Molecular markers have several advantages over the traditional phenotypic markers that are available in silkworm genetic stocks. They are unaffected by environment, detectable in all stages of development and ubiquitous in number covering the entire genome. Development of molecular markers is important in silkworm for construction of linkage map, fingerprinting of strains for breeding, and marker-assisted selection. Many techniques have been employed in silkworm to reveal the genome-wide polymorphism. These are Restriction Fragment Length Polymorphisms (RFLP), Random Amplified Polymorphic DNA (RAPD), Simple Sequence Repeats (SSR) and Inter-Simple Sequence repeats (ISSR) anchored PCR. Three criteria define ideal molecular markers: (i) they should be highly polymorphic, (ii) they should be easy to identify and type, and (iii) they should be amenable to automation.
Microsatellite based genetic markers, which are distributed across genomes of most of the eukaryotes, fulfill these criteria. Microsatellites also known as Simple Sequence Repeats (SSRs) are short stretches of DNA which consist of an array of simple tandemly repeated mono, di-, tri-, tetra-, penta or hexanucleotide repeats such as (A)n, (CA)n, (GA)n, (GTA)n, (ATT)n, (GATA)n, (ATTTT)n, (ACGTCG)n. They are ubiquitous in prokaryotic and eukaryotic genomes and are randomly distributed, both in protein coding and non-coding regions. A unique oligonucleotide on each side of the repeat region is chosen for the production of a primer pair for each of the microsatellite loci. PCR products of different lengths can be amplified using primers flanking the variable microsatellite region. Allelic variations among individuals are based mostly on differences in the number of tandem repeats in a microsatellite array providing a ready source of polymorphism. Thus, the only way in which alleles can be distinguished is by measuring the total length of the microsatellite allele. This is most readily accomplished through PCR amplification of microsatellite itself along with a short stretch of defined flanking sequences on both sides with designed primer pairs for each locus. Details are given in protocols. Cost effectiveness of the assay is achieved by combining two or more loci for simultaneous analysis through multiplex PCR. By using the fluorescent-labelled primers and automated sequencers, the entire process of microsatellite analysis can be automated. Due to these advantages, microsatellites have been used to construct linkage maps. In addition to their clear utility for practical applications, information about the distribution and variability of microsatellite sequences in the genome of a given species can help elucidate its genetic history from the standpoint of evolution and artificial selection.
SSRs are being used extensively in studies involving Forensics, Population Genetic structure analysis, establishment of Kinship, Conservation Genetics, Linkage Mapping, Marker Assisted Breeding etc.
The studies have shown that the silkworm genome is abundantly interspersed with CA/GT and GA/CT repeats. The (GT)n repeats occur at every 49 kb while (CT)n repeats occur, on an average, at every 104 kb in the silkworm genome. These frequencies compare favourably with the well-characterized mouse and human genomes where intensive maps of microsatellite markers have been accomplished. Initial studies have shown that distinct allelic differences exist between different strains of silkworm and are inherited in Mendelian pattern. In the thirteen silkworm populations analysed using 14 microsatellite loci, the number of alleles ranged from 3 to 17 with PIC values of 0.66 to 0.90. Many microsatellite loci (upto 4-5 loci) could be multiplexed based on allelic sizes in an automated sequencer. Thus microsatellite markers could be mapped quickly using the appropriate silkworm mapping population.
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