterozygosity. No loci are in linkage disequilibrium and only two loci show deviations from Hardy-Weinberg expectations. Our results further highlight the utility of next-generation sequencing for discovering molecular markers in non-model taxa and suggest that sequencing on a 454 GS Junior system is a rapid and cost-effective approach for microsatellite characterization.

Comparison between Normalised and Unnormalised 454-Sequencing Libraries for Small-Scale RNA-Seq Studies

Next-generation sequencing of transcriptomes (RNA-Seq) is being used increasingly in studies of nonmodel organisms. Here, we evaluate the effectiveness of normalising cDNA libraries prior to sequencing in a small-scale study of the zebra finch. We find that assemblies produced from normalised libraries had a larger number of contigs but used fewer reads compared to unnormalised libraries. Considerably more genes were also detected using the contigs produced from normalised cDNA, and microsatellite discovery was up to 73% more efficient in these. There was a positive correlation between the detected expression level of genes in normalised and unnormalised cDNA, and there was no difference in the number of genes identified as being differentially expressed between blood and spleen for the normalised and unnormalised libraries. We conclude that normalised cDNA libraries are preferable for many applications of RNA-Seq and that these can also be used in quantitative gene expression studies.

Comparison of random and SSR‐enriched shotgun pyrosequencing for microsatellite discovery and single multiplex PCR optimization inAcacia harpophyllaF. Muell. Ex Benth

Streamlining the development and genotyping of microsatellites in species for which no genetic information is available represents an important technical challenge to overcome in order to enable mainstream application of state-of-the-art population genetic analysis techniques in nonmodel organisms. Using the example of Acacia harpophylla, an acacia tree endemic of north-eastern Australia, we show that high-throughput shotgun pyrosequencing technology, so-called second-generation sequencing, reduces time and cost of microsatellite marker discovery in nonmodel organisms and of their large-scale typing in natural populations. We found that 0.5% of short sequence reads generated on 454 Genome Sequencer FLX Titanium from random genome sampling and 2.2% of reads generated with prior microsatellite enrichment yielded microsatellite markers with designed polymerase chain reaction (PCR) primers, suggesting that enrichment increases efficiency of pyrosequencing when microsatellite discovery is the primary goal. Using stringent selection criteria to facilitate downstream PCR multiplex design, we identified 1435 microsatellite loci with designed primers from a total of 200,908 short sequence reads. From a subset of 96 loci tested for amplification, 38 were validated for population genetics applications, leading to the optimization of a cost-effective multiplex PCR protocol for the simultaneous typing of nine microsatellites in natural populations of A. harpophylla.