Genetic Analysis of Izoenzymes Polymorphisms in Silkworm ( Bombyx mori L . ) Strains

This work carried out to evaluates the polymorhism in the silkworm of different lineages using the isoenzymes electrophoresis to detect biochemical markers and to investigate the genetics of populations for those lineages. They were used as samples individual extracts of silk glands of second day old larvas of the fifth instar, originating from seven Japanese lineages and eight pure Chinese lineages maintained by the Cocamar-Cooperativa Agroindustrial de Maringá. The isozymes acid phosphatase (ACP), alkaline phosphatase (AKP) and carbonic anhydrase (CA) they were submitted to the electrophoresis in starch gels 14%. The esterases (EST) were analyzed in polyacrylamide gels to 10% and stained with α and β-naphtyl acetate. The total of 21 loci was detected, and 04 (19.05%) they are polymorphic, Est-11, Acp, Akp, Ca. The fixation index (Fis) for the analyzed isozymes it was 0.0751, indicating excess of homozygotes. The value of Fst (0.6128) it shows that the lineages are well differentiated. The dendrogram obtained with the values of genetic distance didn't separate the Chinese and Japanese lineages analyzed totally. That preliminary evaluation of the lineages of B. mori shows that they present genetic material that it can be used in breeding programs that have the purpose of producing hybrid for silk production.


Introduction
The world production of silk is around 635 thousand tons, only the China produces 70%, following by India with 20% (ANN, 2008).Brazil participates in 3.2% of that production, arriving to 10.300 tons.Paraná State is the largest producing of Brazil silk, it was responsible for 89% of the harvest 2006/2007(IAPAR, 2008)).
The silkworm silk can be classified according to their geographical origin, such as Japanese, Chinese, Indian or European.This insect has morphological and physiological differences correlated with the production of silk because of its origin and, therefore, different breeds ent origins have been, used in crosses, called arrays, as a source of characters for selection in favor of sericulture (PORTO et al., 2004).
The silkworm has been used as model for genetic studies since the discovery of the Mendelian inheritance in the beginning of the last century, due to its large size, easy of maintenance in the laboratory, and the economic importance (NAGARAJU, 2000).According to this author, Bombyx mori is, after Drosophila melanogaster, the second most important model insect in genetic studies.Over 400 mutations have been mapped in 28 linkage or chromosomes groups in successful genetic studies with the species (NAGARAJU, 2000).Besides, hundreds of geographical races and genetically improved strains are maintained in different countries where sericulture is in vogue.These races and strains differ not only in well characterized Mendelian traits but also in not so well-studied complex or quantitative traits such as body size, feeding duration, thermal tolerance, and disease resistance (NAGARAJU, 2000).
Since 1995, different molecular markers such as RAPD (PROMBOON et al., 1995) AFLP (TAN et al., 2001) and microsatellites (MIAO et al., 2005) are being used to detect polymorphisms and construct linkage maps for silkworm.
Aside from the different molecular markers available nowadays, biochemical markers such as isoenzymes have generated a broad array of practical information for the identification of hybrid species and natural and cultivated populations of diverse live organisms.Compared with other markers, these analyses are relative simple, fast and cheap (TEIXEIRA et al., 2004).
Polymorphism of nonspecific esterases from pupal haemolymph was analyzed, as well as of phosphoglucomutase from different organs of larvae, pupae and imago, from eight introduced from Bulgaria were analysed by Staykova (2008).
The observation of genetic polymorphisms and molecular markers of those lineages can contribute with an improvement in the handling and consequently with the silk production in the northwest of Paraná State.
This study aimed to evaluate the genetic polymorphism in larvae of Japanese and Chinese silkworm (B.mori L.) lineages through isoenzymes, to detect molecular markers and investigate the population genetics of these strains.

Material and methods
Two-day-old larvae of the 5 th instar of different Bombyx mori lineages were provided by ACESP (Associação dos Sericicultores de Nova Esperança e Regiões Sericícolas), Paraná, Brazil.Of the 15 lineages under study, seven were of Japanese and eight of Chinese origin (Table 1).The larvae were deep-frozen at -20ºC in duly closed and identified flasks for the laboratory analysis.
For the electrophoresis the silkworms were dissected and the silk glands removed under a stereomicroscope in a dissection dish.
Electrophoresis was performed for 4.5 hours at 4ºC, at a constant voltage of 200 V.Running buffer was 0.1M Tris-glycine, pH 8.3.
The samples were homogenized in centrifuge tubes (1.5 mL) in 150 μL β-mercaptoethanol and 40 μL carbon tetrachloride with a glass stick and immediately centrifuged at 12000 xG for 15 minutes to 2ºC.Of the supernatant, 20 μL were used with 20 μL of 0.1 M Tris-HCl solution pH 8.8 containing 10% glycerol.This solution (15 μL) was then applied on the gel.
After the electrophoresis the gel was incubated for 30 min. in 50 mL sodium phosphate buffer solution at 0.1 M pH 6.2.Then the gel was incubated for ± 1 hour at 38°C in staining solution (50 mL phosphate buffer 0.1 M pH 6.2; 5 mL Npropanol; 0.06 g Fast Blue RR Salt; 0.03 g αnaphthyl acetate and 0.02 g β-naphthyl acetate) (LAPENTA et al., 1995).

Starch gel
Horizontal electrophoresis was carried out using the starch gel technique according Ruvolo-Takasusuki et al. (2002).After the electrophoretic running the starch gel was cut in three homologous parts, used for the staining of three enzyme systems (Acid phosphatase, alkaline phosphatase and carbonic anhydrase).The buffer system used for the gel was Tris-HCl pH 7.5 0.02M and for the run Tris-HCl at 0.3 M pH 7.5.
Carbonic anhydrase (CA -EC 4.2.1.1)Fluorescein diacetate (10 mg) was dissolved in 0.5 mL acetone and mixed in 100 mL sodium phosphate buffer 0.1 M, pH 6.5.Gel was soaked in that solution and incubated at 37ºC for 40 minutes, and was observed under long wave ultraviolet light.

Analysis of population genetics
After the revelation of the isozyme activity regions, the phenotypes of the discovered loci were recorded.The gene frequencies as well as the measure of the genetic variability were directly estimated, to determine the deviations from the expected genotypic frequency according to the Hardy-Weinberg equilibrium.Furthermore the similarity coefficients and genetic distances for all analyzed strains were calculated, using software Popgene 1.31 (YEH et al., 1999) and Genepop 4.1.1(RAYMOND; ROUSSET, 1995).
The mean heterozygosity observed for all loci was 0.0350, while the expected heterozygosity was 0.0929 (Table 3).These results indicate low heterozygosity for the analyzed loci and suggest that these lineages can be crossed with others for genetic improvement.
The fixation index (F is ) for the analyzed isozymes was 0.0751, indicating homozygous excess and that there is probably no inbreeding in the lineage (Table 4).The high F st value (0.6128) allows the conclusion that the lineages are differentiated (Table 4).Silkworm hybrids are obtained by single crosses of two silkworm parent races, normally Japanese crossed with Chinese races, to obtain the so-called direct hybrids (KRISHNASWAMI et al., 1979).Another form of hybrid production is the mating of two F 1 hybrids produced by different combinations of Japanese and Chinese B. mori races, in other words, double cross hybrids or tetra-parental hybrids (NAGARAJU, 2002).Shows the Japanese and Chinese strains with lowest variability, which are considered pure and are therefore indicated for the development of hybrids with promising results regarding the performance in the silk production.
The values obtained by the genetic distance of Nei (1978) show that the strains are distinct.The results showed that the strain 5 and strain 15 are identical and can be considered a single lineage.
The dendrogram based on the distance of Nei (1978) by the UPGMA method (Figure 1) shows that the Japanese and Chinese strains could not be totally separated by the isoenzyme system analysis.The results indicate that in spite of the genetic distance and differentiation among the lineages, they cannot be separate just with the isozymes alleles.Nei (1978) between Chinese and Japanese Bombyx mori lineages.
The matrices that compose these strains are maintained by crosses that may reduce the genetic variability.It would therefore be important to continuously evaluate the polymorphism degree of the strains, to avoid a marked increase of homozygosis.This would result in the expression of deleterious genes that can cause high matrix mortality.
Several studies have been developed with molecular markers in silkworm to detect the polymorphism degree, develop linkage maps for genes related to silk production and to map economic relevant genes.
The first linkage map by the Amplified Fragment Length Polymorphisms (AFLP) marker in B. mori was developed by Tan et al. (2001).For the AFLP mapping 47 progenies of a backcross population were genotyped.The authors found a total of 1248 (60.7%) polymorphisms detected with 35 primer combinations with adaptor PstI/TaqI.Since each one of the primer combinations generated an average of 35.7 polymorphic markers, a total of 44% of the polymorphic markers was consistent with the segregation ratio of 1:1 at a significance level of P = 0.05.The total AFLP map length was 6512 cM.The genetic distances between two neighboring markers varied from 0.2 to 47 cM, with a mean of 18.2 cM.The od linked gene to the sex was located between the markers P1T3B40 and P3T3B27 at the end of group 3, indicating that the linkage group AFLP 3 was on the Z chromosome (sexual).
The domesticated silkworm, B. mori, is a herbivorous insect and a model organism for Lepidoptera.Chinese and Japanese scientists made great efforts to accomplish the sequencing project of the whole silkworm genome, which offers Bombyx researchers an opportunity to identify peptides using proteomic method (MITA et al., 2004).
The mtDNA of some insects has been completely sequenced, including the mtDNA-arrays of four races of B. mori: (1) Xiafang (LU et al., 2001), (2) Backokjam (LEE et al., 2002) (3) C108 (YUKUHIRO et al., 2002), (4) Aojuku (ITOH et al., 2002).These sequences were used to determine the average size of mtDNA from B. mori in 15,650 bp.Prasad et al. (2005) evaluated the microsatellite frequency and distribution in 21.76-Mb random genome sequences, 0.67-Mb in BAC sequences of the Z chromosome, and 6.3-Mb EST ("Expressed Sequence Tags") sequences of B. mori.In this study microsatellites were estimated in 0.31% of the silkworm genome.
In 13 B. mori strains analyzed by Prasad et al. ( 2005), 23 polymorphic microsatellite loci were found with variation of 2 to 14 alleles, with a mean heterozygosity value of 0.54.Only 36 (18.2%) of 198 microsatellite loci were polymorphic between the two divergent silkworm populations and 10 (5%) loci had no alleles.The map based on these polymorphic markers presented eight linkage groups.The microsatellite loci were most conserved in the closest ancestor, B. mandarina, followed by the wild silkworm, Antheraea assama.
The analysis of 15 microsatellite loci of 13 B. mori strains revealed 3 to 17 alleles, with a heterozygosity between 0.66 and 0.90 (REDDY et al., 1999).These microsatellites enabled the authors to obtain alleles for strains with and lineages without diapause.Chatterjee and Pradeep (2003) identified molecular markers associated with silkworm growth and yield parameters. RAPD profiles were generated with 7 UBC primers for 14 silkworm lineages originated from China, Japan, India, and Russia and statistically analyzed.By multiple regressions analysis a significant association of 45 markers was detected with the larval development, the growth indices and the four relevant cocoon yield parameters for silk production.Miao et al. (2005) constructed a dense linkage map of B. mori using 518 Simple Sequence Repeat (SSR) sequences or microsatellites, as part of an international genome program.Six representative strains and 2500 (CA)n and (CT)n microsatellite markers were used in the study.
These markers revealed a polymorphism of 17 -24%, indicating the high homozygosity degree resulting from the long-standing history of inbreeding of silkworm.Those studies with molecular markers indicate that the genome of the silkworm is being studied broadly, for the development of better lineages of those insects.
The analyzed isozimas showed that the 15 Japanese and Chinese lineages maintained by ACESP-Brazil are genetically differentiated, possess a high polymorphism degree and can be used for production of hybrid and to do part of a future improvement program for those lineages.
Besides the improvement the isozymes may contribute to the understanding of the expression of functional genes, in different tissues or organs and in different phases of the development in B. mori.

Conclusion
Through the analysis of isoenzyme found an excess of homozygotes, results showed that the line 5 and line 15 are identical and can be considered a single lineage.
The genotypes of the study strains can be used in future studies of genetic improvement to develop superior hybrids for silk production.

Figure 1 .
Figure 1.Dendrogram of genetic distance constructed by the UPGMA method based onNei (1978) between Chinese and Japanese Bombyx mori lineages.

Table 1 .
Bombyx mori lineages and their origin.

Table 2 .
Allelic frequency of the polymorphic loci of Bombyx mori lineages.

Table 3 .
Observed and expected heterozygosity of isoenzymes for 15 loci of Bombyx mori lineages.

Table 4 .
Values of the Fixation index (F is ) and differentiation degree (F st ) of Bombyx mori strains.