A molecular approach for estimating of genetic identify and relationship in sixteen genotypes of mulberry silkworm.
IntroductionSilkworm breeding programs are based on the development and selection of outstanding hybrids from inbred lines [35]. Developing and selecting inbred lines for performance is mainly based on morphological characters although time consuming. But hybrid performance is evaluated from extensive yield trials that are costly and time consuming. In any hybrid program, a large number of crosses are made, while only a few good hybrids are obtained. This process is extremely labour intensive, time consuming and tedious and often alternative methods are explored for analyzing the potential parental material. It is already an established fact that the amount of yield obtained by hybrids (Heterosis) depends largely on the genetic divergence of the populations from which the parental lines have been extracted [22]. The level of genetic diversity between the two parents being used for crossing has been proposed as a possible predictor of F1 performance [39]. [D.sup.2] statistics by Mahalanobis [20] has been extensively used in predicting the hybrid performance on the basis of morphological traits. However, these methods require extensive field tests and crossing and hence, a need is now felt to determine genetic distance at the molecular level and thus to predict hybrid performance.
Biochemical markers are useful for screening germplasm with the minimum cost in time and labour [24]. The most important factor in biological genetic resource conservation regimes is to maintain pure strains of each species and establish accurate genetic relationship among species [30]. The Polyacrylamide gel electrophoresis is probably the most highly resolving electrophoretic method yet developed for separating proteins. PAGE is a high resolution method and one of the best available technique for separating complex mixtures of proteins, while using simple equipment. Thus, the study of polymorphic proteins of Bombyx mori is significantly important for selection and hybridization. The establishment of suitable biochemical markers for analyzing the degree of genetic heterogeneity may be used for a preliminary evaluation and selection of the different breeds and families when making optimal variants for cross-breeding with reference to improving the most important economic traits and increasing the effectiveness of heterozygous selection. With this regard different polymorphic enzyme groups have been studied [1,9,16,21,34]. When studying different breeds raised mainly in Japan, China, Korea, India and the former Soviet Union, in the group of nonspecific esterases from different tissues, a genetically determined polymorphism has been ascertained [14,15]. It is important to note that the application of protein and enzyme polymorphism opened up a new approach to the problem of obtaining genetic handle for use in studying proteins and enzymes [6]. Diversity between Bombyx mori can be analyzed with the help of haemolymph protein pattern using SDS-PAGE [5,7,12,27].
This investigation was planned with the objective to record the genetic differences in terms of protein profiles for the better understanding of the genetic variability among the silkworm races and their hybrids so that the knowledge gained may serve as an important yardstick in silkworm breeding.
Materials and methods
Silkworm rearing:
The Nistari, Pure Mysore, [NB.sub.4][D.sub.2] and [C.sub.108] races were drawn from the germplasm bank of the Department of Studies in Sericulture Science, Manasagangotri, University of Mysore, Mysore, India. After incubation of eggs at 25 [+ or -]10[degrees]c and relative humidity of 80[+ or -]5 %, block box was carried out on 8th day to achieve uniformity in hatching. Three layings of each of the pure races were selected. The larvae hatched from each laying were reared separately under uniform laboratory conditions as described by Yokoyama [38] and Krishnaswami [18].
Regular and reciprocal crosses were prepared. They were crossed in different combination of 4 x 4 diallel and the experimental was treated as a completely randomized design. All the four pure races and twelve F1 hybrids were reared during the monsoon season of the year during 2008-2009.
Haemolymph Collection:
Haemolymph was collected in a pre-chilled eppendorf tube containing a pinch of crystal of thiourea by cutting the first pro-leg of larvae. The haemolymph was centrifuged at 5000 rpm for 10 min to remove haemocytes and debris and the supernatant was collected and used. The estimation was made on 5th day of 5th instar. From the collected haemolymph samples the total soluble protein (mg of protein/ml of haemolymph) was estimated by following standard procedure [19] and crystalline Bovine Serum Albumin (BSA) used as standard.
Protein Estimation:
Protein determination is necessary to estimate the amount of protein in the sample, to normalise against the protein concentration or during purification procedure. Depending on the amount of sample, accuracy and presence of interfering agents, one needs to decide on the method to be used. For accurate quantification, the sample protein is compared with a known amount of a standard protein which could either be the commonly used bovine serum albumin (BSA) or it could sometimes be immunoglobulin G (IgG).
Polyacrylamide Gel Electrophoresis:
100 [micro]l of sample buffer (4X) was added to 10 [micro]l of haemolymph and mixed thoroughly in Eppendorf tube. The samples were kept in a float of water bath at 95[degrees]C for 3 minutes. 10 [micro]l of the samples prepared is injected into each slot of the gel, which are then carefully layered with runing (tray) buffer [0.05 M Tris, 0.384 M Glycine buffer (PH 8.3) aontaining 0.1% SDS]. Haemolymph were analyzed through slab type SDS-PAGE using 8.25 % polyacrylamide gel. The haemolymph protein bands were separated upon application of an electric field based on molecules size and charges. The samples were run initially at 15 mA for 1 hour up to stacking gel and loud for 3h at 100 volts till the dye mark reached the bottom of the gel. Standard molecular weight marker was used to compare the approximate molecular weight of the haemolymph proteins separated from sample. The gels were stained in 0.3% Coomassie brilliant blue R-250 in mixture of distilled water, methanol and acetic acid (9:6:3) for over night. After appropriate destaining with mixture of distilled water, methanol and acetic acid (9:6:1 v/v), the gels were photographed on Transilluminator.
Data analysis:
To estimate significant differences among mean value of haemolymph protein of parents and F1 hybrids, the data were subjected by analysis of variance using SAS software version 9.1. The cluster analysis was carried out using the UPGMA method (Unweighted Pair Group Method Algorithm) developed by Sokal and Michener [31] with help of computer software Alphaview version 3.0 For SDS-PAGE.
Results and discussion
Table-1 represents the quantitative estimation of proteins through standard Lowery et al. method. Perusal of the data clearly indicates that between the four pure races the total amount of haemolymph proteins is significantly different. Similarly, among the hybrids significant difference in the total amount of the proteins were recorded by [C.sub.108] x Pure Mysore, [NB.sub.4][D.sub.2] x Pure Mysore, Nistari x [NB.sub.4][D.sub.2] and Pure Mysore x Nistari. It is important to note that the hybrids of [C.sub.108] x Nistari revealed 31.09 [+ or -] 1.355 mg/ml proteins in the haemolymph compared to a highest of 52.00 [+ or -] 0.389 mg/ml protein in the hybrids of Pure Mysore x Nistari. None of the hybrids and pure races exhibited uniform quantity of proteins (mg/ml). The table also clearly indicates a CD value of 0.744 at 5% level.
After staining the electrophorased gels with commassie brilliant blue peak areas (optical density/mm) of various protein fractions were measured with the help of Alpha Innotech gel documentation system, USA and the protein fractions were arbitrarily classified in to "Major fractions" whose peak area measured [greater than or equal to] 0.02 and in to "Minor fractions" whose peak area measured [less than or equal to] 0.02.
Figure-1 presents the protein banding pattern in the four pure races namely [C.sub.108], [NB.sub.4][D.sub.2], Pure Mysore and Nistari. Based on the electrophoretic pattern it is evident that a total of 22 bands were observed in [C.sub.108] race, 21 bands in [NB.sub.4][D.sub.2], 19 bands in Pure Mysore and 24 bands in Nistari. Thus, the number of bands encountered in four races ranged from 19-24. It is important note that most of the major bands are observed between 45-66 kDa. It is also clear from the data that each race in unique in the total number of bands and also in the intensity of the bands.
Figure-2 presents the electrophoretic pattern among the three F1hybrids namely [C.sub.108] x [NB.sub.4][D.sub.2], [C.sub.108] x Pure Mysore and [C.sub.108] x Nistari. It is evident from the figure that the expression of the individual band is different among the three hybrids. Though major number of the protein bands falls between 14 to 55 kDa it is important to know that every hybrid combination is unique in the total number of bands present. Based on the scanning of the gels utilizing gel documentation system an highest of 22 bands were recorded in [C.sub.108] x [NB.sub.4][D.sub.2] and a lowest of 16 bands were observed in the hybrids of [C.sub.108] x Pure Mysore, whereas [C.sub.108] x Nistari recorded 18 bands. Figure-3 shows the protein-banding pattern in the three F1 hybrids namely [NB.sub.4][D.sub.2] x [C.sub.108], [NB.sub.4][D.sub.2] x Pure Mysore and [NB.sub.4][D.sub.2] x Nistari. A maximum number of 17 bands with different intensities are recorded in the hybrids of [NB.sub.4][D.sub.2] x Pure Mysore followed by 16 bands in [NB.sub.4][D.sub.2] x [C.sub.108] and a minimum of 15 bands with different intensities are noticed in the hybrids of [NB.sub.4][D.sub.2] x Nistari. The protein banding clearly demonstrates that [NB.sub.4][D.sub.2] x [C.sub.108] and [NB.sub.4][D.sub.2] x Pure Mysore recorded higher intensity bands compared to other two hybrids. In all the three hybrids the bands of different molecular weights are observed.
Figure-4 represent the observation of the protein pattern in the fifth instar haemolymph among the three hybrids Pure Mysore x [C.sub.108], Pure Mysore x [NB.sub.4][D.sub.2] and Pure Mysore x Nistari. It is important that Pure Mysore x Nistari revealed a number of bands (19) with different intensities followed by Pure Mysore x [NB.sub.4][D.sub.2] (18 bands) and Pure Mysore x [C.sub.108] (15 bands).
Figure-5 represents the protein profiles through SDS-PAGE in the three multi x bi hybrids namely Nistari x [C.sub.108], Nistari x [NB.sub.4][D.sub.2] and Nistari x Pure Mysore. A maximum 19 bands with differential intensity was observed in the hybrid Nistari x [NB.sub.4][D.sub.2] followed by Nistari x [C.sub.108] (18 bands) and Nistari x Pure Mysore (17 bands).
The cluster analysis based on the UPGMA statistical package to establish relationship using haemolymph proteins (Figure-6) clearly indicated that [C.sub.108] and [NB.sub.4][D.sub.2] falls in the same cluster with a similarity coefficient of 0.18, Whereas Nistari and Pure Mysore being multivoltine falls in one cluster with a similarity coefficient of 0.03.
The UPGMA analysis based on Nei [25] algorithm has clustered (Figure-7) twelve hybrids in to three groups. The first group contained the hybrids of [C.sub.108] x Pure Mysore, Pure Mysore x [NB.sub.4][D.sub.2], Nistari x [NB.sub.4][D.sub.2] and Nistari x Pure Mysore. In group two only Pure Mysore x [C.sub.108] is distinctly represented based on UPGMA clustering. All the remaining seven hybrids categorized under group III.
The graphical representation (Figure-8) indicating the manifestation of heterosis calculated based on the banding pattern and total haemolymph proteins clearly indicated the positive heterosis for total quantity of protein in all the hybrids except the hybrids of [C.sub.108] x Nistari, [C.sub.108] x [NB.sub.4][D.sub.2], Nistari x Pure Mysore and Pure Mysore x [C.sub.108]. In regard to the total number of proteins bands through PAGE and calculation of heterosis, it is clear that all the hybrids exhibited negative heterosis which is statistically significant (P<0.05).
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Discussion:
The studies on proteins are of paramount importance in the growth and development of organisms and haemolymph proteins in insects plays an important role not only the specific transport functions but also in their enzyme action. The synthesis and utilization of haemolymph proteins are conditioned by genetic and hormonal control [2,3,34]. SDS-PAGE popularly called sodium dodecyl sulfate polyacrylamide gel electrophoresis is a technique used in biochemistry, genetics and molecular biology to separate proteins according to the electrophoretic mobility.
The differential quantity of soluble protein among the four races clearly indicates the racial differences between four races. Saranghi [28], Nagata and Yashitake [23] in their detailed investigations utilizing haemolymph of silkworm Bombyx mori, demonstrated that haemolymph proteins which functions as a specific transport media plays a vital role in the growth and development of larvae and it is variable among different breeds. Our present results corroborate with the results of the above workers, wherein the amount of soluble protein content in the haemolymph in the four races and twelve hybrids are differently expressed.
Observations on the protein patterns in fifth instar have revealed that the total number of bands in the gel which differs between pure races, between different hybrids and between pure races and hybrids (Table-2). Among the pure races a maximum of the 24 bands with different intensities are noticed in Nistari race followed by 22 in [C.sub.108], 21 in [NB.sub.4][D.sub.2] and 19 in Pure Mysore. Similarly among the hybrids a minimum of 15 bands is observed each in [NB.sub.4][D.sub.2] x Nistari and Pure Mysore x [C.sub.108]. Such variations in the mobility of proteins have been reported to be due to the post-translational modifications [8]. But Noel et al. [26] and Seeberg et al. [29] reported that variations in protein fractions are due to change in the sequence of even a single amino acid. In regard to the silkworm, Bombyx mori, Gamo [10] demonstrated that three lipoproteins according to their mobility on Polyacrylamide gel electrophoresis during different developmental stages and he has demonstrated that co-dominant allelic genes control these lipoproteins. Further, electrophoretic variation in the fibroin and sericin of the silkworm larvae was also reported by Gamo [11]. In addition, Gamo [12] in his studies using a mutant strain obtained three different fractions of sericin proteins linked to "K" gene of the chromosome 11 thereby revealing that major sericin proteins are located on single chromosome. Similarly haemolymph protein profiles in different multivoltine and bivoltine races of silkworm have been demonstrated by Somasundaram, et al. [32] and Koundinya et al. [17] among Indian races.
Figure 6 showed a UPGMA Dendrogram based on the cluster analysis of Nei [25] unbiased genetic similarity haemolymph proteins bands. The Dendrogram revealed the genetic variations that could be possible to derive from haemolymph proteins with similarity co-efficient varying between 0.03-0.42. The UPGMA model separated multivoltine and bivoltine races. The data has clearly demonstrated two bivoltine races [C.sub.108] and [NB.sub.4][D.sub.2] falls at the similarity co-efficient of 0.18 thereby indicating that the two bivoltine races comes under one cluster, whereas the two multivoltine races of tropical origins namely, Nistari and Pure Mysore deviates from 0.03 clustering indicating the genetic differences between the two voltine groups. Thus, the author in the present investigation reiterates that analysis haemolymph protein through UPGMA can be conveniently is utilized to understand the genetic distance between the pure races. From the clustering analysis in the twelve hybrids it is possible to distinctly separate three groups. The results have clearly indicated how twelve hybrids are different in their genetic constitution. The data distinguish based on the UPGMA how one could discriminate different hybrids. Similar observations are made by Vijayan et al., [36,37] for mulberry and Srivastava et al. [33] for silkworm Bombyx mori both in protein and DNA profiles.
A close scrutiny of the results of the banding pattern indicated that differential bands with different intensity and generally three distinct groups differing in their molecular weight are observed in the gel. Thus, from the consideration of the pertinent data presented in the graph (Figure-8), a definite pattern emerges on the heterosis concept. In a detailed work on heterosis hypothesis, Gordon Whaley [13] indicated that heterosis concept can be developed with the ability of the hybrid to synthesis or to utilize one or several specific substances involved in the fundamental growth process of the organisms. Further, Comstock [4] demonstrated that primary heterotic effect is concerned with growth substances which are predominant in plants. Thus, the present studies using protein polymorphism give us a clue on the heterosis expression for selected traits and can be used as an index in silkworm breeding. The evaluation of heterosis of total protein concentration from quantitative assay clearly indicated the heterosis in eight out of twelve hybrids. Based on the qualitative estimation through PAGE all the twelve hybrids established negative heterosis. Thus, from the results of the present findings of the author it is clear that there is an antagonistic heterotic expression in the twelve hybrids between quantitative and qualitative assay and such results can be used as an index in silkworm breeding.
Acknowledgments
The authors wish to express sincere thanks to the Chairman, Department of Studies in Sericulture Science, University of Mysore, Manasagangotri, Mysore for extending the facilities.
References
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(1) E. Talebi, (1) M. Khademi, (2) G. Subramanya
(1) Islamic Azad University, Darab Branch, Darab, PO Box: 74817-83143, Fars, Iran.
(2) DOS in Sericulture Science, University of Mysore, Manasagangotri, Mysore, India.
Corresponding Author
E. Talebi, Islamic Azad University, Darab Branch, Darab, PO Box: 74817-83143, Fars, Iran.
E-mail: [email protected]; Tel: +98-732-6233990 to 5
Table 1: Mean value ([+ or -] SE) of parents and F, progenies
for haemolymph protein (mg/ml)
Genotypes Haemolymph protein [+ or -] SE
46.39 [+ or -] 0.619 (cde)
[C.sub.108] x [NB.sub.4][D.sub.2] 40.13 [+ or -] 1.315 (ghi)
[C.sub.108] x Nistari 31.09 [+ or -] 1.355 (j)
[C.sub.108] x Pure Mysore 49.13 [+ or -] 0.600 (abc)
[NB.sub.4][D.sub.2] 43.43 [+ or -] 0.218 (efg)
[NB.sub.4][D.sub.2] x [C.sub.108] 50.82 [+ or -] 0.376 (ab)
[NB.sub.4][D.sub.2] x Nistari 44.56 [+ or -] 0.096 (def)
[NB.sub.4][D.sub.2] x Pure Mysore 50.54 [+ or -] 0.280 (ab)
Nistari 36.41 [+ or -] 1.107 (i)
Nistari x [C.sub.108] 46.45 [+ or -] 1.338 (cde)
Nistari x [NB.sub.4][D.sub.2] 48.83 [+ or -] 0.540 (abc)
Nistari x Pure Mysore 37.54 [+ or -] 0.103 (hi)
Pure Mysore 40.22 [+ or -] 0.085 (gh)
Pure Mysore x [C.sub.108] 41.20 [+ or -] 0.185 (fgh)
Pure Mysore x [NB.sub.4][D.sub.2] 47.35 [+ or -] 0.245 (bcd)
Pure Mysore x Nistari 52.00 [+ or -] 0.389 (a)
CD 5% 3.744
Means having the same superscript letters do not differ significantly
(P<0.05).
Table 2: Electrophoretic variation in haemolymph protein bands of four
races and their hybrids.
Genotypes Total number of
bands in the gel
[C.sub.108] 22
[NB.sub.4][D.sub.2] 21
Pure Mysore 19
Nistari 24
[C.sub.108] x [NB.sub.4][D.sub.2] 22
[C.sub.108] x Pure Mysore 16
[C.sub.108] x Nistari 18
[NB.sub.4][D.sub.2] x [C.sub.108] 16
[NB.sub.4][D.sub.2] x Pure Mysore 17
[NB.sub.4][D.sub.2] x Nistari 15
Pure Mysore x [C.sub.108] 15
Pure Mysore x [NB.sub.4][D.sub.2] 18
Pure Mysore x Nistari 19
Nistari x [C.sub.108] 18
Nistari x [NB.sub.4][D.sub.2] 19
Nistari x Pure Mysore 17
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| Title Annotation: | Original Article |
|---|---|
| Author: | Talebi, E.; Khademi, M.; Subramanya, G. |
| Publication: | Advances in Environmental Biology |
| Article Type: | Report |
| Geographic Code: | 9INDI |
| Date: | May 1, 2011 |
| Words: | 4547 |
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