«Corresponding author: M.A. Fernandez E-mail: aparecidafernandez Genet. Mol. Res. 12 (2): 2138-2147 (2013) Received July 26, 2012 Accepted ...»
Biological and molecular characterization
of silkworm strains from the Brazilian
germplasm bank of Bombyx mori
N.C. Pereira1, R.E.F. Munhoz1, T.S. Bignotto1, R. Bespalhuk1, L.B. Garay2,
C.R.N. Saez1, V.A. Fassina1, A. Nembri2 and M.A. Fernandez1
Departamento de Biologia Celular e Genética,
Universidade Estadual de Maringá, Maringá, PR, Brasil
Seda y Fibras S.R.L, Hernandarias, Alto Paraná, Paraguay
Corresponding author: M.A. Fernandez
Genet. Mol. Res. 12 (2): 2138-2147 (2013) Received July 26, 2012 Accepted March 2, 2013 Published June 28, 2013 DOI http://dx.doi.org/10.4238/2013.June.28.1 ABSTRACT. Brazil has only one public genetic pool of Bombyx mori strains, which was established in 2005 at Universidade Estadual de Maringá, Maringá, Paraná State. This genetic bank has been maintained, and the strains have been characterized using genetic and morphological tools. The quantitative and qualitative traits, directly or indirectly related to productivity, were evaluated in 14 silkworm strains. In addition to biological and productivity analyses, DNA markers related to susceptibility to the B. mori nucleopolyhedrovirus (BmNPV) were analyzed. BmNPV is a major cause of production loss and is a serious problem for Paraná sericulture. The silkworm strains from diverse geographic origins were found to have different characteristics, including body weight, larval stage duration, cocoon weight, and other biological traits. In terms of productivity, the raw silk percentages were almost uniform, with an overall average of 16.28%.
Overall, the Chinese strain C37 gave the best performance in many of the quantitative traits, and it surpassed the other strains in productivity Genetics and Molecular Research 12 (2): 2138-2147 (2013) ©FUNPEC-RP www.funpecrp.com.br 2139 Genetic characterization of Brazilian B. mori germplasm bank traits. Therefore, it can be used as one of the strains that compose the elite germplasm for silkworm breeding programs. Additionally, genetic
INTRODUCTIONBombyx mori L. was described by Linnaeus in 1758 and belongs to the order Lepidoptera and the family Bombycidae. B. mori has been domesticated for approximately 5000 years using a practice called sericulture. The silkworm is a well-known lepidopteran, and it has been used as a model system to provide a rich repertoire of information related to genetic mutations that affect morphology, development, and behavior (Arunkumar et al., 2006). This species has been used as a source of silk production and has lost some features through many years of breeding under artificial conditions and the domestication process.
Silkworm specimens are no longer observed in nature and are totally dependent on human intervention for their survival. Genetic stocks of this species are found only in germplasm banks. Silkworm germplasm banks are important reservoirs of biodiversity and a source of alleles that can be easily retrieved for the genetic enhancement of popular strains. Accurate estimation of genetic diversity is becoming more important in the conservation of silkworm genetic resources. The use of molecular markers provides a much more accurate analysis of genetic diversity than the evaluation of morphological traits (Jingade et al., 2011). In 2006, the Universidade Estadual de Maringá (UEM) initiated the Program for Silkworm Genetic Breeding. This genetic wealth has thus been consolidated in the only public B. mori germplasm bank in Brazil.
Economic interest in this lepidopteran has promoted great advances in domestication and genetic breeding. The genetic improvement of B. mori is important in obtaining hybrids with increased productivity and lower susceptibility to various diseases. According to Fonseca and Fonseca (1988), a silkworm breeding program requires a survey of all the important sericulture characters related to the nature of the egg, larva, cocoon, and moth.
Many pathogens can affect B. mori strains, but their susceptibility to baculovirus infection is the major cause of production loss in sericulture and accounts for approximately 70% of cocoon losses (Brancalhão, 2002). One of the most important agents that cause this lethal silkworm disease is the B. mori nucleopolyhedrovirus (BmNPV). Therefore, the prediction of strain susceptibility or resistance to BmNPV is an important criterion in the evaluation of strains from the genetic bank. Yao et al. (2003) reported that random amplified polymorphic DNA (RAPD) markers could be associated with these characteristics.
Genetic and biological analyses of B. mori strains from germplasm banks are one of the most important steps in the maintenance, conservation, and use of these genetic resources (Chen, 2003). Based on the characterization of germplasm data and the identification of resistant strains, it is possible to develop commercial and experimental silkworm hybrids for industrial and scientific research (Srivastava et al., 2005).
In our study, 14 B. mori strains maintained at the UEM Brazilian Germplasm Bank were evaluated for biological and productivity traits, as well as for molecular genetic markers, including possible susceptibility to BmNPV.
MATERIAL AND METHODS
Silkworm larvae from each strain were reared on the same nutritional diet of mulberry leaves in rearing houses under semi-controlled temperature. The biological traits analyzed were egg shape (ES), color of the newly hatched larva (CNL), larval markings (LM), cocoon shape (CS), cocoon color (CC), pupa color (PC), pupa shape (PS), and color of the female and male moths. The 16 quantitative variables for evaluated production performance were cocoon weight (CW, g), cocoon shell weight (CSW, g), cocoon length (CL, mm), cocoon width (CWd, mm), raw silk percent [RSP = shell weight of 30 cocoon shells (g)/30 cocoon weights (g) x 100; 24% was deducted from this value due to silk wiring loss], number of eggs (unit)/moth, rate of hatching (%), total larval duration (days), duration of the 5th instar (days), larva weight on the 5th day of the 5th instar (g), silk gland pair weight of larva on the 5th day of the 5th instar (g), pupa weight (g), malformed cocoon rate (%), ambushing rate (%), cocoons per liter and pupa mortality rate (%).
The experimental delineation was completely randomized, and the data obtained from qualitative and quantitative evaluations were submitted to analysis of variance, followed by the Scott-Knott test (0.05%) using the SISVAR statistical software.
Genetic analysis Genomic DNA was extracted from the silkworm moth according to Monesi et al. (1998),
with modifications. Briefly, silk moths were first frozen with liquid nitrogen and ground using a cooled mortar and pestle. The homogenate was dissolved in 750 μL extraction buffer (10 mM Tris-HCl, pH 7.5, 60 mM NaCl, 10 mM EDTA, pH 8.0, 5% sucrose, 0.15 mM spermine, 0.15 mM spermidine), centrifuged at 10,000 rpm for 15 min and transferred to a new tube containing proteinase K buffer (0.2 M Tris-HCl, pH 9.0, 30 mM EDTA, pH 8.0, 5% sucrose, 2% SDS, 100 μg/mL proteinase K). The samples were incubated at 50°C for 2 h, centrifuged at 12,000 rpm for 15 min and extracted with chloroform and phenol/chloroform. The DNA-containing phase was ethanol-precipitated in the presence of 0.2 M NaCl and resuspended in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) containing 10 mg/mL RNase. The DNA was re-extracted with phenol/chloroform and ethanol-precipitated as described above and was quantified using a UVPC UV-visible spectrophotometer (Shimadzu) and diluted to 60 ng/μL for RAPD analysis.
RAPD analysis was carried out by the amplification of genomic DNA in a thermal cycler (Eppendorf) using 20 mL 10X Taq polymerase buffer, 200 mM dNTPs, 1.0 mM MgCl2, 25 µM primer, 1 U Taq polymerase, and 60 ng template DNA. The amplification conditions were 38 cycles at 94°C for 30 s, annealing at 36°C for 60 s and extension at 72°C for 120 s, followed by a final extension at 72°C for 10 min.
The primers OPW-11 (5ꞌ-CTGATGCGTG-3ꞌ) and OPA-18 (5ꞌ-AGGTGACCGT-3ꞌ) were described by Yao et al. (2003) and were used in the analysis. PCR products of the OPW-11 primer were analyzed by electrophoresis on a 1.5% agarose gel run for 1 h at 100 V; PCR products generated by the OPA-18 primer were analyzed on an 8% polyacrylamide gel run for 17 h at 110 V.
RAPD analyses were conducted using the BioNumerics software (version 6.5, Applied Maths, Kortrijk, Belgium). The polymorphisms generated by the OPW-11 and OPAprimers were used to determine the Jaccard coefficient, which was used to construct the UPGMA dendrogram.
RESULTS AND DISCUSSIONBiological and productivity analyses The evaluation of qualitative and quantitative traits helps characterize and differentiate between different strains from the UEM Brazilian Germplasm Bank. In addition to helping to maintain the germplasm bank, these analyses are crucial in choosing the parents involved in the production of superior hybrids in breeding programs. Moreover, the biological performance of B. mori reflects maximum final silk production potential (Porto et al., 2004). Qualitative traits from 14 silkworm strains were evaluated, including ES, CNL, presence of LM, CS, CC, PC, PS, female moth color, and male moth color; the results are listed in Table 2.
ES varied according to the geographical origin of the strain; Japanese strains had elliptical-shaped eggs, whereas Chinese strains had oval-shaped eggs. Most of the eggs were small in size, except for AS3 and C75 strains. Many of the CNL were brown in color. Exceptions included the AS31, C36, C37, and JK strains, whose CNL were black. Two patterns of LM, plain and multilunar, were observed. Plain larvae did not have any markings, whereas multilunar larvae had 2 different characteristic spots. In the literature, different types of LM have been described, including zebra, speckled, quail, multistars, and others (Tazima, 1978).
LM is a useful variable as it helps prevent the mixing or contamination of larvae from different strains.
CS can be either elliptical or oval and can also exhibit variations in the intensity of constriction at the center of the cocoon (Bojan et al., 2007). Although the intensity of constriction varied between strains, the Chinese strains produced oval cocoons and Japanese strains produced elliptical or peanut cocoons in this study. CS is an extremely relevant variable in commercialization, since automated wiring admits only elliptical cocoons into the machines.
In general, silkworm CC can vary greatly. There are descriptions of green, pink, and blue cocoons (Basavaraja et al., 2005). However, the strains evaluated in the present study did not show any variation related to color. All of the strains produced white cocoons that varied in brightness and opacity as indicated in Table 2.
Important strain features such as PC, PS and moth color were uniform (Table 2), demonstrating that these variables were not indicative of the qualitative distinction between the strains analyzed. This homogeneity hampers pupa and moth morphological characterization;
therefore, researchers should take care when performing crosses between individuals for strain maintenance or breeding.
The quantitative variables analyzed, including CW, CSW, CL, CWd, and RSP, are shown in Table 3. CW and CL varied greatly, with values ranging from 0.73 to 1.66 g for CW and from 28.5 to 35.2 mm for CL. In general, Chinese strains performed the best regarding CW, except for the AS31 strain. In contrast, most Chinese strains demonstrated low CL values, with the C37 strain being an exception. CSW variations were observed for the C37 and M11 strains of Chinese and Japanese origin, respectively (Table 3). In contrast, only minor variation was observed for cocoon width, with values ranging from 13.14 mm for the M8 strain to
20.85 mm for the C37 strain.
The RSP is a more uniform trait among the strains analyzed, with an overall average of 16.28%. This value was lower than the 18.21% reported by Zanatta et al. (2009) in a study of other silkworm strains of the same UEM Brazilian Germplasm Bank. On the other hand, Porto et al. (2004) observed an average RSP of 16.37%, which is closer to that observed in the present study. However, a lower value (14.99%) than that observed in this study was reported by Kumaresan (2007). Other strains from a Pakistani germplasm bank (Hussain et al., 2010) and a polyvoltine germplasm bank (Rao et al., 2006) also revealed lower RSP values of 11.6
and 11.1%, respectively. Overall, the C37 strain demonstrated the best performance in most of the quantitative variables analyzed (Table 3) and differed from other strains in terms of productivity traits.
Table 3. Mean values of quantitative traits for 14 strains of Bombyx mori from the UEM Brazilian Germplasm Bank.
For a more complete characterization of the 14 strains, 11 additional traits were evaluated, and the results are shown in Table 4. In this analysis, a large variation was observed in the number of eggs per laying. The best performance was observed with the JK strain with an average of 644 eggs per laying, and the worst performance was observed with the B106 strain with an average of 270 eggs per laying. Overall, 9 strains laid more eggs than the average value of 409.79 reported by Porto et al. (2004).
Table 4. Quantitative traits related to 14 Bombyx mori strains from the UEM Brazilian Germplasm Bank.