Introduction

Mulberry silkworm Bombyx mori (Linnaeus, 1758) is the model organism for Lepidoptera, including many races with different geographical origin and phenotypic characterization. More than 4000 univoltine, bivoltine and polyvoltine strains are maintained in the world germplasm of B. mori (Bindroo & Moorthy, 2014). The economic, cultural and scientific significance of this species have made it the subject of intensive studies. Genetics researches of silkworm have a great impact in improving sericulture. The traditional breeding activities, involving hybridizations between members of elite groups, are adding new varieties every year (Reddy, Abraham, & Nagaraju, 1999). A number of quantitative, qualitative and ethological traits are used for selection of parental strains for breeding programs, but many of silkworm varieties cannot be differentiated by the use of conventional characteristics, only. Isoenzymes have become a highly informative class of genetic markers for breeding programs and marker-assisted selection, as well as for conservation strategies of silkworm Bombyx mori L. Allozyme markers are powerful molecular tools that have facilitated the study of genetic diversity. Allozyme electrophoresis is a technique that has proved useful for determining the genetic variations within and among populations (Egorova, Naletova, & Nasirillaev, 1985; Eguchi, Takahama, Ikeda, & Horii, 1988; Marcato, Trevisan, Cappellozza, & Bisol, 1990; He, 1995; Staykova, 2008; Staykova et al., 2015).

Silk producing countries have collected number of silkworm strains suitable for a wide range of agroclimatic conditions (Bindroo & Moorthy, 2014). A large number of strains (more than 250) with different origin compose Bulgarian germplasm resources of Bombyx mori (Petkov, Tzenov, Petkov, Natcheva, & Vasileva, 2006). They have been described mainly on the base of their morphological characteristics that are highly variable and environment dependent. Isoenzymic variations and phylogenetic relatedness among strains have been slightly studied (Shabalina, 1990; Staykova, 2008; Staykova et al., 2010; Staykova, Ivanova, Grekov, & Avramova, 2012; Staykova et al., 2015). Due to this fact the purpose of the present study was to assess genetics variations within and among twelve strains with different origin belonging to Bulgarian germplasm bank of silkworm and to establish their relationships using isoenzyme markers.

Material and methods

The silkworm resources used in the present investigation include a total of twelve strains with different geographical origin and phenotype characteristic (Table 1), which were obtained from the Sericulture Experiment Station germplasm bank in Vratza, Bulgaria. All individuals were nourished at a standard regime of silkworm breeding.

Totally 477 larvae (male and female) on the fifth day of the fifth instar were studied. Larvae were selected randomly from each strain and were submitted to electrophoretic analysis of haemolymph, silk glands and midgut tissues (mean sample size per locus ranged from 33 to 41, Table 3).

The tissue extracts were prepared according to Stoykova, Popov, and Dimitrov (2003), Staykova, Grekov, and Panayotov (2004), Staykova et al. (2012) and Staykova (2013). The individual samples were studied by 7.5% polyacrilamide gel electrophoresis (PAGE) (Davis, 1964) for nonspecific esterases (EST, ЕС 3.1.1), malate dehydrogenase (MDH, ЕС 1.1.1.37) and acid phosphatase (ACP, ЕС 3.1.3.2) - from hemolymph; hexokinase (HK, EC 2.7.1.1) - from silk glands and alkaline phosphatase (ALP, 3.1.3.1) – from midgut. The 6% PAGE was used to analyze phosphoglucomutase (PGM, ЕС 5.4.2.2) from silk glands. This concentration of dividing gel was more appropriate for investigation of PGM polymorphism, as was pointed earlier (Staykova, 2008). The staining mixtures for the enzymatic activities tested were taken from Shaw and Prasad (1970), Staykova et al. (2010), Spencer, Hopkinson, and Harris (1964), Eaton, Brewer, and Tashia (1966), Schmidtke and Engel (1972) and Boyer (1961). All the reagents were high purity grade, provided by Sigma-Aldrich™.

After the revelation of the isozyme activity regions, the phenotypes of the discovered loci were recorded. Allele frequencies, mean number of alleles per locus, proportion of polymorphic loci, observed (H_o) and expected (H_e) heterozygosity, deviation from the Hardy-Weinberg equilibrium, Nei’s genetic distance (D) (Nei, 1972), and Wright's fixation index, F_ST (Wright, 1965) were calculated using BIOSYS-1 (Swofford & Selander, 1981). Dendrogram was constructed using Nei’s (1972) genetic distance, by UPGMA (Sneath & Sokal, 1973) method using the PHYLIP (Felsenstein, 1993) software package.

Results

A total of nine izoenzymes of nonspecific esterases, tree allozymes of phosphoglucomutase, malate dehydrogenase and acid phosphatase, and two allozymes of alkaline phosphatase and hexokinase were detected by PAGE electrophoresis.

The six enzyme systems recorded a total of nine polymorphic loci with 26 heterogeneous alleles (Table 2). Nonspecific esterases, phosphoglucomutase, acid phosphatase and malate dehydrogenase manifested inter- and intra-strain polymorphism. Alkaline phosphatase was polymorphic in AES 1 wh and the hexokinase - in F-13 strain, only.

The analysis conducted showed monomorphic and fixed allele of Bes A locus in all tested strains (Bes A₁ allele), except for AES 1wh (Table 2). In the gene pool of this strain introduced from Spain we found polymorphism with “null” allele - Bes A_o. Homozygous Bes A_o/A_o specimens did not show any bands in the BES A zone of the gel plate. For the Bes B locus three alleles were recorded (Bes B₁, B₂ and B₃). Bes B₂ allele was fixed in the gene pool of Par+ and Daizo, whereas Bes B₃ was fixed in the gene pool of 371 strain (Table 2). In Cislau Tokay, F-13, Sh 3 and E31 we obtained all three alleles of the Bes B locus. Polymorphism with two alleles we recorded in VB1, Shandon and 70-42 (Bes B₁ and B₂ were presented in the gene pools), in Syria 2 (Bes B₁ and B₃) and in AES 1 wh (Bes B2 and B3). Among the strains with polymorphism on Bes B locus, the allele Bes B₁ showed the highest frequency in 70-42, Bes B₂ – in Shandon and Bes B₃ – in Cislau Tokay. Polymorphism with four alleles we found on the Bes D locus (Tab. 2). Bes D₁ and D₂ alleles were presented in the gene pool of 70-42, AES 1wh and E31, Bes D1 and D3 – in 371, Sh3 and Syria2, Bes D₂ and D₃ – in VB1, Bes D₃ and D_o – in F-13. Bes D₂ allele was fixed in the gene pool of Cislau Tokay and Par+ and Bes D3 – in Shandon. For Daizo we obtained Bes Do alele, only. Homozygous Bes Do/Do specimens did not show any bands in the corresponding esterase zone BES D. Frequency of the allele Bes D1 was the highest in Syria 2, of Bes D₂ – in AES 1wh and of Bes D₃ – in F-13 (except for strains with fixing Bes D alleles). Three allele polymorphism were found in Bes E locus (Bes E1, E₂ and E_o alleles) in VB1, Cislau Tokay, 70-42, AES 1wh and Syria 2. Two of these alleles we obtained in Shandon (Bes E₁ and E_o), F-13 and E31 (Bes E₂ and E_o). All tested individuals from Daizo were homozygous of Bes E₂ allele, whereas the individuals from 371, Par+ and Sh3 were homozygous of Bes E_o allele. This “null” allele demonstrated the highest frequency in all polymorphic strains tested, except for Cislau Tokay and Shandon (Table 2).

The electrophoretic separation of allozyme forms of phosphoglucomutase from silk glands exhibited variations with three Pgm alleles - Pgm A₁, A₂ and A₃ in VB1, 371 and 70-42 strains. Pgm A₁ and A₂ were presented in the gene pool of Par+ and Pgm A2 and A3 –in Cislau Tokay, Sh3, AES 1wh, Syria 2 and E31 (Table 2). Pgm A₁ allele had the highest frequency in Par+, Pgm A2 – in 70-42 and Pgm A₃ – in Syria 2. In Daizo we obtained Pgm A₁ allele, while in F-13 and Shandon strains - Pgm A₂ allele, only.

For the Mdh locus, three alleles were recorded in strain 371 (Mdh A₁, A₂ and A₃). Two of them (Mdh A₂ and A₃) were found in the gene pool of Cislau Tokay, F-13 and Par+ (Table 2). Mdh A₃ was the most common allele among these four strains with polymorphism of the Mdh locus. Mdh A₂ allele was fixed in the gene pools of the rest eight strains.

Electrophoresis of acid phosphatase allozymes from hemolymph showed total of five alleles in tested strains (Bph A, B, C, D and 0) (Table 2). The allele symbolized as Bph 0 was “null” allele. Three of the Bph alleles we found in AES 1wh (Bph A, B and 0), E31 (Bph A, B and D), VB1 (Bph B, C and 0). Two alleles were recorded in 371, F-13, Par+ (Bph A and B), Cislau Tokay (Bph A and D), Sh3, 70-42 and Syria 2 (Bph A and 0). Bph A allele was fixed in Shandon and Daizo strains. The Bph 0 allele was the most common allele in five of the tested strains with extremely high frequency in Syria 2. Bph A was the most expressed allele in three of strains with extremely high frequency in 371. Bph D was more frequently allele in two of the strains – Cislau Tokai and E31.

Polymorphism of the Alph locus with two alleles (Alph A₁and A₂) was recorded in AES 1wh strain, only (Table 2). All the rest strains were monomorphic on this locus with fixed Alph A1 allele.

Among the twelve strains included in the present study, we found polymorphism with two alleles on the hexokinase (Hk A) locus. Hk A₁ and A₂ alleles were recorded simultaneously in F-13, only. Hk A₁ allele was fixed in Shandon, whereas Hk A₂ allele was fixed in all the rest ten strains.

The number of alleles, allele and genotype frequencies of the loci of nonspecific esterases, phosphoglucomutase, malate dehydrogenase, acid phosphatase, alkaline phosphatase and hexokinase tested manifested strain specificity.

In the silkworm strains analysed using nine enzyme loci, the number of alleles per locus calculated with BIOSYS-1 software package, ranged from 1.0 (Daizo) to 2.0 (AES 1wh) (Table 3). The degree of polymorphism (according to the criterion 0.99) was the highest for strain AES 1wh (77.80%), and the lowest – for Daizo (0% - no polymorphic loci). The heterozygosity (H_o) by polymorphic loci observed varied from 0.000 (for Daizo) to 0.280 (for Cislau Tokay). The expected heterozygosity (H_e) was higher than the observed one (H_o) in all strains tested except Cislau Tokay, in which the value of H_o exceeded the value of He insignificantly (Table 3). Significant differences (P ˂ 0.05) in genotype frequencies were seen at the most loci in strains studied. Chi-Square test (DF=1÷3) showed that the deviations from the Hardy-Wein­berg equilibrium were in result of excess of homozygotes and deficiency of heterozygotes (Table 3).

Summarized results of F-stastistics are presented in (Table 4). The mean FST value over all loci, which is associated with the level of inter-strain differentiation, was 0.4903 and shows that 49.03% of the overall genetic diversity observed was among strains. 50.97% of genetic variations were within strains. The higher level of genetic diversity among strains we found for the Hk A locus (0.8604) and the lower one – for the Bes A locus (0.2839). For three loci (Hk A, Bes D and Mdh A) we established a level of intra-strain differentiation over than 50%. The mean value of F_IТ coefficient (0.6298) shows that there was a deficit of heterozygotes in the tested strains and correlates with the obtained lower level of heterozygosity observed compared to the expected one and with deviations from the Hardy-Wein­berg equilibrium as well. This fact proved the inbreeding effect.

The values of genetic distance (Nei, 1972) were calculated using the allele frequencies and ranged from 0.072 (between the strains Sh3 and Syria 2) to 0.651 (between strains Cislau Tokai and Daizo).

The tree drawn by UPGMA (Sneath & Sokal, 1973), based on isoenzyme results, showed that Romanian strain Cislau Tokay is formed one main clade, while the rest strains studied are formed the other, which is differentiated in two subclades (Figure 1). One of the subclade includes two Japanese strains Shandon and Par+ and the second one includes all others. Tree strains (AES 1 wh, Syria 2 and E31) were grouped to form one subclade and six strains (Sh3, F-13, 371, VB1, 70-42 and Daizo) were grouped to form another subclade. Five of them F-13, 371, VB1, 70-42 and Daizo strains were grouped together.

Figure.1.
UPGMA dendrogram

Discussion

Any attempt to improve silkworm breeds needs knowledge of their genetic variability and relationships. Silkworm breeding is the selection of superior genotypes and/or phenotypes over a period of time (Bindroo & Moorthy, 2014). The selection of best genotypes depends on a number of characters and diversity among them (Moorthy & Chandrakanth, 2015). Genetic variability can be analyzed by different methods and markers. According to Bindroo and Moorthy (2014) application of isoenzymes and other molecular markers helps to estimate genetic diversity much more accurately than that of morphological traits. Isoenzyme analysis appears to be useful in studing of intra- and inter-strain polymorphism of mulberry silkworm and determining the level of genetic changeability and genetic relationships (Egorova & Nasirillaev, 1993; Staykova, 2008; Staykova et al., 2010; Staykova et al., 2012; Staykova et al., 2015). Isozymes like esterase, acid phosphatase, alkaline phosphatase, malate dehydrogenase, phosphoglucomutase have been used by various researchers to study diversity in silkworm genotypes (Bindroo & Moorthy, 2014). Among them nonspecific esterases were most preferred because of its polygene control and polymorphic expression. The results in this study described indicate that esterase isozyme markers used could be successfully applied to examine the genetic variability in mulberry silkworm Bombyx mori. A total of twelve alleles of four esterase loci were recorded. Among strains tested “null” alleles of three esterase loci (Bes A_o, Bes D_o and Bes E_o) were found. Polymorphism with “null” allele of the Bes A and Bes E locus was described earlier in other strains of Bulgarian germplasm resourses of silkworm by Staykova et al. (2015). He (1995) also described polymorphism with a “deletion” type esterase (Bes A_o) in the corresponding zone of electrophoretic spectra in some races and hybrids in China. Polymorphism with five alleles was determined of the acid phosphatase. One of them was found as “null” type. Four electrophoretic variants (A, B, C, D) of hemolymph acid phosphatase in silkworm and a “null” mutant (0) controlled by total of five codominant alleles found Yoshitake and Akiyama (1964) and Eguchi et al. (1988). By means of linkage analysis Fujimori, Shimizu, and Enokijima (1984) located these alleles at 18.2 map unit on the chromosome 23 from silkworm karyotype. Polymorphism with five codominant alleles of the Bph locus was described earlier by Staykova et al. (2010) and Staykova et al. (2015) in other strains with different geographical origin, belonging to Bulgarian germplasm bank of silkworm. In this study polymorphism with three or two alleles of the phosphoglucomutase and malate dehydrogenase, and alkaline phosphatase and hexokinase loci, respectively was recorded. These results add new information about genetic variability of silkworm germplasm resources in Bulgaria (Staykova, 2008; Staykova et al., 2015). Some of the alleles of the polymorphic loci demonstrated strain specificity. For instance Bes Ao and Alp A2 alleles were presented only in AES 1 wh strain, Mdh A1 was presented in strain 371 and Hk A1 was presented in F-13, only.

The dendrogram generated by UPGMA analysis based on isoenzyme polymorphism resolved twelve strains into two main clusters: one comprising strain Cislau Tokay and the other the rest strains. Classification of silkworm genotypes is very important in breeding programs, because commercial rearing of this species is based on hybrids crossed from pure lines (Moorthy & Chandrakanth, 2015). Isoenzyme and allozyme markers utilized in this study resulted in differentiating of some strains with different geographical origin in the same subclades and also strains with common origin in different subclades. According to Etebari, Mirhoseini, and Matindoost (2005) and Chandrakanth, Anusha, and Moorthy (2014) this may be due to the changes in biological and developmental process and strains’ adaptation to environmental conditions. In our opinion genetically determined biochemical variances among tested strains from Bulgarian germplasm resources could be in result of founder effect provoked through introducing, also.

Almost all tested strains (eleven from twelve) had mean heterozygote deficiencies compared with Hardy-Weinberg expectation, which corresponds with high average value of FIT coefficient. According to Bindroo and Moorthy (2014), reduction in genetic diversity in silkworm, might be mainly due to domestication, breeding systems, selection, genetic drift and inbreeding. The low level of heterozygosity among tested strains observed in this study, probably results from low effective number of reproductive individuals, which provokes genetic drift, selection process and inbreeding effect. According to Lande (1994) and Whitlock (2002), the effects of inbreeding can accumulate over many generations. Li et al. (2010) pointed out that the low level of mt sequence polymorphism in the domesticated varieties of B. mori is most likely caused by inbreeding or population bottlenecking (by Bindroo & Moorthy, 2014). In silkworm germplasm maintenance centers, at every cycle only 40-60 cocoons are selected from each strain from the next generation (Bindroo & Moorthy, 2014). This size of population is small and may be led to bottleneck effect and to change the allelic frequencies. Bottleneck may restrict genetic diversity and breeding flexibility. On the other hand artificial selection for target characteristics has been widely utilized in the silkworm breeding programs (Bindroo & Moorthy, 2014). Breeders use the best genotypes available and select superior progeny, which narrows the gene pool variability to only those alleles that are available from the elite parent’s genotypes and therefore tends to decrease the genetic variation of the population (Brown, 1988; by Bindroo & Moorthy, 2014). Systematic selection of parents’ results in reduced genetic variation among their offspring. According to Bindroo and Moorthy (2014), to broaden the genetic diversity of silkworm, exotic elite strains were required to be introduced from various countries. Maintaining proper population size can prevent the loss of genetic variability due to genetic drift (Guzman & Lamkey, 2000). Genetic diversity is very important for selection of suitable parents required for successful development of improved varieties and hybrids of silkworm that have high adaptive potential lines (Bindroo & Moorthy, 2014). The results obtained in this study make use of breeding programs. Genetic variability, based on isoenzymes, could be also used during breeding of disease resistant silkworm strains (Mahesha & Thejaswini, 2014).

Conclusion

The present study is a step towards analyzing the genetic diversity in order to identify suitable parents for breeding programs with a view to cross different genotypes for yield improvement. The enzymes used in this investigation are further able to characterize genetic variability of silkworm strains and their relationships. Haemolymph esterases, acid phosphatase and malate dehydrogenase, as well as phosphoglucomutase and hexokinase from silk glands are appropriate markers for examination the genetic diversity and differentiating the silkworm strains. Allozyme data depicted substantiate amount of diversity among strains tested and hence, they can be utilized as parents in breeding programs.

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Notas de autor