COMAERATIVE PERFORMANCE AND GENETIC POLYMORPHISM IN SOME POTENTIAL SILKWORM BOMBYX MORI L. GENOTYPES

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1 Journal of Cell and Tissue Research Vol. 16(3) (2016) (Available online at www. Tcrjournals.com) ISSN: ; E-ISSN: Original Article COMAERATIVE PERFORMANCE AND GENETIC POLYMORPHISM IN SOME POTENTIAL SILKWORM BOMBYX MORI L. GENOTYPES BUHROO, Z. I., 1? MALIK, M. A., 1 GANAI, N.A., 2 KAMILI, A. S. 1 AND BHAT, B. A. 3 1 Temperate Sericulture Research Institute, 2 Division of Animal Breeding and Genetics, Faculty of Veterinary Sciences and Animal Husbandry, 3 Division of Statistics, Faculty of Fisheries, Sher-e- Kashmir University of Agricultural Sciences and Technology of Kashmir, Srinagar (J &K). E. mail: zafar.buhroo1985@gmail.com, Cell: Received: October 4, 2016; Revised: October 18, 2016; Accepted: November 10, 2016 Abstract: The study was undertaken to evaluate twelve potential bivoltine silkworm Bombyx mori L. genotypes viz., SKAU-R-1, SKAU-R-6, SKUAST-28, SKUAST-31, CSR 2, CSR 4, CSR 18, CSR 19, NB 4 D 2, SH 6, DUN 6 and DUN 22 for their performance during spring and summer seasons of 2012 and The data generated in respect of different traits was pooled separately and analyzed statistically. During spring, significantly higher estimates for larval weight, cocoon weight, shell weight, shell ratio and filament length was registered by SKAU-R-1. However, during summer significantly higher estimates for larval weight, cocoon weight, shell weight, shell ratio and filament length was recorded for SKAU-R-6. The results of the study revealed that SKUAR-1 and SKUAR-6, performed significantly higher for all the traits under study in both spring and summer seasons. The study also revealed that irrespective of genotypes the values for these traits were marginally higher in spring than summer season. Based on the extent of DNA polymorphism, RAPD markers were effective in assessing the genetic variability within genotypes of silkworm Bombyx mori, given the large number of markers identified and the higher polymorphism of 88.88% found with mean PIC value of per loci were sufficient to discriminate between all genotypes. The study has demonstrated that 117 markers obtained with RAPD molecular marker technique suggest that this population has a wide genetic base suitable for rational utilization in designing the breeding programs to boost up bivoltine silk production in the temperate sericulture industry. Key words: Genotype, Silkworm, Genetic divergence INTRODUCTION Jammu and Kashmir, the most sparsely populated states of India, is basically an agrarian economy where in agriculture occupies predominant position in terms of dependence for livelihood and employment. Sericulture plays an important role in transformation of rural economy as it assures regular employment and periodic returns round the year [1]. Sericulture has also an important place in the economy of Jammu and Kashmir as more than 30,000 rural families which belong to economically backward sections of the society are generating their employment through this vocation [2]. Being one of the traditional and eco-friendly agro-based labor intensive industries of the state, helps in improving the economic conditions of landless farmers by providing subsidiary employment and supplementing the income of rural farmers especially the economically weaker section of the society [3]. From the past several years, sericulture has shown signs of recovery in the valley of Jammu 5911

2 J. Cell Tissue Research and Kashmir. The overall numbers of silk farmers, silk production and other realms associated with the sericulture sector of the state have shown steady recovery during this short span of time [4]. Though, the state presents an ideal and fertile land for the growth and development of bivoltine sericulture. However, production of quality bivoltine silk is still a challenge in J & K having enormous potential to produce bivoltine silk of international grade, which can help to reduce the import of bivoltine silk in the country. Traditional breeding methods employed during the last few decades has resulted in the development of many productive silkworm breeds which have contributed significantly in maximizing the silk production in India in general and Jammu and Kashmir state in particular. Efforts made in this direction during the 90 s have lead to the evolution of highly productive CSR bivoltine breeds which have the potential to produce international grade silk [5]. However, these new breeds continue to suffer badly in adverse conditions of low/high temperature, humidity, poor leaf quality and low management practices prevalent with the small and marginal farmers in Kashmir as unlike tropics, temperate sericulture being carried out under highly fluctuating environmental conditions and poor leaf quality urgently needs the development of broad based silkworm breeds with genetic plasticity to buffer the adverse situations. Therefore, it is of paramount importance to know the seasonal performance of silkworm genotypes before formulating any breeding programme. To date, new varieties are usually described on the basis of their morphological characteristics. Thus, requiring reliable techniques for genotype characterization. The selection of parents based on phenotypic traits along with genetic variation (DNA profiles) may be the better option as phenotypic traits expresses variation due to their interaction with the environment. Such varietal DNA profiles would be useful in producing reliable estimates of genetic polymorphism and diversity for the selection of parents to develop elite hybrids [6]. Hence, an urgent need was felt to reorient our approach using the molecular marker approach and available genetic stocks to supplement the conventional practices adopted in breeding programmes. Wide arrays of DNA marker techniques are available for polymorphism and diversity studies. All DNA markers reflect differences in DNA sequences. However, the choice of a particular genetic marker often depends upon the purpose of the study. Randomly Amplified Polymorphic DNA (RAPD) marker techniques have proven powerful in estimation of genetic diversity and polymorphism. [7-12] highlighted the utility of these molecular markers in silkworm fingerprinting for analyzing the genetic diversity and phylogenetic relatedness. The perusal of the literature indicates that most of the RAPD marker studies were carried out on different silkworm varieties in India and abroad. However, in temperate sericulture industry, polymorphism at DNA level among silkworm genotypes have not been taken up. Hence, the use of RAPD marker technique was necessary to be used as a tool to investigate the extent of genetic polymorphism among temperate bivoltine silkworm genotypes. In this context, assessment of DNA based polymorphism using RAPD marker technique was undertaken to evaluate their use in breeding purpose for the development of broad based silkworm breeds to push up productivity levels in temperate bivoltine sericulture industry. MATERIALS AND METHODS In the present investigation, twelve potential bivol tine silkworm genotypes (Table-1) were selected to assess the performance during spring and summer seasons (2012 &2013) and to ascertain the extent of polymorphism among the genotypes using Randomly Amplified Polymorphic DNA (RAPD) markers. The disease free laying s (DFL s) of these selected silkworm genotypes were obtained from the Germplasm Bank of Temperate Sericulture Research Institute (TSRI), Sher-e-Kashmir university of Agricultural Sciences and Technology of Kashmir- India (SKUAST-K) Mirgund, Central Sericultural Germplasm Resources Centre (CSGRC) Hosour, Tamilnadu, India and Central Sericultural Research and Training Institute (CSR&TI) CSB, Pampore, Srinagar Kashmir. The eggs were incubated at 25±1 0C temperature and 75-80% relative humidity for about days till their hatching. The larvae of all the selected bivoltine breeds were reared under standard package of practices [13]. The experiment was laid out in Completely Randomized Block Design with three replications for each treatment. Each replication comprised of 250 silkworms of uniform age and size retained after 5912

3 Buhroo et al. third moult. The larvae and cocoons were assessed for different parameters, recorded replication-wise for all the treatments during spring and summer seasons (2012 and 2103) and subjected to statistical analysis. The parameters studied and observational procedures adopted are given under the following headings: Fecundity (No.): This character represents the number of eggs laid by a healthy female moth. The fecundity was calculated by taking the number of eggs laid by a single moth from each replication. The eggs were counted individually and recorded. Hatching (%): It denotes the number of larvae hatched from the disease free laying s. The hatching percentage is arrived at, after deducting the unhatched, unfertilized and dead eggs from the total number of eggs laid by a single female.it was calculated as: Hatching (%) = ( No.of hatched eggs) (Total no.of eggs laid) 100 Fifth age larval duration (d:h): It measures the time taken from the beginning of fifth instar till the commencement of spinning. It was calculated as the total hours taken from the first day of fifth age up to mounting of the ripe worms. Total larval duration (d:h): This character is the measure of time taken from hatching till the commencement of spinning. The mean larval duration is derived by calculating the larval feeding and moulting periods in hours commencing from I instar to Vth instar. It was calculated as the total hours taken from the date of brushing to the mounting of ripe worms. Weight of ten mature larvae (g Larva-10): It denotes healthiness and robustness of the larvae. It is the measure of ten randomly selected larvae weighed one day before spinning. Ten mature larvae were selected randomly from each replicate of each treatment and weighed separately. Single cocoon weight (g): This is the average weight of a cocoon in grams derived by calculating the weight of a random sample of 10 cocoons. Ten cocoons from each replicate of each treatment were selected randomly and weighed to determine the average cocoon weight. Single shell weight (g): This trait represents the total quantity of silk in a cocoon. It is the average individual shell weight in grams of 10 cocoons selected for assessment of cocoon weight. The cocoons used for determining the average single cocoon weight were cut open and weighed to obtain shell weight. Pupal weight (g): The pupae obtained from the cut open cocoons were weighed to determine the average papal weight. Shell ratio (%): It is the ratio of the shell weight to the cocoon weight. It is calculated in percentage from a random sample of 10 to 25 cocoons from each laying. It was calculated as, Shell ratio (%)= Cocoon yield /10,000 larvae: (Single Shell wt.) (Single cocoon wt.) 100 By Number (No.): This character denotes the survival rate of larvae that spin good cocoons. The unit of 10,000 newly hatched larvae is taken as a standard unit. No.of cocoons harvested from each replicate No.of worms retained after 3rd moult 10,000 By Weight (Kg): It is the total quantity of good cocoons in kilogram obtained for a standard unit of 10,000 larvae. Weight of cocoons harvested from each replicate No.of worms retained after 3rd moult 10,000 Filament length (M): It is the length of the silk filament in meters reeled from a single cocoon. The mean value of the filament length is obtained by reeling ten cocoons collected at random from each laying. Ten randomly selected cocoons from each treatment and replicate were reeled to determine the average filament length. Genomic DNA Extraction: Genomic DNA was extracted from adult silk moths. In each of the genotype, 2-3 moths were frozen with liquid nitrogen and homogenized in pestle and mortar. The powdered content was transferred to fresh/ autoclaved tubes containing DNA extraction buffer 5913

4 J. Cell Tissue Research (50 mm Tris HCI/L, ph 8.0, 100 mm NaCl/L, 20 mm EDTA/L) having 100 μg/ml Proteinase K. After digestion with Proteinase K at 55 C for 1 h, phenol/chloroform extraction was carried out and DNA was recovered by ethanol precipitation. Purified DNA was dissolved in 1X Tris EDTA buffer (ph 8.0). DNA concentration was measured using spectrophotometer. Phenol-chloroform method of DNA extraction [14] was followed for DNA extraction with minor modifications as outlined by Srivastava et al. [11]. RAPD Marker Analysis: The list of RAPD primers which were utilized in the present study are presented in Table-4 along with their sequences. RAPD-PCR was performed in 25 μl of a reaction mixture containing 30 ng DNA, 2.5 μl of 10X Buffer, 2.0 μl of dntp mix (10 mm), 2 mm MgCl2, 1.0 μl of primer (0.5 μm) and 1.0 U of Taq DNA polymerase (Genie). The DNA amplification reactions were performed in a thermal cycler (Applied Biosystems). The RAPD products were analyzed by electrophoresis by resolved on 1.5 % Agarose gel and data was analyzed by counting the banding pattern generated by each primer. The DNA fragments amplified by a primer were scored as present (1) or absent (0) for the genotype studied. POPGENE software program version 1.31 [15] was used to obtain the degree of polymorphism among the genotypes. RESULTS AND DISCUSSION The magnitude in the phenotypic variability is dependent on the responsiveness of the different genotypes to different environmental conditions. Judging the performance of genotypes on the basis of individual traits under different seasons becomes slightly difficult, particularly when more than twenty one component traits determine the yielding ability of a silkworm genotype [16]. The egg laying potential of Bombyx mori L. has been noticed to be a heritable character expressed within the genotypic limitations of the insects like Bombyx mori. In this context, the perusal of the data on fecundity under temperate conditions revealed that the maximum fecundity was recorded for genotype SKUAST-31 (635) while it was minimum in SH6 (613) during spring (Table 2). In summer season, same genotypes displayed superiority for this trait where maximum fecundity was exhibited by SKUAST-28 and minimum SH6 (609) during summer season (Table-3). The superior fecundity of these genotypes in both the seasons indicated their genetic constitution since, the fecundity of Bombyx mori varies due to variation in the genetic makeup of silkworm race [17] which is influenced by number of physiological and ecological factors [18]. Hatching percentage is an important component reflecting viability of the eggs and the vigor of an organism. Hence the reduced hatching percentage is not favored by natural selection. However, increased hatching percentage reflects the genetic background and physiological state of the female moth [19]. The present study revealed that during spring season the maximum hatching percentage of 96.05% was exhibited by genotype SKAU-R-6, while it was minimum in DUN6 (94.98) during the same season. While in summer the maximum mean hatching percentage was exhibited by SH6 (95.02%) and it was minimum in CSR18 (94.10). The maximum hatching percentage of these genotypes reflected their genetic background for this trait. The survival and development of insects are at the mercy of nature and the biological and developmental activities are restricted in accordance with the prevailing ecological conditions and to a certain extent to their genetic build up [20]. Larval duration is an important attribute of economic value in sericulture as the reduction in larval duration would help in minimizing the quantum of the total food consumption and labour requirement, besides completion of larval period in desirable time period [21]. In the present study, CSR19 recorded significantly the shorter Vth age and total larval durations in both the seasons among all the genotypes evaluated for this trait. During spring significantly short Vth age larval duration of 6.19 days in summer was recorded for this genotype. Total larval duration for the genotype (CSR19) in spring and summer was and days, respectively (Tables 2,3). Short larval duration of days in respect of CSR19 silkworm genotype irrespective of the season clearly reflects the genetic constitution of this genotype for this trait [22]. Daniel et al. [23] reported that rate of development depends on both genetic and environmental factors. The larval duration varies depending on the prevailing temperature. High ambient temperature during summer tends to reduce the larval duration 5914

5 Buhroo et al. Table 1: Characteristic features of different bivoltine silkworm genotypes under study Genotype Voltinism Parental Source Larval pattern Cocoon colour Cocoon shape Origin/ Evolution Source SKAU-R-1 Shunrei Shogetsu Marked White Constricted TSRI, SKUAST- SKAU-R-6 Shogetsu Hoshu Plain White Slightly oval Kashmir- Mirgund SKUAST-28 Evolved Under Broad Based Germplasm Complex, Comprising 10 Breeds With Marked Larvae SKUAST-31 Evolved Under Broad Based Germplasm Complex, Comprising 10 Breeds With Plain Larvae Marked White Short dumbell Marked White Oval CSR 2 Shunrei Shogetsu Plain bluish Bright white Oval CSR&TI, 4 CSR (BN18 BCS25) NB 4 D 2 Plain bluish Bright white Dumbell Mysore-India CSR 18 B201 BCS12 Plain & marked Creamish white Oval Bivoltine CSR 19 B201 BCS12 Plain & marked Creamish white Dumbell NB 4 D 2 (Kokko Seihaku) (N124 C124) Plain faint bluish White Elongated,constricted SH 6 Shogetsu Hoshu Moderately marked White Oval RSRS, Majira, Dehradun- India DUN 6 CC1 NN6D Plain White Oval CSR&TI, DUN (KS NB D ) (AT NB 4 D 2 ) Marked White Oval Pampore-Kashmir Table 2: Mean performance of twelve silkworm genotypes during spring (Data pooled over same seasons of 2012 and 2013) Silkworm Germplasm Bank, TSRI, SKUAST-K, Mirgund Silkworm Germplasm Bank, CSGRC, Hosur- Tamilnadu, India Silkworm Germplasm Bank, CSR&TI,CSB- Pampore Genotype Fecundity (no.) Hatching ( %) V th age larval duration (d:h) Total larval duration (d:h) Weight of Ten mature larvae(g) Single cocoon weight (g) Single shell weight (g) Pupal weight (g) Shell ratio %) Filament length (m) Cocoon yield/10,000 larvae by number by weight (kg) SKAU-R SKAU-R SKUAST SKUAST CSR CSR CSR CSR NB 4 D SH DUN DUN Mean S.D CD p

6 J. Cell Tissue Research Table 3: Mean performance of twelve silkworm genotypes during summer (Data pooled over same seasons of 2012 and 2013) Genotype Fecundity (no.) Hatching ( %) V th age larval duration (d:h) Total larval duration (d:h) Weight of Ten mature larvae(g) Single cocoon weight (g) Single shell weight (g) Table 4: Randomly Amplified Polymorphic DNA (RAPD) Primer sequences and degree of polymorphism. Single letter abbreviations for mixed base positions: R = (A, G); Y = (C, T) Abbreviations: A= Adenine, G= Guanine, C= Cytosine, T= Thymine; *bp= base pairs Pupal weight (g) Shell ratio (%) Filament length (m) Cocoon yield/10,000 larvae by number by weight (kg) SKAU-R SKAU-R SKUAST SKUAST CSR CSR CSR CSR NB 4 D SH DUN DUN Mean S.D CD p Primer Primer Sequence No. of Alleles Allele size range (bp)* Polymorphic Alleles Polymorphism ( %) PIC UBC CCTTCCCTCC UBC GTGTGGTGGG UBC CTCTCCTCCC UBC ACCCACCACC UBC GGGTGGTGGG UBC CCACACCACA UBC GGGAAGAAGG UBC GGTGGGTTGT UBC CACCCAACCA UBC GGAAGGGAGA Total Average

7 Buhroo et al. by several hours to one or two days, where as lower temperature during spring and autumn season extends the larval duration. Larval weight is one of the important parameter which determines not only the health of the larvae, but also the quality of the cocoons spun [24]. Present study revealed that there was significant variation in larval weight of different genotypes of silkworm in both the seasons. SKAU-R-1, SKAU-R-6, SKUAST-31, NB4D2 and SKUAST-28 recorded higher larval weight of about 50.19g, 49.47g, 49.17g, 48.87g, 48.27g in spring season (Table 2) and in summer SKAU-R-6, SKAU-R-1, NB4D2, SKUAST-31 and SKUAST-28 recorded the higher larval weight of 45.17g,43.65g, 43.36g 42.70g and 42.23g (Table 3) over the other genotypes evaluated. It is well known that the environmental conditions prevailing in different seasons affects the growth and development as well as expression of economic traits in different genotypes of silkworms. Current studies revealed that genotypes under study have good genetic variability and thus significant variation in larval weight was recorded. The present findings confirm the results of Masarat et al. [25] who reported higher larval weight of 52.46g in SKAU-R-1, 49.53g in SKAU-R-6, 49.56g in SKUAST-31 and 49.20g in SKUAST-28. Pal and Moorthy [26] have also reported highest larval weight of 3.53g in NB14, 3.24g in SK4C and 2.43g in CSR19. Higher larval weight in SKAU-R-1, SKAU-R-6, SKUAST-31, NB4D2 and SKUAST-28 reflected that these breeds have the potential to be used in breeding programmes. The difference in the larval weight among the genotypes studied could be attributed to the racial character, difference in degree of assimilation that differ from one genotype to another and the quality and quantity of food consumed by the larvae which has a direct bearing on the performance on the growth and development of larvae. Cocoon weight, shell ratio and filament length are highly heritable traits and are significantly important as these determine the quality, quantity and efficiency of the reeling process [27]. In present study, SKAU-R-1 registered the highest cocoon weight of 2.09g while SH6 recorded minimum cocoon weight of 1.84g during spring season (Table-2). In summer, SKAU-R-6 recorded the highest cocoon weight of about 1.76g and SKUAST-28 was found to register a minimum cocoon weight of 1.70g during the same season (Table 3). However, the cocoon weight of all the genotypes evaluated was relatively lower in summer season than spring which could be attributed to poor quality of mulberry leaf available in summer season thereby affecting the cocoon weight. The higher cocoon weight recorded in SKAU-R-6, SKAU-R-1, NB4D2, SH6, SKUAST-31 and SKUAST-28 possibly indicated a clear difference in nutrient utilization by these genotypes during 5th instar. The present findings are in agreement with the results of Masarat et al. [25] who reported higher cocoon weights for these genotypes during spring and summer seasons under temperate climatic conditions. Present findings are also in conformity with the reports of Singh et al. [28] who concluded that environmental factors influence the physiology of insects and also have deleterious effect on economic traits such as cocoon weight and shell weight. Present findings reflected that SKAU-R-6-and SKAU-R-1 are potential genotypes with higher cocoon weight thus, producing more silk qualitatively and quantitatively. The present findings also corroborate with the results of Kumar and Shamitha [29] who noticed the deleterious effect of adverse temperature and humidity on economic traits. Cocoon and shell weights are the major traits evaluated for productivity in sericulture. Cocoon weight is an important commercial characteristic used to determine approximately the amount of raw silk that can be obtained. Shell weight gives a better measure, but cannot be determined in commercial cultures because it requires damaging the cocoon. The difference between the two measures is the weight of the pupa [30]. Cocoon shell weight is an important character in determining the silk weight. Our results are in conformity with the findings of Basavaraja et al. [31] who have reported that cocoon shell weight shows variability under different environmental conditions. The variations observed in the present findings in shell weight and shell ratio might also be due to racial character. The difference between the weight of cocoon and shell is the weight of the pupa [30]. The findings of this study revealed that no significant difference was found in pupal weight among the genotypes during both the seasons. In the present study, significantly higher pupal weight of 1.63g, 1.60g, 5917

8 J. Cell Tissue Research 1.50g and 1.49 g was recorded by SKAU-R-1, SKAU-R-6, SKUAST-31 and NB4D2 genotypes during spring. In summer SKUAST-31, NB4D2 and SH6 recorded the highest pupal weight of 1.39g, followed by SKAU-R-6 (1.38g), SKAU-R-1 and SKUAST-31 (1.37g). This could be attributed to better larval growth period and reduced meltage during pupal development. Rayar [22] found pupal weight 1.38g in CSR18, 1.34 g and 1.33 g in CSR19 and NB4D2 breeds, respectively. The pupal weight of Bombyx mori has been noticed to be influenced by the variation in the level of secreted hormones [32] and genotype variation [33,34]. Higher pupal weight attained by these genotypes also indicates their better feed consumption hence good larval growth during the larval periods [35]. Filament length is one of the important attributes of the silkworm breed. The silk filament length is different in silkworm breeds under different set of rearing conditions and rearing seasons [36]. In present study, the highest average filament length of m was observed in SKAU-R-1, in spring season (Table 2) while as in summer season SKAU-R-6 registered the longest filament length of m (Table 3). Longest filament length in SKAU-R-1 and SKAU-R-6 implied the superiority of these genotypes over the other genotypes evaluated. The filament length was relatively higher in spring than summer. The results of the present findings are in conformity with the findings of Masarat et al. [25] who have identified SKAU-R-1 and SKAU-R-6 as the genotype with higher filament length during spring and summer seasons under temperate climatic conditions of Kashmir. Cocoon yield /10,000 larvae by number is an important trait of studying the probability of survival of the breeds/genotypes. Higher values for this trait are indicative of higher silk productivity and a good cocoon crop. Cocoon yield /10,000 larvae by number in spring was significantly higher in SKAU-R-1 (9367/10,000 larvae) while in summer, cocoon yield/10,000 larvae was significantly higher in SKAU-R-1 (9296/10,000 larvae). The higher cocoon yield by number could be attributed to the higher survivability of these genotypes due to favorable hygrothermic conditions for the rearing and availability of quality mulberry leaf. These findings are well supported by Legay [37] who reported that cocoon production is chiefly dependent on nutritive value of mulberry leaves and the conversion efficiency of the larvae which is affected by weather conditions [38,39]. This indicated that SKAU-R-1, DUN6 and CSR18 have disease resistance which led to better survival and hence higher cocoon yield in these genotypes. Highest cocoon yield /10,000 larvae by number for CSR18 during summer could be attributed to the better nutrient utilization of the genotype and the potential for its high temperature tolerance. Similar kind of studies have been carried out by Naseema Begam et al. [40] who reported that bivoltine breeds have high effective rate rearing (ERR) under adverse climatic conditions of summer season. Cocoon production is chiefly dependent on larval nutrition and nutritive value of mulberry leaves and conversion efficiency of larvae which is affected by weather conditions [38,41,42] Genetic Polymorphism Based on RAPD Markers: The data regarding RAPD molecular markers applied on selected silkworm genotypes revealed considerable polymorphism among the genotypes studied (Table 4). Amplification products yielded a total of 117 scorable bands out of which 104 (88.88 %) were polymorphic with an average of 10.4 per primer. The number of bands produced by each primer varied from 6 to 23 with an average of 12 bands per primer. The highest number of bands (23) was obtained with primer UBC-778, while the lowest number (6) was obtained with primers UBC-769. The highest percentage of polymorphism was 100%, generated by UBC- 760, UBC-762, UBC-778, UBC-782 and UBC- 785 while as lowest percentage of polymorphism was 75.00%, generated by UBC-783. Ten RAPD primers generated PCR products in the range of bp generated by UBC-783 and UBC- 782, respectively. The Polymorphism information content (PIC) ranged from to with a mean PIC value of per loci. The lowest (0.295) and highest (0.493) PIC values were recorded for UBC-778 and UBC-767, respectively (Table-4). All the ten RAPD primers used in the study produced unambiguous markers and revealed considerable polymorphism among the silkworm genotypes studied The present study using these reliable markers generated higher polymorphism percentage of %. This kind of higher polymorphism was also reported by Nagaraja and Nagaraju [8] utilizing 5918

9 Buhroo et al. two races of silkworm Bombyx mori. [43] reported 93.00% polymorphism with 12 different RAPD markers among silkworm stocks. Similar kind of polymorphism 90 % was observed by Srivastava et al. [44] using same primers while evaluating genetic diversity among silkworm stocks. Srivastava et al. [11] found considerable polymorphism among silkworm genotypes using these markers. The present findings are also in agreement with the findings of Moorthy et al. [12] who have obtained considerable polymorphism utilizing these markers on twenty silkworm varieties from tropical origin. The degree polymorphism detected in the present study corrobates with the phenotypic difference among the genotypes illustrating their genetic distinctness as also reported among other varieties of silkworm by Srivastava et al. [44]. CONCLUSION The selection of parents based on phenotypic traits along with genetic variation (DNA profiles) may be the better option as phenotypic traits expresses variation due to their interaction with the environment. Such varietal DNA profiles would be useful in producing reliable estimates of genetic diversity, for the selection of parents to develop elite hybrids. Based on the performance and better physiological compensatory mechanisms in response to prevailing climatic conditions during spring and summer seasons, eight genotypes viz., SKAU-R-1, SKAU-R-6, SKUAST-28, SKUAST-31, NB4D2, SH6, and DUN6 seem to be more suitable for rearing in both seasons than other productive genotypes. However, SKUAR-1 and SKUAR-6 will be more suitable for rearing under temperate climatic conditions owing to their best performance in terms of maximum economic traits during both seasons. Based on the level of DNA polymorphism, RAPD markers were effective in assessing the genetic variability within genotypes of silkworm Bombyx mori, given the large number of markers identified were sufficient to discriminate between all genotypes and the higher polymorphism found. The study has demonstrated that 117 markers obtained with this molecular technique suggest that this population has a wide genetic base suitable for rational design of breeding programs in modifying the yield potentials of silkworms to boost up bivoltine silk production in the temperate sericulture industry. ACKNOWLEDGEMENTS The first author wishes to thank Directorate of Research, Sher-e- Kashmir University of Agricultural Sciences and Technology of Kashmir, India for financial support. The author sincerely acknowledges Director, CSGRC Hosur, Director, CSR&TI Pampore (Central Silk Board, Ministry of Science & Technology, Govt. of India) and Head, TSRI, SKUAST-K, Mirgund for proving silkworm germplasm. REFERENCES [1] Malik, M.A.: Intern. J. Agricul. Sci., 5(2): (2009). [2] Economic Survey, J&K. Directorate of Economics and Statistics, Govt., of Jammu and Kashmir, Srinagar. pp ( ). [3] Qadri, S.F.A., Malik, M.A. Sbahat, A. and Malik, F.A.: Intern. J. Agricul. Statist. Sci., 6(1): (2010). [4] Dhar, A., Farooq, M. and Nisar, M.: Technical compendium of workshop held at SKICC, Srinagar, October 29 (2011). [5] Datta, R.K.: CSR & TI, CSB (Ministry of Textiles) Govt. of India, Srirampura, Mysore-India (2000). [6] Chandrakanth, N., Moorthy, S.M., Anusha, P., Dayananda., Ashwath., S.K., Kumar. V. and Bindroo, B.B.: Intern. J. Biotechnol. Allied Fields. 2(3): (2014). [7] Williams, J.G.K., Kubelik, A.R., Kenneth, J.K., Rafalski, J.A and Tingey, S.V.: Nucleic Acid Res., 18(22): (1990). [8] Nagaraja, G.M and Nagaraju, J.: Electrophoresis, 16: (1995). [9] Chatterjee, S.N. and Pradeep, A.R.: Russian J. Genet. 40: (2003) [10] Pradeep, A.R. Chatterjee, S.N. and Nair, C.V.: J. Appl. Genet., 46(3): (2005). [11] Srivastava, P.P., Vijayan K., Kar, P.K. and Saratchandra, B.: Intern. J. Tropical Insect Sci., 31(3): (2011). [12] Moorthy, S.M., Chandrakanth, N., Ashwath, S.K., Rao, V.K. and Bindroo, B.B.: Annals Biol. Res., 4(12): (2013). [13] Krishnaswami, S.: Central Sericultural Research and Training Institute, Mysore, India, pp 23 (1978). [14] Suzuki, Y., Gage, L.P. and Brown, D.D.: J. Mol. Biol. 70: (1972). [15] Yeh, F.C. and Yang, R.C.: Popgene version 1.31 Microsoft window based freeware for population genetic analysis: A joint project development by, University of Alberta and Tim Boyle, Centre for International Forestry Research (1999). 5919

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