Thousands Of Microsatellite Loci Research Articles

Genetic resources of macroalgae: Development of an efficient method using microsatellite markers in non-model organisms

Red and brown seaweeds are species with high ecological and economic importance. Here we report the feasibility of cost-effective molecular marker development in 6 species from different phyla. Microsatellite markers of two brown seaweed species Alaria esculenta and Pylaiella littoralis, and of four red seaweed species Calliblepharis jubata, Gracilaria gracilis, Gracilaria dura and Palmaria palmata were identified and characterized in genomic sequences obtained using Double-Digest Restriction site Associated DNA (ddRAD). A total of 64,623,186 reads were generated from two runs of multiplexed Illumina Miseq sequencing, from which 30,636 reads containing microsatellite motifs including 15,443 where primer pairs could be designed. Five hundred seventy-six primers pairs were selected for amplification trials to determine levels of polymorphism. Of these 576 loci, 338 could be amplified and 142 of the 338 loci were polymorphic. A total of 28 usable polymorphic markers were developed in A. esculenta, 18 in P. littoralis, 11 in C. jubata, 14 in G. gracilis, 21 in G. dura and 13 in P. palmata. The overall number of alleles per locus ranged from 2 to 22. These 105 new microsatellite markers will be useful for further studies of population genetics, breeding programs and conservation genetics of these species. Compared with traditional approaches, our study yielded thousands of microsatellite loci for six different species in a short time and with affordable costs. This study, based on the use of ddRADseq, provided preliminary data about the genetic structure and reproduction mode of these six non-model species based on a small number of individuals from two geographically-distant populations and on the genetic structure and reproduction mode of two non-model species, i.e. detection of clonality for two red algae, C. jubata and G. dura and detection of highly genetically divergent populations corresponding probably to different cryptic species under the name P. littoralis.

Low STR variability in the threatened marsh deer, Blastocerus dichotomus, detected through amplicon sequencing in non-invasive samples.

Blastocerus dichotomus is the largest deer in South America. We have used 25 microsatellite markers detected and genotyped by Next Generation Sequencing to estimate the genetic variability of B. dichotomus in Argentina, where most of its populations are threatened. Primer design was based on the sequence of a shallow partial genome (15,967,456 reads; 16.66% genome coverage, mean depth 1.64) of a single individual. From the thousands of microsatellite loci found, even under high stringency selection, we chose and tested a set of 80 markers on 30 DNA samples extracted from tissue and feces from three Argentinean populations. Heterozygosity levels were low across all loci in all populations (H=0.31 to 0.40). Amplicon sequencing is a fast, easy, and affordable technique that can be very useful for the characterization of microsatellite marker sets for the conservation genetics of non-model organisms. This work is also one of the first ones to use amplicon sequencing in non-invasive samples and represents an important development for the study of threatened species.

Characterization of a New Set of Microsatellite Markers Suggests Polygyny and Polyandry in Atta cephalotes (Hymenoptera: Formicidae).

The leaf-cutting ant, Atta cephalotes L. (1758), is a major herbivore with great economic impact in the Neotropics. Because of its broad range and human-mediated dissemination, the ecology of this ant has received considerable attention; however, questions concerning its population genetics, dispersal, and social structure remain unexplored. Here, we aimed to identify and provide information on molecular and statistical performance of a suite of polymorphic microsatellite markers for A. cephalotes while demonstrating their utility for further genetic studies. We designed primer sequences targeting thousands of microsatellite loci and then screened 30 of these for amplification and polymorphism. Fifteen of these loci were selected and used to evaluate their polymorphism using 74 ants sampled from 15 different nests of the same location. This set of 15 loci exhibited variation of 2-20 alleles, with a mean heterozygosity of 0.57. All loci followed Hardy-Weinberg expectations with no evidence of linkage disequilibrium, while two loci showed evidence of null alleles. Our preliminary analysis suggested substantial nest differentiation with no population viscosity within the sampled location, as well as colonies with multiple queens (polygyny) and paternity (polyandry). Our newly identified microsatellites have proven to be highly informative to investigate gene flow, social structure and reproduction of this key agricultural pest.

Detection of Microsatellite Instability Biomarkers via Next-Generation Sequencing.

A high level of microsatellite instability (MSI-H+) is an emerging predictive and prognostic biomarker for immunotherapy response in cancer. Recently, MSI-H+ has been detected in a variety of cancer types, in addition to the classical cancers associated with Lynch Syndrome. Clinical testing for MSI-H+ is currently performed primarily through traditional polymerase chain reaction (PCR) or immunohistochemistry (IHC) assays. However, next-generation sequencing (NGS)-based approaches have been developed which have multiple advantages over traditional assays. For instance, NGS has the ability to interrogate thousands of microsatellite loci compared with just 5-7 loci that are detected by PCR. In this chapter, we detail the biochemical and computational steps to detect MSI-H+ from analysis of paired tumor and normal samples through NGS. We begin with DNA extraction, describe sequencing library preparation and quality control (QC), and outline the bioinformatics steps necessary for sequence alignment, preprocessing, and MSI-H+ detection using the software tool MANTIS. This workflow is intended to facilitate more widespread usage and adaptation of NGS-powered MSI detection, which can be eventually standardized for routine clinical testing.

A broad genomic panel of microsatellite loci from Brycon orbignyanus (Characiformes: Bryconidae) an endangered migratory Neotropical fish

A broad panel of tens of thousands of microsatellite loci is unveiled for an endangered piracema (i.e. migratory) South American fish, Brycon orbignyanus. Once one of the main fisheries resources in the Platine Basin, it is now almost extinct in nature and focus of intense aquaculture activity. A total of 178.2 million paired-end reads (90 bases long) were obtained through the use of sequencing-by-synthesis (from a primary genomic library of 500 bp DNA fragments) and is made available through NCBI’s Sequence Read Archive, SRA accession SRX3350440. Short reads were assembled de novo and screening for perfect microsatellite motifs revealed more than 81 thousands unique microsatellite loci, for which primer pairs were proposed. A total of 29 polymorphic microsatellite markers were already previously validated for this panel. A partial genomic assembly is hereby presented and these genomic resources are publicly made available. These data will foster the rapid development of hundreds of new DNA markers for genetic diversity studies, conservation initiatives and management practices for this important and depleted species. The availability of such preliminary genomic data will also be of use in the areas of bioinformatics, ecology, genetics and evolution.

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.

Highly efficient identification of thousands of microsatellite loci in the pearl oyster (Pinctada martensii) from RNA-Seq

High-throughput RNA-Seq affords a cost and time effective means of obtaining large numbers of genetic markers for aquatic genomics. Here, we present thousands of novel microsatellite loci developed for the pearl oyster, Pinctada martensii from the Illumina HiSeq™ 2000 library of the pearl sac. Free user-friendly bioinformatics tools were employed to screen for microsatellite loci and design appropriate primers in 102,762 unigenes with 7216 microsatellite loci identified in total, 4862 of which had flanking sequences suitable for polymerase chain reaction primer design. The 50 randomly chosen primer pairs were tested in two populations of pearl oyster (base population (POP1) and selected population (POP2), with 30 individuals of each population). All the primer pairs were amplified successfully in two populations. All loci were polymorphic in POP1, while there were 3 loci showing monomorphism in POP2. In POP1 and POP2, observed heterozygosity from 0.033 to 1.000 and 0.000 to 1.000, 19 and 16 microsatellite loci deviated significantly from Hardy–Weinberg expectations including a Bonferroni correction (P < 0.001). Thirteen loci were highly informative content (PIC ≥ 0.5) in both populations. These identified loci will be useful for potential application for evolutionary, population genetic and chromosome linkage mapping research on pearl oyster.

Microsatellites of the self-compatible nemertean Prosorhochmus americanus (Hoplonemertea)

Thousands of microsatellite loci were identified in the nemertean Prosorhochmus americanus through enrichment-isolation and next-generation sequencing protocols. We tested 48 loci for amplification and subsequently genotyped 17 loci in 30 individuals from three North American populations. Most loci were polymorphic within and across populations; six were monomorphic. The number of alleles per locus ranged from 1 to 9 (mean 4.35, SD 2.37). Cross-species amplification in four nemertean species revealed extended marker utility. These loci will enable investigations of colonization potential and natural history in this selfing, putatively introduced species.

Identification of thousands of microsatellite loci from 454 sequencing in non-model organisms

Identification of thousands of microsatellite loci from 454 sequencing in non-model organisms