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Comparison of Multilocus Rflps and PCR-Based Marker Systems for Genetic 1 Analysis of the Silkworm, Bombyx Mori

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Comparison of Multilocus Rflps and PCR-Based Marker Systems for Genetic 1 Analysis of the Silkworm, Bombyx Mori Heredity 86 (2001) 588±597 Received 1 November 2000, accepted 23 January 2001 Comparison of multilocus RFLPs and PCR-based marker systems for genetic 1 analysis of the silkworm, Bombyx mori J. NAGARAJU* , K. DAMODAR REDDYà, G. M. NAGARAJAà 2 & B. N. SETHURAMANà Laboratory of Molecular Genetics, Centre for DNA Fingerprinting and Diagnostics, ECIL Road, Nacharam, Hyderabad, 500 076, India and àSeribiotech Research Laboratory, No. 8, West of Chord Road, Bangalore-560 086, India The utility of multilocus RFLPs and three PCR-based techniques, Random Ampli®ed Polymorphic DNA (RAPD), Inter-Simple Sequence Repeat-PCR (ISSR-PCR) and simple sequence repeats (SSRs) for genetic characterization was examined using 13 diverse silkworm strains. All four approaches successfully discriminated the 13 silkworm varieties but diered in the amount of polymorphism detected. The usefulness of each system was examined in terms of number of loci revealed (eective multiplex ratio, EMR) and the amount of polymorphism detected (diversity index, DI). For example, the six multilocus RFLP probes produced 180 products of which 97% were polymorphic; 15 SSR loci gave rise to an average of 8 alleles each, of which 86% were polymorphic. The ISSR-PCR produced 39 fragments of which 76.98% were polymorphic. The highest diversity index was observed for ISSR- PCR (0.957) and the lowest for RAPDs (0.744). The RAPD, ISSR-PCR and RFLP assays clearly separated the diapausing and non-diapausing silkworm varieties. These results are discussed in terms of choice of appropriate marker technology for dierent aspects of silkworm genome analysis. Keywords: EMR, ISSR-PCR, non-diapausing, RAPD, RFLP, SSR. Introduction of radioactivity, and are amenable to automation. The RAPD method described by Williams et al. (1990) and The detection and exploitation of naturally occurring Welsh & McClelland (1990) generates PCR products by DNA sequence polymorphisms are among the most annealing to randomly distributed homologous target signi®cant developments in molecular biology. Poly- sites of the template DNA. This technique mostly morphic genetic markers have wide potential applica- generates dominant markers, although length polymor- tions in animal and plant improvement programmes as a phisms caused by insertions/deletions can also occur means for varietal and parentage identi®cation, evalu- at low frequencies. Because of its relative simplicity, ation of polymorphic genetic loci aecting quantitative RAPD technology is being extensively used in genetic economic traits, and genetic mapping. RFLPs have been analysis of various plant and animal species. However, used extensively for genetic studies (Bishop & Skolnick, limitations in the applications of RAPDs such as the 1980; Botstein et al., 1980), molecular mapping and dominant nature of the markers, chance co-migration of genetic improvement programmes. However, the tech- bands at dierent loci, and requirement for stringent nical complexity of performing RFLP analysis coupled protocol standardization to ensure reproducibility have with the widespread use of short-lived radioisotopes for also been encountered (Black, 1993). detection have prompted researchers to look for alter- Another class of PCR-based markers, the micro- native methods. satellites, takes advantage of the abundant and ubiqui- The advent of the PCR has resulted in the develop- tously distributed simple sequence repeats (SSRs) in the ment of a large number of molecular techniques, which eukaryotic genome (Hamada et al., 1982; Weber & oer an eective alternative to the hybridization meth- May, 1989; Dietrich et al., 1992). The variation in repeat ods of RFLP analysis. PCR-based approaches use only number can be visualized as dierences in the length of small quantities of DNA, avoid DNA blotting and use PCR-ampli®ed products (Tautz et al., 1986; Tautz & Renz, 1984). Analysis of SSRs requires prior character- *Correspondence. E-mail: [email protected]; jnagaraju@www. ization of sequences ¯anking the repeats to allow the cdfd.org.in design of primers for PCR ampli®cation. SSRs are 588 Ó 2001 The Genetics Society of Great Britain. COMPARISON OF MOLECULAR MARKERS IN BOMBYX MORI 589 codominant markers and can reveal multiple alleles at a single locus. The technique is robust in that it is highly reproducible and the primer sequence information can (m) length be exchanged between laboratories. Filament As PCR technology ®nds increased use in genetic % ratio analysis, novel variations of this technique are emerging Shell which promise precision, economy and speed (Wu et al., 31994; Zietkiecwicz et al., 1994; Vos et al., 1995). One such alternative is SSR-anchored PCR (or ISSR-PCR (gm) Shell for Inter-Simple Sequence Repeat PCR) in which a weight stretch of microsatellite sequence, which also contains a short oligonucleotide `anchored' sequence either at the (gm) weight 5¢ or 3¢ end, is targeted to the template DNA in question Cocoon and upon PCR ampli®cation, inter-repeat genomic regions can be visualized as discrete products (Ziet- kiecwicz et al., 1994). The technique generates a large Larval (D : H) number of markers by simultaneously targeting multiple duration microsatellite loci and allows screening of a large number of samples in a single gel. The silkworm, Bombyx mori, is an excellent model genetic system and an important insect economically. shape Genome analysis has been initiated in the silkworm Cocoon with the objectives of obtaining genetic maps using dierent marker systems: RFLPs (Shi et al., 1995), RAPDs (Promboon et al., 1995) and RAPD double primers (Yasukochi, 1998), and to characterize genetic diversity by DNA ®ngerprinting using ISSR-PCR (Reddy et al., 1999a), a heterologous minisatellite probe, Bkm (Nagaraju et al., 1995; Sharma et al., 1998), microsatellite loci (Reddy et al., 1999b) and RAPDs (Nagaraja & Nagaraju, 1995). In addition, more than 3000 silkworm strains which represent a repertoire of genetic dierences for various complex traits such as silk ®bre length, larval growth and Larval pattern Cocoon colour resistance to disease, are being maintained in dierent countries. Molecular genetic analysis of such complex traits using DNA markers would provide valuable tools for economic improvement of this insect. In the present study, we examined multilocus RFLPs and three PCR-based marker assays, RAPDs, SSRs and ISSR-PCR for evaluating the genetic diversity in certain representative strains of silkworm. We report results here using 13 diverse silkworm strains with a view to comparing their eectiveness in detecting genetic vari- ation and to determine the utility of each system with respect to technical and ®nancial considerations. Materials and methods Silkworm strains and DNA extraction Characteristics of silkworm strains used in the present study Thirteen diverse silkworm genotypes were used in the present study. The characteristics of the genotypes are listed in Table 1. All DNA extractions were performed Table 1 Strains Voltinism Origin *Also lays diapausing eggs. HU204KANB1 DiapausingNB7NB18NB4D2 Diapausing Diapausing ChinaC.nichi DiapausingGungnong Diapausing DiapausingMoria India Non-diapausing Non-diapausing JapanNistari IndiaPure China Mysore India India IndiaDaizo* Non-diapausing Non-diapausing Non-diapausingSarupat Plain Karnataka Assam (India) (India) West Bengal Non-diapausing (India) Plain Non-diapausing White Plain Marked Japan Plain Assam Plain (India) Marked Golden Plain yellow Greenish Plain Plain yellow White Plain Cream White Cream White White White Plain White Spindle Spindle Marked Peanut Cream Dark greenish 28 22 yellow : : 00 00 Oval Short oval Oval 24 Spindle : Spindle 00 Oval 1.00 1.10 Peanut 20 Peanut : Peanut 00 1.43 20 0.138 0.151 : 24 22 00 : : 25 Spindle 00 00 : 00 1.07 0.240 21 13.8 13.7 25 : 25 25 : 00 : : 00 00 00 1.18 1.71 16.7 1.09 0.173 1.95 425 450 22 : 0.92 00 1.93 0.170 1.78 1.99 15.0 0.289 0.136 760 0.410 14.7 0.103 0.402 17.0 1.00 0.364 12.5 0.420 480 21.0 11.1 20.8 380 20.5 21.1 0.131 804 375 970 350 904 13.1 906 934 375 Ó The Genetics Society of Great Britain, Heredity, 86, 588±597. 590 J. NAGARAJU ET AL. on liquid nitrogen frozen silk glands using the method 10 min at 72°C. Aliquots of ampli®ed DNA from already described (Nagaraja & Nagaraju, 1995). individual PCR reactions were mixed with formamide stop solution (95% formamide, 20 mM EDTA, 0.05% Bromophenol blue and 0.05% xylene cyanol in the ratio RFLP analysis of 3 : 2). Samples (4 lL) were denatured at 75°C for High molecular weight genomic DNA was isolated from 2 min, chilled on ice and electrophoresed on standard posterior silk glands of instar 5 larvae pooled from 10 sequencing gels (6% acrylamide, 8 M urea, 1´ TBE individuals per strain. 10±12 lg of DNA was digested to (89 mM Tris-borate, 2 mM EDTA, pH 8.3). An M13 completion with restriction enzymes, EcoRV or BamHI sequence ladder was used on each gel as a size marker. (New England Biolabs) depending on the probe After electrophoresis, the gels were ®xed for 2 ´ 20 min (Table 2). Digested samples were fractionated by 0.8% with 10% acetic acid, dried at room temperature and agarose gel in TAE buer and southern transferred on exposed to X-ray ®lm for 4±12 h. to a nylon membrane (Hybond-N, Amersham) by vacuum blotting (Pharmacia Biotech). The membranes SSR-anchored PCR were baked at 80°C for 2 h. The anonymous multilocus probes used in the present study were obtained from A range of primers was synthesized on an Applied a PstI subgenomic library. The probes were labelled Biosystems DNA synthesizer based on the core with a32P dCTP using a random primer labelling repeats, anchored either at the 5¢ end or 3¢ end kit (Amersham, UK). All other operations including (Table 2). These primers were used to screen the pre-hybridization, hybridization and washing were silkworm strains as described by Reddy et al.
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