Genetic characterization of silkworm (Bombyx mori L.) strains (Lepidoptera: Bombicidae) with different geographical origin on the basis of isozyme markers

The aim of the present study was to assess genetics variations within and among strains with different geographical origin, belonging to Bulgarian germplasm bank of mulberry silkworm (Bombyx mori L.) and to establish their relationships using isoenzyme markers. Polyacrilamide gel electrophoresis (PAGE) was used to study the isoenzymes of nonspecific esterases (EST), malate dehydrogenase (MDH) and acid phosphatase (ACP) from haemolymph, phosphoglucomutase (PGM) and hexokinase (HK) from silk glands and alkaline phosphatase (ALP) from midgut of mulberry silkworm (Bombyx mori L.). Variability was evident in all of these enzyme systems among twelve strains from Bulgarian germplasm resources of B. mori. Total of nine loci were detected. All of them (100%) were polymorphic. “Null” alleles in four loci were determined. Intraand inter-strain polymorphism was obtained. The degree of polymorphism ranged from 0% to 77.80%. Low levels of observed heterozygosity in comparison with the expected one have been calculated in almost all of strains as well as deviations from Hardy-Weinberg equilibrium in almost all analyzed loci, resulting of excess of homozygotes. The value of FST was 0.4903. The dendrogram constructed with the values of genetic distance manifests that Romanian strain Cislau Tokay is formed one main clade while the rest strains studied (from Bulgaria, Japan, China, Vietnam, Spain, Syria and Egypt) are formed the other clade with subclades. The genetic data of the tested strains from different geographical regions, would be used for identifying suitable parents for breeding programs with a view to yield improvement in this species with economic significance.


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. Par + Uni-bivoltine strain, introduced in 1998. The egg serosa color is gray, chorion color is white and eggs are sticky. The larvae are white in color and plain with translucent yellow haemolymph. The cocoons are yellow in color and elongated.

Shandon
Three moulter strain, introduced in 1998. The egg serosa color is grey brown, chorion color is white and eggs are sticky. The larvae are white in color and plain. The cocoons are spindle with cream color.

Daizo
Poly -bivoltine strain, introduced in 1998.The egg serosa color is green brown, chorion color is yellow and eggs are sticky. The larvae are white in color with marking and with translucent haemolymph. The cocoons are green yellow in color and spindle.

Sh 3 China
Uni-bivoltine strain, introduced in 1990.The egg serosa color is gray or green, chorion color is white or yellow and the eggs are sticky. The larvae are white in color with marking or plain. The cocoons are white in color and oval-elongated in shape.

70-42 Vietnam
Uni-bivoltine strain, introduced in 1989.The egg serosa color is gray, chorion color is white or yellow and the eggs are sticky. The larvae are white in color with marking. The cocoons are white in color, elongated with low constriction.

AES 1 wh Spain
Uni-bivoltine strain, introduced in 2008. The eggs serosa color is green and gray, chorion color is white or yellow and eggs are sticky. The larvae are plain and with zebra marking. The cocoons are elongated and spindle with yellow color.

Syria 2 Syria
Uni-bivoltine strain, introduced in 1991.The egg serosa color is green, chorion color is yellow and eggs are sticky. The larvae are white in color and plain. The cocoons are white in color with oval shape.

E 31 Egypt
Uni-bivoltine strain, introduced in 2001.The egg serosa color is gray, the chorion color is white the eggs are sticky. Larvae are white in color, with markings. The cocoons are white in color, elongated with low constriction.
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).

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 A 1 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 B 1 , B 2 and B 3 ). Bes B 2 allele was fixed in the gene pool of Par+ and Daizo, whereas Bes B 3 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 B 1 and B 2 were presented in the gene pools), in Syria 2 (Bes B 1 and B 3 ) and in AES 1 wh (Bes B 2 and B 3 ). Among the strains with polymorphism on Bes B locus, the allele Bes B 1 showed the highest frequency in 70-42, Bes B 2 -in Shandon and Bes B 3 -in Cislau Tokay. Polymorphism with four alleles we found on the Bes D locus (Tab. 2). Bes D 1 and D 2 alleles were presented in the gene pool of 70-42, AES 1wh and E31, Bes D 1 and D 3 -in 371, Sh3 and Syria2, Bes D 2 and D 3 -in VB1, Bes D 3 and D o -in F-13. Bes D 2 allele was fixed in the gene pool of Cislau Tokay and Par+ and Bes D 3 -in Shandon. For Daizo we obtained Bes D o alele, only. Homozygous Bes D o /D o specimens did not show any bands in the corresponding esterase zone BES D. Frequency of the allele Bes D 1 was the highest in Syria 2, of Bes D 2 -in AES 1wh and of Bes D 3 -in F-13 (except for strains with fixing Bes D alleles). Three allele polymorphism were found in Bes E locus (Bes E 1 , E 2 and E o alleles) in VB1, Cislau Tokay, 70-42, AES 1wh and Syria 2. Two of these alleles we obtained in Shandon (Bes E 1 and E o ), F-13 and E31 (Bes E 2 and E o ). All tested individuals from Daizo were homozygous of Bes E 2 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 A 1 , A 2 and A 3 in VB1, 371 and 70-42 strains. Pgm A 1 and A 2 were presented in the gene pool of Par+ and Pgm A 2 and A 3 -in Cislau Tokay, Sh3, AES 1wh, Syria 2 and E31 (Table 2). Pgm A 1 allele had the highest frequency in Par+, Pgm A 2 -in 70-42 and Pgm A 3 -in Syria 2. In Daizo we obtained Pgm A 1 allele, while in F-13 and Shandon strains -Pgm A 2 allele, only.
For the Mdh locus, three alleles were recorded in strain 371 (Mdh A 1 , A 2 and A 3 ). Two of them (Mdh A 2 and A 3 ) were found in the gene pool of Cislau Tokay, F-13 and Par+ (Table 2). Mdh A 3 was the most common allele among these four strains with polymorphism of the Mdh locus. Mdh A 2 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 A 1 and A 2 ) was recorded in AES 1wh strain, only ( Table 2). All the rest strains were monomorphic on this locus with fixed Alph A 1 allele.
Among the twelve strains included in the present study, we found polymorphism with two alleles on the hexokinase (Hk A) locus. Hk A 1 and A 2 alleles were recorded simultaneously in F-13, only. Hk A 1 allele was fixed in Shandon, whereas Hk A 2 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 H e 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).
Acta Scientiarum. Biological Sciences, v. 42, e47970, 2020 Summarized results of F-stastistics are presented in (Table 4). The mean F ST 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 A o and Alp A 2 alleles were presented only in AES 1 wh strain, Mdh A 1 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  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 F IT 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.