Analysis of Allelic frequencies of microsatellite loci among 13 Bombyx mori strains

In order to determine the levels of allelic polymorphism of microsatellite loci, we analyzed 13 diverse silkworm populations which include six diapausing (HU₂₀₄, KA, NB₁, NB₇, NB₁₈ and NB₄D₂) and seven non-diapausing (C.nichi, Moria, Nistari, Pure Mysore, Daizo, Gungnong and Sarupat) strains. In each of the strains, DNA sample was pooled from 10 individuals except NB₄D₂ in which DNA from only 5 individuals was pooled.

Considering the microsatellite repeat motifs of di-, tri-, and tetra-nucleotides, 26 microsatellite loci (11 loci from EST database, 12 from GenBank and 3 from genomic library) were chosen for the study. PCR amplification was performed and the PCR products were resolved on PAGE followed by silver staining. The allelic distribution for a representative locus Bmsat062 is shown in Figure 1. Polymorphism was detected for 23 of the 26 loci among the 13 silkworm strains. Two microsatellite loci (Bmsat012 and Bmsat015) which were present in the coding region and one locus (Bmsat 109) with an unknown location were found to be monomorphic.

Allelic frequencies at microsatellite loci

For the 23 polymorphic microsatellite loci, a total of 89 alleles were detected in 13 diverse silkworm strains. The number of alleles scored at each locus in 13 strains varied from as few as 2 to as many as 14 alleles (Table). The heterozygosity values varied from 0.2 to 0.87. In five microsatellite loci (Bmsat010, Bmsat013, Bmsat016, Bmsat018, Bmsat063) null alleles were observed. Allele frequencies for some of the representative loci are shown in Figure 2.

Three loci, Bmsat132, Bmsat066 and Bmsat110 revealed alleles specific to diapausing and non-diapausing strains. For example, 215 bp allele in locus Bmsat066 is specific only to diapausing strains except Hu₂₀₄, which shares the allele with non-diapausing strains. Likewise, 95 bp allele of Bmsat110 is specific only to diapause strains. Such diapause and non-diapause strain-specific markers are useful in silkworm genetic analysis and breeding programs since these two groups of silkworm strains differ in a large number of qualitative and quantitative traits. The possible linkage of such specific markers with the traits of interest could be established using appropriate populations for focused and rapid practical breeding programs.

Heterozygosity values

To assess the level of polymorphism, 13 silkworm strains were analysed with the 26 primer pairs. The average heterozygosity value for the 23 microsatellite loci was 0.54, ranging from 0.2 to 0.87. The most polymorphic locus, Bmsat103, which is a dinucleotide repeat, showed the highest heterozygosity value of 0.87. Since the silkworm strains chosen for the present study are quite diverse it is likely that we have examined a reasonable representation of the alleles of the SSR loci used in the present study and the heteozygosity values given are realistic estimates for these loci.

The relationship between the length of the microsatellite motif and degree of polymorphism was also examined. Although the number of loci used for each class for such an observation was low, the available trend shows that there is no clear relationship between the repeat length and the degree of polymorphism in the present study. For example, locus Bmsat071 (TCA)₁₃, the largest perfect repeat used in this study, revealed a heterozygosity value of 0.58 with 3 alleles, and locus Bmsat007, with the perfect (TCA)₉ repeat, has a heterozygosity of 0.76 with 6 alleles.