INTERNATIONAL JOURNAL OF BIOTECHNOLOGY RESEARCH • July-December 2011 • Volume 4 • No. 2, pp. 53-57

IJBR

 International Science Press

DNA Profiling of Bivoltine Silkworm Germplasm Races Through Microsatellite Markers T. Thiyagu and C.K. Kamble Central Sericultural Germplasm Resources Centre, Central Silk Board, Hosur-635109 Tamil Nadu, India.

ABSTRACT DNA Fingerprinting of 10 Bivoltine silkworm Germplasm Races from different origin, silk yield, Voltinism, Morphological differences were studied using simple sequence repeat (SSR) markers. Ten primer pairs flanking microsatellite sequences in the silkworm genome were assessed. A total of 139 alleles were detected in the 10 silkworm Germplasm races, which ranged from 100 to 200 bp and minimum of one allele and maximum of three alleles were observed. SPSS cluster analysis of Nei’s genetic distance grouped silkworm strains based on their origin. Four major ecotypic silkworm groups were analyzed. Principal components analysis (PCA) for SSR data supports their SPSS clustering. The results indicated reasonable grouping of high productive and Low productive silkworm races and these kind of specific markers can be used by breeders/researchers for the selection of better parental stock for introgenesis of higher genetic value to augument silk productivity Keywords: Silkworm, SSR, Bombyx mori, microsatellite Suspension Cell Culture, Chilli Callus Capsaicinoids.

1. INTRODUCTION

The silkworm Bombyx mori has emerged as lepidopteron molecular model system for diverse biological studies, including genetics, development and physiology (Goldsmith, 1995), in addition to retaining its economic importance in silk production. The silkworm has a large number of geographical races and inbred lines, which show substantial variation for a large number of quantitative traits. The traditional breeding activities, involving hybridizations between members of elite groups, are adding new varieties every year. At present, in the silkworm traits such as cocoon size and shape, cocoon colour, Filament length, larval duration, together with many other ethological traits, are used to differentiate varieties. The selection of parental strains for breeding programme is based on these characteristics. But the silkworm varieties, particularly those which have been bred from crosses involving many varieties, cannot be distinguished un ambiguously by the use of conventional characteristics. It is thus, apparent that the use of molecular markers could provide a solution to the problem, by providing unique DNA profiles. Such varietal DNA profiles would be useful in producing reliable estimates of genetic diversity, for the selection of parents for the development of elite hybrids, and to protect silkworm breeder’s rights. Varietal-specific DNA markers could also provide additional markers for the ongoing silkworm genome mapping programme. If economically important traits are found to have close linkage with the DNA markers, the latter could also be used in marker-assisted selection. Attempts have already been made to Fingerprint the diverse silkworm genotypes and

establish their genetic relationships using defective transposons (Tamura et al., 1993), RAPDs (Nagaraja & Nagaraju, 1995), a Bkm-derived probe (Nagaraju et al., 1995) and SSRs (Reddy et al., 1999). Simple sequence repeats (SSRs), known as microsatellites, are tandemly repeated DNA sequence motifs (usually2-5 bp long) that occur at multiple sites of eukaryotic genomes. The repeats are abundant and highly polymorphic markers in human and other mammalian genomes, as well as in plant genomes (Tautz 1989; Weber and May 1989; Broun and Tanksley 1996). The important feature of this class of repetitive DNA is hyper variability, mainly expressed as variation in the copy number of tandem repeats at a particular locus. Due to their hyper variability, relative ease of scoring by Polymerase chain reaction (PCR), co dominant nature, and high reproducibility, they are now considered to be the most powerful genetic markers The micro satellite markers consist of an array of simple tandemly repeated mono, di-, tri- tetra-,penta or hexa nucleotide repeats such as (A)n, (CA)n, (GA)n,(GTA)n, (ATT)n, (GATA)n, (ATTTT)n, (ACGTCG)n which are distributed across the genomes. These are ubiquitous in prokaryotic and eukaryotic genomes, randomly distributed both in protein coding and noncoding regions; these become highly informative and versatile classes of genetic markers (Tautz, 1989). The advantages of SSRs over other molecular markers are viz (i) multiple SSR alleles may be detected, (ii) SSRs are evenly distributed all over the genome, (iii) they are co-dominant, (iv) very small quantities of DNA are required for screening

54 / T. Thiyagu, and C.K. Kamble

and (v) analysis may be semi-automated. Therefore, it is an imperative that these molecular markers are utilized for broad range of applications, such as genome mapping ,characterization and phenotype at a single gene locus using a simple PCR-based mapping (Powell et al., 1996;Robinson et al., 2004). Recently, this microsatellite PCR-SSRs and ISSR marker has enabled researchers to investigate genetic linkage, polymorphism, homozygosity and heterozygosity analysis in gene loci among silkworm populations (Reddy et al., 1999a, Rao and Chandrashekharaiah, 2003; Shen et al., 2004; Li et al., 2005, 2006). Recently, RAPD markers linked to nsd-Z had been screened (Li et al., 2001) and the molecular linkage map of nsd-Z by SSR markers had been constructed in silkworm on monogenic trait against denso nucleosis virus (Li et al., 2006; Muwang et al., 2007).

h, phenol-chloroform extraction was carried out. The DNA was recovered by isopropanol precipitation and purified DNA molecule was dissolved in Tris-EDTA buffer (pH 8.0) and concentration was seen in 0.8%Agarose Gel electrophoresis

Microsatellite or simple sequence reapts are considered useful because of their highly polymorphic nature and relative abundance in the genome (Wang et al., 1994, Tautz, 1989). Microsatellites are powerful DNA marker for quantifying genetic variations with in and between populations of the species (O’Connell and Wright, 1997). Micro satellites (SSRs) have been used in different plant and animal genetic resources for fingerprinting, varietal/line identification. Frame work/region specific mapping, genetic maps, F1 identification, Breeding, bulk segregant analysis, diversity studies, conservation genetics, phyllogeography, novell allele detections, marker assisted selection and map-based gene clomnning (Sunnucks, 2000). Information about the distribution and variability of Microsatellite sequences in the genome of the species can elucidate its genetic history from the stand point of evolution and artificial selection

The basic program used to amplify PCR-SSR DNA was performed on a thermal cycler PTC 100 (MJ Research). Polymerase chain reaction cycles for the SSR micro satellite loci included (i) an initialdenaturation step at 95°C for 3 min, an annealing step at 63°C for 1min and an extension step at 72°C for 1 min followed by (ii) 14cycles of 94°C for 30s denaturation, a 14-step touch down decreasing by 0.5°C at each step to 56°C (30 s) and an extension step at 72°C for 1 min. (iii) conditions for the last 24 cycles were94°C for 0.5min, 56°C for 30 s, and 72°C for 1 min followed by (iv) a final elongation step at 72°C for 10 min extension. The PCR was performed in a final volume of 15 ?l containing 10 mmol Tris-HCl/L(pH 8.4), 50 mmol KCl/L, 1.5 mmol MgCl2/L, 0.2 mmol each dNTP/L, 0.2 _mol each primer/L, approximately 20 ng of each silkworm parental breeds / hybrids genomic DNA, 0.5 U of Taq polymerase and distilled de-ionized water.

2. MATERIALS AND METHODS

4. ELECTROPHORESIS OF PCR PRODUCTS

Selection of Silkworm Races In the present study Ten silkworm races collected from CSGRC Hosur were selected based on silk yield, Geographical Origin, Voltinism, Morphological differences and their significance to the sericulture industry shown in Table-1. DNA Extraction Genomic DNA from the 10 silkworm Germplasm Races was extracted from single moth using the phenol chloroform method and purified (Nagaraja and Nagaraju, 1995; Nagaraju.et al., 1995). These 10 silkworm Races were pulverized with a mechanical homogenizer in a microcentrifuge tube and suspended in DNA extraction buffer (50 mmol/L Tris-HCI (pH 8.0), 100 mmol/L NaCl, 20 mmol/LEDTA) that contained 150 _g/ml proteinase K. After digestion with the proteinase K at 50°C for 8 - 10

Microsatellite SSR Primers Ten primer sequences of PCR-SSR repeat motif in silkworm were selected from the previously wellcharacterized microsatellite repeats represented different gene loci viz. NS0101, NS0101-1, NS0101-3, NS0104, NS0106-1, NS0201, NS0207, NS0207-2, NS0217, NS0211, (Li et al., 2005, 2006). 3. PCR AMPLIFICATION

The following PCR amplified product was mixed with 5µl TE buffer and 2µl 40% sucrose containing 0.5% bromophenol blue was loaded on to a 2.5% agarose gel along with the 100 bp ladder molecular weight marker (Genei) and run at constant voltage (100 V) for 2 hour. After electrophoresis, the gels were stained with Ethidium Bromide, clear bands were UV visualized and photographed with digital scientific camera in gel documentation system (Hou et al., 2005). To authenticate repeatability of the results obtained, the PCR amplification and gel electrophoresis was repeated twice. 5. STATISTICAL METHOD

The presence and Absence of band variation was revealed against each SSR marker and Race. They were calculated in Excel Sheets for comparision of number of loci and allele identification. The loci and alleles were analyzed through Tools for population Genetic analysis Program (SPSS) and the clusters were made for comparative study

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DNA Profiling of Bivoltine Silkworm Germplasm Races Through Microsatellite Markers / 55

on trait specific clusters groups. Polymorphism will be scored for presence or absence of bands/alleles. The data were analyzed using Nei and Li coefficients (Nei and Li, 1979) for cluster analysis. The allelic status, homozygosity

etc. were calculated utilizing appropriate statistical tools. Gel profiles for isozymes were scored for presence (1) and absence (0) of bands and similarity or dissimilarity was calculated as per Standard methods (Nei and Li., 1979).

Table 1 List of Silkworm Races, Quantitaative & Quantitative Traits of the Silkworm, Bombyx Mori S.No.

SMGS Acc-N0

Race Name

Origin

Parentage

Fec (no)

Hat%

TLD

VLD

SCW

SSW

S.R%

Filament Length (M)

1 2

BBE-0267

14M

JAPAN

NA

495.50

91.75

628

169

1.95

0.43

22.10

1177.00

BBI-0290

CSR-2

INDIA

NA

484.00

86.90

562

157

1.66

0.38

23.97

747.14

3

BBI-0338

DD-1

INDIA

Daizo, NB4D2 & Japanese be races

583.00

93.70

555

153

1.89

0.38

20.36

739.67

4.

BBI-0343

NK-4

INDIA

NB4D2, Hugo, S. koriyean & chienese bv races

564.00

96.50

555

153

1.78

0.35

19.88

940.44

5.

BBI-0344

NP-4

INDIA

NP-2, NB4D2 & 646.00 Japanese bv races

90.20

603

153

1.94

0.39

20.17

750.64

6.

BBE-0195

6P

JAPAN

7.

BBE-0209 CN × C140 JAPAN

8.

BBE-0213

9. 10.

NA

458.29

85.48

602

158

1.42

0.29

20.35

921.15

CN.C140

528.64

89.49

621

174

1.78

0.34

19.30

640.60

FCC2(P)

JAPAN

original

590.00

93.59

641

181

1.72

0.34

19.51

687.35

BBE-0220

1-15

FRANCE

NA

437.17

76.36

627

169

1.21

0.19

15.91

883.72

BBE-0298

PY-1

INDIA

NA

510.00

92.00

623

176

1.62

0.34

20.94

675.00

Fec = Fecundity, Hat=Hatching, TLD = Totla larval duration, VLD = Vth instar larval duration, SCW = Single Cocoon weight, SSW = Single weight, S.R% = Shell ratio. * Sl No. 1-5 are high productivity and 6-10 lower productivity Table 2 Primer Sequence for the Microsatellite Loci S.no

Original site

Forward Primer

Reverse Primer

Mgcl2

T°C

1

NS0101

ATAATCAGCCAAAACGGTCC

CAGAAGCCAGATAATCCAAAGA

2.5

46

2

NS0101-1

3

NS0101-3

GGGCAAGGTAGAAGGGAA

GCCGAAACAAACAGACAGAC

2.0

48

ATTGAGGAGTGTTTTCGGTGT

CGGTTTTAGCGAGTAGCAGA

3.0

4

50

NS0104

ACGGTTCAGTTATTTCAGA

TAGGATGTTATTGCGAGTA

2.5

52

5

NS0106-1

TTGTTAGTTTAGATTGTCTGTGCCC

CACCTCGTCACGCTTATCC

2.5

52

6

NS0201

CTGCCTACAAGGGTAATAAA

GTCGGGAAGAGTCATAAAGT

2.0

49

7

NS0207

CTGAATCTTCGCACCTTACAA

CAACGACTGCCTTACCCTT

2.5

47

8

NS0207-2

CCTGCCTGTTGACTTATTT

TGAGTTCTTACCGCCTTT

2.0

54

9

NS0217

GATGCTTCTTGCTCTTGTGGT

TGGGTCGTAAGTTCGTTTCTG

2.5

52

10

NS0211

TGGGCAAGAGTGGTGAAGG

GGTTTGGGTCCTTGATTGGTA

2.0

50

6. RESULTS AND DISCUSSIONS

Ten Bivoltine races that are conserved in the Central Sericultural Germplasm Resources Centre (CSGRC), Hosur, of Central Silk Board, Bangalore were collected for the study. These ten races were originally collected from different states of India like Karnataka and Tamil Nadu and few races also belong to Japan and France (Table 1). Some of the races are evolved through continuous breeding and the remaining races belong to the original parentage. All the ten races show distinct phenotypic diversity as far as cocoon colour, cocoon shape and size etc. The color of the cocoons is white and creamy white and the shape of the cocoons is either elongated or

oval. There is significant diversity in the quantitative characters in all the races (Table 1). The total larval duration (TLD) varied from 555 hours (DD-1) to 641 (FCC2 (P)). The cocoon weight (SCW) varied from 1.21g (I-15) to 1.95g (14 M). The single shell weight (SSW) ranges from 0.19g (I-15) to 0.43 (14 M). The shell ratio (SR %) is between 15.91% (I-15) to 23.97% (CSR-2). Through BBE-0220 race has the lowest cocoon weight, shell weight and shell ratio, it is hardy silkworm race and commercially exploited in rain fed areas, hottest regions and unfavorable climates of India. The race 14 M is having high cocoon weight, shell weight and highest shell ratio and highest hatching % is improved Bivoltine race evolved through continuous

International Journal of Biotechnology Research • July-December 2011 • Volume 4 • Issue 2

56 / T. Thiyagu, and C.K. Kamble

breeding. Whereas highest filament length was found in 14 M, i.e., 1177 m among the GRS taken for this study. In order to analyze the genetic divergence among ten races, SSR markers were used. The representative DNA profile of silkworm races amplified with microsatellite primer NS0211 is depicted in Fig. 1 Similarity coefficients among ten races were estimated from the binary data by Hierarchical cluster analysis using Ward method Fig. 2.

Figure 1: Representative DNA Profile of Silkworm Breeds Amplified with Microsatellite Primers NSO211

Low productive having similar values among themselves. DD-1, NK-4, NP-4 have been placed together as they are highly productive. Thus, the UPGMA clustering has clearly shown meaningful groupings of the selected silkworm Germplasm races in the present study substantiating their geographical origin. The present study has clearly proved the utility of microsatellite markers in exploring the genetic diversity in silkworm Germplasm races with regard to the polymorphism of repetitive DNA. Similarly 10 SSR primers are used to differentiate higher and lower productivity and out of 10 Silkworm Germplasm races there is no collision between the high productive and low productive. Hence, it would be worthwhile to take up a systematic and through investigation using more number of microsatellite loci to identify the high productivity and low productivity of the Silkworm Germplasm races. This will lead to successful use of microsatellite markers as molecular tags in silkworm breeding through the Strategy of MAS for improvement of commercial traits. This New Strategy of MAS has already been successfully used in Silkworm breeding using Digestive amylase as a Marker (Ashwath et al., 2001) by transferring high activity amylase genes from low yielding multivoltine silkworm Germplasm races into the genetic background of productive multivoltine breeds for improving digestibility and survival. ACKNOWLEDGEMENTS

The authors are thankful to the Director, Central Sericultural Germplasm Resources Centre Hosur, for providing the lab facilities and also acknowledge with thanks the scientists for their help and guidance. Figure 2: Dendrogram of Silkworm Races Amplified with Microsatellite Primers

Preliminary studies on assessment of genetic diversity and DNA profiling of silkworm Germplasm races based on RAPD and Bkm minor satellite probes has shown that the genotypes can be conveniently grouped based on yield, geographical origin, voltinism and morphological differences (Nagaraju and Singh, 1997). Subsequently, microsatellites were isolated and characterized from the silkworm genome (Reddy et al., 1999 a) and the study has shown that the silkworm genome is abundantly interspersed with CA/GT and GA/CT repeats. The (GT)n repeats occur at every 49 kb while (CT)n repeats occur at about every 109 kb in the silkworm genome. The Dendrogram generated based on genetic similarity matrices of microsatellite alleles has clearly differentiated the 10 silkworms Germplasm races into 4 Cluster groups 14M and CSR-2 have been placed together as they are highly productive having similar values among themselves. I-15, and PY-1 have been placed together as they are Low productive races. 6P, CN x C140 and FCC2 (P) have been placed in one cluster as they are

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