The domesticated silkworm is the foundation of the silk industry in many countries, including China, India, and Brazil. More than 1000 inbred lines of the domesticated silkworm Bombyx mori are maintained worldwide. In Korea, 340 silkworm strains are preserved by the National Academy of Agricultural Science (NAAS; http://www.genebank.go.kr/), and approximately 78 strains originate from Japan. These strains do not have particular larval skin markings, but some strains show quail marking. Generally, the larvae of the Japanese strains, which are nearly bivoltine, are strong and resistant to adverse environments, but the larval period is comparatively long. Cocoons of these strains are peanut-shaped and frequently white in color (although some are green or yellow), and have a slightly short and thick filament (Lim et al., 1996; Table 1).

[Table 1.] General information for the Japan-origin silkworm strains utilized in this study

General information for the Japan-origin silkworm strains utilized in this study

Microsatellites are simple sequence repeats (SSR) of one to six bases that are abundant in both coding and non-coding regions of all eukaryotic nuclear and some prokaryotic genomes (Tautz and Renz, 1984). Due to the allelic hyper-variability of these markers, conservation in flanking sequence, and co-dominant mode of inheritance and recent advances in PCR technology, microsatellites are useful markers in several fields of sciences where detection of fine-scale genetic structure is required (Weber and May, 1989; Meglécz et al., 2007; Li et al., 2002).

Numerous microsatellite loci have been identified and described in the silkworm (e.g., Dharma Prasad et al., 2005; Li et al., 2005). Previously, Kim et al. (2010) genotyped 54 silkworm strains preserved in Korea using nine polymorphic microsatellite loci, and described the importance of these markers for strain discrimination. Subsequently, Kim et al. (2012) genotyped 85 silkworm strains originating from China (but preserved in Korea) using eight polymorphic microsatellite loci.

In this study, we selected eight microsatellite markers that were used in previous studies (Kim et al., 2010; 2012), and genotyped 78 silkworm strains of Japanese origin that were preserved in Korea. The goal of this investigation was to determine the utility of the markers in detecting DNA polymorphism, and to assess their potential for use in strain discrimination.

Seventy-eight B. mori silkworm strains originating from Japan were analyzed for this study. The voltanism, moltinism, egg color, blood color, and cocoon color/shape are presented in Table 1. These strains are under preservation at NAAS, Republic of Korea.

Approximately 100 eggs of each strain were crushed in a glass grinder in liquid nitrogen, and genomic DNA was extracted using the DNA Extraction Kit, in accordance with the manufacturer’s instruction (Qiagen, USA). Eight microsatellite loci were successfully amplified using primers previously designed using published and unpublished B. mori genomic DNA sequences (Table 2). In order to verify the presence of simple sequence repeats in the microsatellite loci, PCR amplicons of each locus from one or two strains were cloned and sequenced. Characteristics of the eight microsatellite loci, including the primer sequences, are presented in Table 2.

[Table 2.] Information of the 8 microsatellite loci analyzed in 78 Japan-origin silkworm strains

Information of the 8 microsatellite loci analyzed in 78 Japan-origin silkworm strains

PCR was carried out in a 25 μL reaction volume containing ~30 ng of genomic DNA, 200 nM of each reverse and forward primer, 200 μM of each dNTP, 2.5 μL of 10× PCR buffer [50 mM KCl, 10 mM Tris-HCl (pH 8.8), 150 nM KCl, 1.5 mM MgCl₂], and 1 unit of FR-Taq DNA polymerase (Biomedic, Korea), using an ABI 2720 Thermal Cycler (Applied Biosystems, USA). The PCR cycling conditions were as follows: initial denaturation at 95℃ for 3 min, followed by 30 cycles of 94℃ for 30 s, annealing at 46℃~68℃ for 30 s, extension at 72℃ for 1 min, and a final extension step at 72℃ for 6 min. Forward primers were labeled with 6-FAM fluorescent dye (Yue et al., 2000) for subsequent peak detection using fluorescence-based DNA electrophoresis. To verify successful DNA amplification, samples were electrophoresed through a 1.4% agarose gel for 1 h. For size analysis, 0.2 μL of PCR product was mixed with 9.8 μL of Hi-Di Formamide (Applied Biosystems) and 0.2 μL of LIZ-500 size standard (Applied Biosystems). The samples were then denatured at 95℃ for 5 min, stored on ice, and analyzed using an ABI 3730xl DNA Analyzer (capillary sequencer, Applied Biosystems, USA). GeneMapper® version 4.0 (Applied Biosystems) was used to determine allele sizes. To verify the accuracy of size determination, electrophoresis was carried out a minimum of three times, generally using two independent PCR products.

Observed heterozygosity (H_o; Weir, 1996), expected heterozygosity (E_o; Nei, 1987), Polymorphic Information Content (PIC; Bostein et al., 1980), and the allelic and genotypic frequencies at each locus were calculated using PowerMarker ver. 3.25 (Liu and Muse, 2005). Allelic PIC was calculated using the following formula: PIC = 1- Σ (Pi)², where Pi is the proportion of the strain carrying i^th allele, calculated for each microsatellite locus (Bostein et al., 1980). The relationships among silkworm strains were inferred using Neighbor-Joining (NJ) analysis of the distance matrix based on the shared allelic methods (Jin and Charkaborty, 1993); these analyses were also carried out using PowerMarker ver. 3.25 (Liu and Muse, 2005).

Fragment analysis of the 78 silkworm stains in eight microsatellite loci were largely successful; however, analysis of one strain at locus D49370, five strains at locus Bmsat127, two strains at locus D49948, and four strains at locus Bmsat129, one strain at locus X17219, and one strain at locus AF005384 were not successful, either due to amplification failure or multiple amplification. Thus, 76 of the 78 strains were genotyped on average at each locus (Table 3). It is likely that the unsuccessful amplification was due to null alleles, in which a mutation occurs within the primer region, resulting in a failure to amplify the complete product, or reduction of the allele number to a single allele (Kwok et al., 1990).

[Table 3.] Genotypes of 78 Japan-origin silkworm strains at each microsatellite locus

Genotypes of 78 Japan-origin silkworm strains at each microsatellite locus

We detected 73 alleles at eight loci, and the average allele number at each locus was 9.1, ranging in number from five (locus D90454) to 16 (locus Bmsat129) (Table 4). Although the dinucleotide repeat locus Bmsat129 (Reddy et al., 1999) provided the highest allele number, the dinucleotide repeat loci K02 and X17219 (Unpublished, GenBank accession number DE376976; Michaille et al., 1990) provided only seven alleles . Thus, the allele number at each locus is unlikely to have a direct relationship with the length of the repeat motif. Previous analysis of silkworm strains from China showed that the loci Bmsat129 and AF005384 exhibited the highest allele numbers, which were as high as 14 (Kim et al., 2012); however, the strains we examined from Japan only exhibited eight alleles at the locus AF005384, thus indicating somewhat different alleles frequencies between the strains from China and Japan. The trinucleotide repeat locus D90454 (Itoh et al., 1991) exhibited the lowest number of alleles, only five, which is consistent with the previous study of silkworm strains from China (Kim et al., 2012).

[Table 4.] Summary statistics of the 8 microsatellite loci

Summary statistics of the 8 microsatellite loci

In accordance with the allelic diversity, the number of genotypes was generally proportional to the allele number (Table 4). For example, the locus D90454, which exhibited only five alleles, resulted in only six genotypes in 78 strains, whereas the locus Bmsat129, which exhibited 16 alleles, resulted in 23 genotypes in 74 strains (Table 3). The frequency of the most common allele at each locus ranged from 0.32 (Bmsat129) to 0.78 (Bmsat127) (Table 4). Thus, some loci exhibited particular alleles at very high frequencies, but others did not. For example, allele 180 found at locus D90454 occurred in 75 of the 78 strains, either as a homozygote or heterozygote, but allele 177 was observed along with allele 180 in 42 strains, and mostly as a heterozygote,. The remaining four alleles, 171, 174, 177, and 183 were found along with allele 180 in only one strain, and either as a heterozygote or homozygote (Table 3). This pattern is identical to that observed in the strains from China, where the same loci were genotypes (Kim et al., 2012).

The expected (H_e) and the observed heterozygosity (H_o) over all microsatellite loci ranged from 0.38 to 0.79 and from 0.13 to 1.00, respectively (Table 4). The loci D90454, D49948, X17219, Bmsat129, and AF005384 exhibited somewhat higher or equivalent estimates of H_o versus H_e, but the remaining loci K02 (0.64 vs. 0.13), D49370 (0.71 vs. 0.56), Bmsat127 (0.38 vs. 0.25), and Bmsat129 (0.79 vs. 0.65) exhibited substantially lower estimates of H_o, potentially indicating inbreeding in these strains. Silkworm strains from China showed a similar pattern, in which the loci D90454, D49948, Bmsat129, and X17219 exhibited somewhat higher or equivalent estimates of H_o versus H_e. However, the remaining loci K02 (0.37 vs. 0.07), D49370 (0.56 vs. 0.45), Bmsat127 (0.35 vs. 0.17), and AF005384 (0.84 vs. 0.56) exhibited substantially lower estimates of H_o. Strains from Japan generally showed higher H_o than the strains from China, indicating that the latter has higher genetic diversity than the former . The PIC value of the Japanese strains we examined ranged from 0.36 to 0.77, with an average of 0.58 per locus (Table 4). The locus Bmsat129, which exhibited the high number of alleles, also exhibited the highest PIC value of 0.77, and K02, D49370, D49948, Bmsat129, X17219, and AF005384 all exhibited PIC values higher than 0.50, indicating the high power of these loci to discriminate among strains. The previous analysis of strains from China reported PIC values ranging from 0.34 to 0.82, with an average of 0.54 per locus (Kim et al., 2012). Furthermore, Kim et al. (2010) reported that 54 silkworm strains originating from several countries exhibited a mean PIC value of 0.47 over all loci (nine loci, including the eight loci used in this study). The PIC values at each locus ranged from 0.06 to 0.86, which is slightly higher than the values we calculated for the strains from Japan.

Phylogenetic analysis failed to detect any clear grouping among strains based on known characteristics, such as voltinism, moltinism, egg color, blood color, and cocoon color/shape (Fig. 1). For example, JP1, Hwangyu, N19, W il2, BN, Woosuk, SK-1, and 11 formed a clade in the NJ tree; although these strains share characteristics such as bi-voltinism, tetra-moltinism, white-colored blood, and white-colored cocoon, the egg color is either brown (most strains) or white (SK-1). Further, the cocoon shape is either peanut shaped (N19, SK-1, Hwangyu, and 11), long and peanut-shaped (Woosuk and W il2), or oval (JP1), indicating no known character-based grouping. Similar examples can be found in many other branches of the tree. Instead, the clustering pattern indicates that the microsatellite loci typed in this study may reflect genetic differences among strains that can be utilized for the discrimination of silkworm strains. Previous studies of silkworm strains originating from China also support this notion (Kim et al., 2010, 2012).

[Fig. 1.] Phylogenetic tree constructed using Neighbor-Joining analysis that illustrates the genetic relationships among 78 silkworm strains originating from Japan. The genetic distances between strains were calculated based on the shared allelic methods (Jin and Chakraborty, 1994) using the PowerMarker v3.25 program. The scale bar indicates the branch length.

The eight microsatellite loci exhibited the presence of strainspecific alleles (Table 3). Locus D49370 provided the alleles 192, 199, 214, and 224, which were unique to strains N44 (no. 16), JF (no. 175), Suwonjam (no. 232), and J137 (no. 211), respectively (Table 3). Similarly, the locus D90454 exhibited alleles 171, 174, and 183, which were unique to strains zepere (no. 327), Kicho N27 (no. 194), and N27 (no. 10), respectively. The locus Bmsat127 exhibited alleles 186 and 228, unique to strains Xze (no. 332) and Kumkwangju (no. 197), respectively. Locus D49948 exhibited alleles 196 and 235, unique to strains W109 (no. 48) and Zepere (no. 327), respectively. Locus Bmsat129 exhibited alleles 151, 165, 167, 183, 193, 195, 179, and 185, unique to strains W109 (no. 48), N80 (no. 26), Spil (no. 329), Artificial diet line (no. 330), 11 (no. 173), N19 (no. 7), Il 111 (no. 174), and Artificial diet line (no. 330), respectively (Table 3). In total, 19 apomorphic alleles were observed, which discriminated 16 of the 78 silkworm strains. These strain-specific alleles can thus be utilized for the discrimination of B. mori strains without further typing of other loci.

Genotyping results showed that a substantial number of strains possessed homozygotic alleles (Table 5). At locus K02, 25 strains were homozygous for allele 104, six strains were homozygous for allele 110, two strains were homozygous for allele 113, 33 strains were homozygous for allele 115, one strain was homozygous for allele 120, and one strain was homozygous for allele 127 (Table 5). Similarly, 34 strains were homozygous at two alleles for locus D49370, 37 strains were homozygous at three alleles at locus D90454, 55 strains were homozygous at six alleles at locus Bmsat127, 20 strains were homozygous at one allele at locus D49948, 26 strains were homozygous at five alleles at locus Bmsat129, and 21 strains were homozygous at four alleles at locus AF005384 (Table 5). Consequently, 27 of the 118 genotypes were homozygous at one or more of the eight loci. Because we used ~100 eggs for the extraction of DNA from each B. mori strain, no hidden alleles are expected within the strains. Therefore, this particular combination of microsatellite loci effectively discriminated among silkworm strains from Japan. For other strains, typing of all eight loci is required for the discrimination of strains originating from Japan.

[Table 5.] Genotype frequency in each microsatellite marker among 78 silkworm strains

Genotype frequency in each microsatellite marker among 78 silkworm strains

Previously, 85 silkworm strains originating from China were genotyped using the same microsatellite loci, and exhibited 22 strainspecific apomorphic alleles, which discriminated 19 of the 85 strains. Furthermore, a substantial number of homozygous strains were observed. The previous and current results support the continued use of these microsatellite loci for the discrimination of silkworm strains originating from China and Japan that are under preservation in Korea. Nevertheless, the isolation of additional microsatellite loci is needed to discriminate among the >300 silkworm strains that are currently preserved in Korea.