Development of Microsatellites: A Powerful Genetic Marker

The tandem repeats, conserved short segments of DNA, which are found in all prokaryotic and eukaryotic genomes, are called microsatellites. It is also known as variable number tandem repeats (VNTRs), simple sequence repeats (SSRs) and short tandem repeats (STRs). Microsatellites present in both coding and non-coding regions of a genome. The high polymorphism of microsatellites makes them powerful genetic markers for genome mapping of many organisms. It is also suitable for ancient and forensic DNA studies for population genetics and conservation of biological resources. The major disadvantage of microsatellites is that for the first time they need to be isolated de novo from most species being examined. The task of microsatellite isolation is quite cumbersome involving in terms of effort and time, because it traditionally involves screening of genomic libraries. Cross-species amplification, Mining microsatellites from nucleotide sequenced data and Genomic library- based method are general methods of microsatellite isolation. Cross-species method may not effective for all species, Data mining is not applicable if there is no or limited data of DNA sequence. Genomic library based method is the best choice. Traditional protocol, primer extension protocol, selective hybridization, and Fast Isolation by AFLP of Sequences containing repeats (FIASCO) are the protocols of microsatellite development based on genomic library. FIASCO is the best protocol ever developed.The Agriculturists 2015; 13(1) 152-172

Isolation and identification of a new set of microsatellite loci from Ucides cordatus genome

A new set of microsatellite loci (simple sequence repeats, SSRs) from the overexploited mangrove crab Ucides cordatus is described in this study. Microsatellite isolation used a highly simplified and inexpensive protocol based on (i) multiple enzyme digestion/ligation; (ii) mixed biotin-labeled probes and streptavidin-coated magnetic bead hybridization capture strategy, and (iii) a double-repeat-enrichment procedure. A genomic library, double-enriched for inserts containing tetranucleotide repeat motifs [(GACA)6, (GATA)7, (GGAT)5 and (GTAT)5], was constructed to increase the chance of recovering SSR-containing sequences within DNA fragments. Amplified enriched DNA was cloned and transformed into competent E. coli. Then, positive clones were identified by 'white/blue plaque selection?. One hundred and five colonies were PCR-screened for sequencing, and 72 of these were found to have unique SSR inserts. Microsatellite motifs contained more than five repeats, and most loci were found to have perfect tandem repeats (51.4%), of which 94.4% were dinucleotide and 5.5% trinucleotide. Only 20% of all loci were compound and 28.6% were imperfect repeats containing di-, tri- and/or tetranucleotides. The high frequency of perfect repeat motifs after enrichment is additional evidence of the importance of adopting this procedure for the isolation of SSR. The novel 34 SSRs described in this study are expected to be highly polymorphic and, therefore, useful in population/stocks discrimination of this valuable mangrove species throughout its range, currently subjected to excessive fishing efforts.

Successful development of microsatellite markers in a challenging species: the horizontal borer Austroplatypus incompertus (Coleoptera: Curculionidae)

The analysis of microsatellite loci has allowed significant advances in evolutionary biology and pest management. However, until very recently, the potential benefits have been compromised by the high costs of developing these neutral markers. High-throughput sequencing provides a solution to this problem. We describe the development of 13 microsatellite markers for the eusocial ambrosia beetle, Austroplatypus incompertus, a significant pest of forests in southeast Australia. The frequency of microsatellite repeats in the genome of A. incompertus was determined to be low, and previous attempts at microsatellite isolation using a traditional genomic library were problematic. Here, we utilised two protocols, microsatellite-enriched genomic library construction and high-throughput 454 sequencing and characterised 13 loci which were polymorphic among 32 individuals. Numbers of alleles per locus ranged from 2 to 17, and observed and expected heterozygosities from 0.344 to 0.767 and from 0.507 to 0.860, respectively. These microsatellites have the resolution required to analyse fine-scale colony and population genetic structure. Our work demonstrates the utility of next-generation 454 sequencing as a method for rapid and cost-effective acquisition of microsatellites where other techniques have failed, or for taxa where marker development has historically been both complicated and expensive.