Scot

Plant Mol Biol Rep (2009) 27:86–93 DOI 10.1007/s11105-008-0060-5 Start Codon Targeted (SCoT) Polymorphism: A Simple, Novel DNA Marker Technique for Generating Gene-Targeted Markers in Plants Bertrand C Y Collard & David J Mackill Published online: 16 September 2008 # Springer-Verlag 2008 Abstract Random amplified polymorphic DNA (RAPD) markers have been used for numerous applications in plant molecular genetics research despite having disadvantages of poor reproducibility and not generally being associated with gene regions A novel method for generating plant DNA markers was developed based on the short conserved region flanking the ATG start codon in plant genes This method uses single 18-mer primers in single primer polymerase chain reaction (PCR) and an annealing temperature of 50°C PCR amplicons are resolved using standard agarose gel electrophoresis This method was validated in rice using a genetically diverse set of genotypes and a backcross population Reproducibility was evaluated by using duplicate samples and conducting PCR on different days Start codon targeted (SCoT) markers were generally reproducible but exceptions indicated that primer length and annealing temperature are not the sole factors determining reproducibility SCoT marker PCR amplification profiles indicated dominant marker like RAPD markers We propose that this method could be used in conjunction with these markers for applications such as genetic analysis, bulked segregant analysis, and quantitative trait loci mapping, especially in laboratories with a preference for agarose gel electrophoresis Keywords Gene-targeted markers Start codon Genetic diversity QTL mapping B C Y Collard (*) : D J Mackill Plant Breeding, Genetics and Biotechnology Division, International Rice Research Institute (IRRI), DAPO Box 7777, Metro Manila, Philippines e-mail: bcycollard@hotmail.com Present address: B C Y Collard Department of Primary Industries, Bundoora, Victoria 3083, Australia Introduction DNA markers have numerous applications in plant molecular genetic research (Gupta et al 1999; Semagn et al 2006; Winter and Kahl 1995) Two of the most common uses of DNA markers have been the assessment of genetic diversity within crop germplasm and the construction of linkage maps for mapping genes or quantitative trait loci (QTL) controlling agronomically important traits (Collard et al 2005; Farooq and Azam 2002) The use of random amplified polymorphic DNA (RAPD) markers (Welsh and McClelland 1990; Williams et al 1990), inter simple sequence repeat (ISSR) markers (Blair et al 1999), and amplified fragment length polymorphism (AFLP) markers (Vos et al 1995) have been very popular because these marker techniques may generate relatively high numbers of DNA markers per sample and are technically simple These methods have been used extensively in genetic analysis of prokaryotes and eukaryotes In crops, RAPD, ISSR, and AFLP markers have been used extensively for genetic diversity analysis and QTL mapping (Botha and Venter 2000; Dziechciarkova et al 2004; Gostimsky et al 2005; Gupta et al 1999; Kelly and Miklas 1998; Mueller and Wolfenbarger 1999; Rao et al 2002) Each marker system has certain advantages and disadvantages compared to each other In many research labs, the selection of marker systems is largely influenced by crop species, technical expertise, available equipment, and available research funding In recent years, many new alternative and promising marker techniques have emerged These techniques include inter retrotransposon amplified polymorphism, retrotransposon microsatellite amplified polymorphism (Kalendar et al 1999), sequence-related amplified polymorphism (Li and Quiros 2001), and target region amplified polymorphism (TRAP; Hu and Vick 2003) Coupled with the rapid growth Plant Mol Biol Rep (2009) 27:86–93 87 of genomics research, there has been a trend away from random DNA markers towards gene-targeted markers (Andersen and Lubberstedt 2003; Gupta and Rustgi 2004) Genome sequence data offers enormous potential for the development of new markers in diverse plant species (Holland et al 2001) In this study, we describe a novel marker system called start codon targeted polymorphism that is based on the short conserved region in plant genes surrounding the ATG translation start (or initiation) codon that has been well characterized in previous studies (Joshi et al 1997; Sawant et al 1999) DNA markers are produced by polymerase chain reaction (PCR) using single primers that are designed from the short conserved region flanking the ATG start codon that is conserved for all genes Therefore, in principle, this technique is similar to RAPD or ISSR or single primer amplification reaction because a single primer is used as the forward and the reverse primer (Gupta et al 1994; Williams et al 1990) Markers are visualized by standard gel electrophoresis with agarose gels and staining making this technique suitable for the vast majority of plant research labs with standard equipment We have validated this technique using the important crop and model species rice (Oryza sativa) Materials and Methods Plant Material A representative set of ten rice genotypes was used for testing the level of polymorphism These genotypes include popular ‘mega-varieties’, common rice ‘reference’ genotypes, and important donor parents currently used in International Rice Research Institute’s breeding program (Table 1) A random set of 14 BC1 lines were obtained from a larger BC1 population derived from a IR64 (recurrent parent)×FL478 cross DNA was extracted from a 2-week-old seedling tissue following the method by Zheng et al (1995) with minor modifications described by Collard et al (2007) Primer Design Primers were designed from consensus sequences derived from the studies by Joshi et al (1997) and Sawant et al (1999) For primer design, the ATG codon (+1, +2, and +3), ‘G’ at position +4, and A, C, and C at positions +7, +8, and +9, respectively, were fixed (Table 2) All primers were 18-mer and ranged in GC content between 50% and 72% (Table 3) There were no degeneracies Primers were checked for dimers and hairpin loops using the program ‘FAST PCR’ (Kalendar 2007) PCR and Electrophoresis PCR was optimized for testing the SCoT method The final optimized protocol is reported here All PCR reactions were performed within a total volume of 10 μl in 96-well plates using a PTC-100 Thermocycler (MJ Research Model PTC100) PCR reaction mixtures contained PCR buffer (Promega; 20 mM Tris-HCl (pH 8.4), 50 mM KCl), 1.5 mM MgCl2, 0.24 mM of each deoxyribonucleotide triphosphates, 0.5 U of Taq polymerase (Promega), and 0.8 μm of primer Each reaction contained 25 ng of template DNA A standard PCR cycle was used: an initial denaturation step at 94°C for min, followed by 35 cycles of 94°C for min, 50°C for min, and 72°C for min; the final extension at 72°C was held for All PCR amplification products were separated on 1.2% agarose gels in Tris-borate buffer stained with ethidium bromide and visualized under UV light Data Analysis PCR-amplified SCoT fragments detected on gels were scored as absent (0) or present (1) Only clear, reproducible Table List of rice genotypes selected for SCoT genotyping Genotype Subspecies Information IR64 FL478 Swarna Samba Mahsuri BR28 BR11 IR40931 IR36 Nipponbare Azucena indica indica indica indica indica indica indica indica japonica japonica Popular and widely grown rice ‘mega variety’a Salt tolerance donor breeding line derived from Pokkali Popular rice mega variety grown in India Popular rice mega variety grown in India Popular rice mega variety grown in Bangladesh Popular rice mega variety grown in Bangladesh Submergence tolerant donor breeding line derived from FR13 Popular and widely grown rice ‘mega variety’ Reference genotype (genome sequence available) Drought tolerant a The term ‘mega variety’ refers to extremely popular rice varieties that are widely grown by farmers – A/C N – C/T A C C N A/G T/A N C N A G A N bands were scored Pairwise comparisons between accessions, based on the proportion of shared bands produced by the primers, were calculated using the Dice similarity coefficients (expressed as percentages) using the program FreeTree (Pavlicek et al 1999) A dendrogram showing the genetic relationships between accessions, based on the unweighted pair-group method with arithmetic averages, was constructed using TreeView (Page 1996) Results and Discussion C/G C G 11 10 12 13 15 Plant Mol Biol Rep (2009) 27:86–93 14 88 C C N A/G N N G T G C C N G G G G G G T T T A A A b a Dataset I was based on highly expressed genes described in Sawant et al (1999) Dataset II was based on lowly expressed genes described in Sawant et al (1999) C A N C/A C A A/G A G/A C A/C N Joshi et al (1997) Sawant et al (1999) Sawant et al (1999) Monocot consensus Dataset Ia Dataset IIb G T N A/C A N −1 −2 −3 −4 −5 −6 Reference Consensus sequence Nucleotide position Table Consensus sequences flanking the ATG start codon from Joshi et al (1997) and Sawant et al (1999) Rationale and Concept Gene-targeted markers are preferable for numerous applications in plant molecular genetics especially QTL mapping since recombination levels between marker and gene/QTL are generally lower compared with ‘indirect random markers’ such as RAPDs, ISSRs, or SSRs (Andersen and Lubberstedt 2003) The SCoT technique is based on the single primer amplified region principle since it uses a single primer as a forward and reverse primer, like the RAPD or ISSR technique However, PCR amplification using SCoT primers targets gene regions surrounding the ATG initiation codon on both DNA strands as shown in Fig It is possible that some SCoT markers would be codominant due to insertion–deletion mutations; these would be the minority like codominant RAPDs (Davis et al 1995) Due to the basis of SCoT primer design, we expect SCoT markers to be distributed within gene regions that contain genes on both plus and minus DNA strands It is also possible that pseudogenes and (genes within) transposable elements may be used as primer binding sites by SCoT polymorphism technique An important factor is the distance in base pairs between primer binding sites of the template Therefore, a relatively long extension time of the thermal cycle is important and we recommend at least Primer Design and PCR Protocol There are many important parameters that influence the reliability and reproducibility of PCR-amplified markers (Tyler et al 1997) SCoT primers in this study were designed based on a consensus sequence for the flanking region around the ATG start codon derived from the previous studies by Joshi et al (1997) and Sawant et al (1999) Specific nucleotides in the primer sequence were fixed: the ATG codon (at positions +1, +2, and +3), ‘G’ at position +4, ‘C’ at position +5, and A, C, and C at positions +7, +8, and +9, respectively Most primers differed from each other by at least one nucleotide with an emphasis Plant Mol Biol Rep (2009) 27:86–93 89 Table SCoT primer sequences SCoT primer 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 Sequence (5′-3′) %GC 50 56 56 50 50 56 56 50 50 56 50 61 61 67 67 56 61 67 67 67 61 56 61 56 67 61 61 67 72 72 67 67 67 61 72 56 CAACAATGGCTACCACCA CAACAATGGCTACCACCC CAACAATGGCTACCACCG CAACAATGGCTACCACCT CAACAATGGCTACCACGA CAACAATGGCTACCACGC CAACAATGGCTACCACGG CAACAATGGCTACCACGT CAACAATGGCTACCAGCA CAACAATGGCTACCAGCC AAGCAATGGCTACCACCA ACGACATGGCGACCAACG ACGACATGGCGACCATCG ACGACATGGCGACCACGC ACGACATGGCGACCGCGA ACCATGGCTACCACCGAC ACCATGGCTACCACCGAG ACCATGGCTACCACCGCC ACCATGGCTACCACCGGC ACCATGGCTACCACCGCG ACGACATGGCGACCCACA AACCATGGCTACCACCAC CACCATGGCTACCACCAG CACCATGGCTACCACCAT ACCATGGCTACCACCGGG ACCATGGCTACCACCGTC ACCATGGCTACCACCGTG CCATGGCTACCACCGCCA CCATGGCTACCACCGGCC CCATGGCTACCACCGGCG CCATGGCTACCACCGCCT CCATGGCTACCACCGCAC CCATGGCTACCACCGCAG ACCATGGCTACCACCGCA CATGGCTACCACCGGCCC GCAACAATGGCTACCACC Fig Diagram showing principle of SCoT PCR amplification on variations at the 3′ end, which has been shown to critical for primer-template specificity (Kwok et al 1990; Sommer and Tautz 1989) Nonconserved nucleotide positions (i.e., ‘N’) were exploited, by designing primers such that these nonconserved nucleotides typically occurred within the last three or four nucleotides at the 3′ end Previous research studies have indicated that primer length and annealing temperature are important factors for the reproducibility of PCR-based DNA markers Longer primers have been shown to improve reproducibility (Debener and Mattiesch 1998; Tanaka and Taniguchi 2002; Ye et al 1996) The study by Gillings and Holley (1997) suggest that primers between 18 to 24 nucleotides are preferable for producing reproducible markers SCoT primer design was constrained by the number of highly conserved nucleotides within the conversed ATG region, and 18-mer primers were considered to be the maximum length An initial comparison of 12-mer versus 18-mer primers indicated that the latter primer length was superior in terms of reproducibility (data not shown) The optimal primer length in the TRAP technique was also found to be 18 nucleotides (Hu and Vick 2003) Higher annealing temperatures also improve marker reproducibility (Atienzar et al 2000; Johnson and Clabots 2000) A range of annealing temperatures was tested (data not shown) There was a noticeable decrease in the number of scorable markers when annealing temperatures exceeded 55°C, and, therefore, 50°C was selected due to the number of reproducible scorable bands when this annealing temperature was used Magnesium chloride, an important cofactor for Taq DNA Polymerase and for primer-template binding, is another important factor that influences reproducibility (Perry et al 2003) It was reasoned that a standard, relatively low, fixed concentration of 1.5 mM would minimize the likelihood of GENOTYPE #1 , , Gene A SCoT primer , SCoT primer , Gene B PRESENT GENOTYPE #2 , Gene A , SCoT primer , , ABSENT 90 nonspecific binding, even though higher number of markers may be obtained using higher concentrations Generation of Polymorphic DNA Markers Plant Mol Biol Rep (2009) 27:86–93 SCoT 1 SCoT 9 10 11 3 10 11 10 11 1500 1000 1500 1000 250 Preliminary evaluation using a representative set of rice genotypes has indicated that SCoT primers generate DNA fingerprints similar to those generated by using RAPD markers (Fig 2) The size range was typically between 200–1,500 bp Using IR64 and Nipponbare as reference genotypes, between two and six markers were usually scored, the exception was using SCoT which produced only a single marker for most genotypes This is comparable to the number of markers typically generated by RAPD and ISSR techniques in rice (Kaushik et al 2003; Mackill 1995; Parsons et al 1997) The results also indicated the effect of altering a single nucleotide within the last three nucleotides at the 3′ end (Fig 2) For example, SCoT primers 29, 30, and 31 differ only in the last nucleotide at the 3′ end yet produced different profiles SCoT primers 18 and 20 differed only in the second last nucleotide and SCoT primers 12 and 13 differed only at the third last nucleotide yet also produced very different DNA marker profiles (Fig 2) Two primers differed by a single nucleotide at the 5′ end (SCoT and SCoT 11), which also generated different DNA marker profiles (data not shown) Further experiments are needed to determine the full effects of nucleotide substitution on the generation of polymorphism (Table 3) Reproducibility RAPD markers have been notorious for having problems with reproducibility, especially between laboratories (Hallden et al 1996; Jones et al 1997; Penner 1996) Since the SCoT technique was based on the same principle of single primer PCR, reproducibility was thoroughly tested and consisted of two stages: (1) comparing PCR amplification results between duplicate samples of selected genotypes and (2) repeating PCR reactions on different days and using different thermal cyclers Primers were initially prescreened on samples of selected genotypes; only primers that passed stage (i.e., produced reproducible markers) were considered for stage evaluation In general, SCoT markers were reproducible when using duplicate samples, although some primers did not produce reproducible profiles suggesting that primer length and annealing temperature per se not ensure reproducibility Markers that ‘passed’ stage testing were found to be reproducible when PCR was performed on different days and using different thermal cyclers with the exception of SCoT 28 This emphasizes the need to carefully test all markers generated by using this technique, 250 SCoT 12 SCoT 13 10 11 1500 1500 1000 1000 250 250 SCoT 18 10 11 1500 1000 1000 250 250 10 11 1500 1500 1000 1000 250 250 3 10 11 1500 1000 1000 250 250 3 1500 1000 250 7 10 11 10 11 8 10 11 SCoT 34 SCoT 31 SCoT 30 1500 4 SCoT 29 SCoT 22 SCoT 20 SCoT 19 1500 10 11 10 11 1500 1000 250 Fig Examples of SCoT marker profiles Lane IR64, FL478, Swarna, Samba Mahsuri, BR28, BR11, IR40931, IR36, Nipponbare, 10 Azucena, 11 no template control Left most lane— 250 bp DNA ladder (Invitrogen Cat No: 10596-013) like RAPD These results may also imply that further optimization of PCR conditions is required for these primers since the primer length of 18 nucleotides and annealing temperature of 50°C not guarantee reproducibility In this study, it was observed that primers with a higher GC content were generally more reproducible (data not shown) Plant Mol Biol Rep (2009) 27:86–93 91 Conclusion Although we have only tested 36 primers in this study, many additional primers could be designed by minor alterations of the primer sequences described in this study Azucena Nipponbare IR64 Swarna Samba Mahsuri IR36 FL478 BR28 BR11 IR40931 0.1 Fig Radial tree of rice genotypes based on 50 SCoT markers IR64 10 11 12 13 14 1500 1000 250 FL478 SCoT 34 IR64 SCoT primers were used to fingerprint a small diverse set of rice genotypes The 13 primers generated a total of 50 polymorphic SCoT markers, which were scored based on at least two replicates Only markers that were clearly scorable and detected on both gels were considered for diversity analysis Clustering of the rice genotypes was generally consistent with known taxonomic and pedigree information (Fig 3) The two japonica genotypes, Nipponbare and Azucena, clearly clustered together and were more diverse compared to the indica genotypes Samba Mahsuri and Swarna both have Mahsuri as a common parent and clustered together The Bangladeshi varieties, BR11 and BR28, clustered together which is consistent with the common parents IR262 and Peta in their pedigrees The abiotic stress tolerant breeding lines FL478 and IR40931 also clustered together, which is consistent with IR1561 being a common ancestor Genotyping of a small random subset of backcross lines indicated typical segregation of a dominant marker and high reproducibility since identical marker profiles were generated between replications (Fig 4) FL478 SCoT 33 Validation of SCoT Markers: Genetic Diversity Analysis and Testing in a Backcross Population 10 11 12 13 14 1500 1000 250 Fig Validation of SCoT markers in 14 backcross lines or by targeting different regions of the conserved region flanking the ATG start codon Since the region flanking the ATG start codon is highly conserved in all plant species, we predict that the SCoT method will be useful for generating DNA markers in diverse plant species although this has not been experimentally validated We anticipate that there will be three main applications of SCoT markers: QTL mapping, bulked segregant analysis, and genetic diversity studies For QTL mapping, SCoT markers could be integrated into existing framework maps to increase the marker density or to target specific chromosomal regions In another analogy with RAPD, we propose that important SCoT markers, such as those identified to be tightly linked to a gene or QTL of interest, could be converted into sequence characterized amplified regions markers or sequence tagged site markers in order to make the marker single locus and improve robustness (Monna et al 1994; Paran and Michelmore 1993) In summary, a novel DNA marker technique that targets plant gene regions by the design of primers was developed It is agarose-based and therefore simple and relatively cheap to use Furthermore, markers should be associated with gene regions based on their primer design This marker technique should provide an additional option to the RAPD or ISSR techniques for several applications in plant genetic analysis Acknowledgements Technical assistance for gel electrophoresis by Ms Miladie Penarubia is gratefully acknowledged We also thank Dr C Raghavan and two anonymous reviewers for valuable comments on the manuscript 92 References Andersen JR, Lubberstedt T Functional markers in plants Trends Plant Sci 2003;8:554–50 Atienzar F, Evenden A, Jha A, Savva D, Depledge M Optimized RAPD analysis generates high-quality genomic DNA profiles at high annealing temperature BioTechniques 2000;28:52–4 Blair MW, Panaud O, McCouch SR Inter-simple sequence repeat (ISSR) amplification for analysis of microsatellite motif frequency and fingerprinting in rice (Oryza sativa L.) Theor Appl Genet 1999;98:780–92 Botha AM, Venter E Molecular marker technology linked to pest and pathogen resistance in wheat breeding S Afr J Sci 2000;96:233–40 Collard BCY, Das A, Virk PS, Mackill DJ Evaluation of ‘quick and dirty’ DNA extraction methods for marker-assisted selection in rice (Oryza sativa L.) Plant Breed 2007;126:47–50 Collard BCY, Jahufer MZZ, Brouwer JB, Pang ECK An introduction to markers, quantitative trait loci (QTL) mapping and markerassisted selection for crop improvement: the basic concepts Euphytica 2005;142:169–96 Davis TM, Yu H, Haigis KM, McGowan PJ Template mixing: a method of enhancing detection and interpretation of codominant RAPD markers Theor Appl Genet 1995;91:582–8 Debener T, Mattiesch L Effective pairwise combination of long primers for RAPD analysis Plant Breed 1998;117:147–51 Dziechciarkova M, Lebeda A, Dolezalova I, Astley D Characterization of Lactuca spp germplasm by protein and molecular markers—a review Plant Soil Environ 2004;50:47–58 Farooq S, Azam F Molecular markers in plant breeding-II Some prerequisites for use Pak J Biol Sci 2002;5:1141–7 Gillings M, Holley M Amplification of anonymous DNA fragments using pairs of long primers generates reproducible DNA fingerprints that are sensitive to genetic variation Electrophoresis 1997;18:1512–8 Gostimsky SA, Kokaeva ZG, Konovalov FA Studying plant genome variation using molecular markers Russ J Genet 2005;41:378–88 Gupta M, Chyi YS, Romero-Severson J, Owen JL Amplification of DNA markers from evolutionary diverse genomes using single primers of simple-sequence repeats Theor Appl Genet 1994; 89:998–1006 Gupta PK, Rustgi S Molecular markers from the transcribed/expressed region of the genome in higher plants Funct Integr Geonomics 2004;4:139–62 Gupta PK, Varshney RK, Sharma PC, Ramesh B Molecular markers and their applications in wheat breeding Plant Breed 1999; 118:369–90 Hallden C, Hansen M, Nilsson NO, Hjerdin A, Sall T Competition as a source of errors in RAPD analysis Theor Appl Genet 1996;93:185–92 Holland JB, Helland SJ, Sharopova N, Rhyne DC Polymorphism of PCR-based markers targeting exons, introns, promoter regions, and SSRs in maize and introns and repeat sequences in oat Genome 2001;44:1065–76 Hu J, Vick BA Target region amplification polymorphism: a novel marker technique for plant genotyping Plant Mol Biol Report 2003;21:289–94 Johnson JR, Clabots C Improved repetitive-element PCR fingerprinting of Salmonella enterica with the use of extremely elevated annealing temperatures Clin Diagn Lab Immunol 2000;7:258–64 Jones CJ, Edwards KJ, Castaglione S, Winfield MO, Sala F, vandeWiel C, et al Reproducibility testing of RAPD, AFLP and SSR markers in plants by a network of European laboratories Mol Breed 1997;3:381–90 Joshi C, Zhou H, Huang X, Chiang VL Context sequences of translation initiation codon in plants Plant Mol Biol 1997;35:993–1001 Plant Mol Biol Rep (2009) 27:86–93 Kalendar R FastPCR: a PCR primer design and repeat sequence searching software with additional tools for the manipulation and analysis of DNA and protein Available at www.biocenter helsinki.fi/bi/programs/fastpcr.htm; 2007 Kalendar R, Grob T, Regina M, Suoniemi A, Schulman A IRAP and REMAP: two new retrotransposon-based DNA fingerprinting techniques Theor Appl Genet 1999;98:704–11 Kaushik A, Saini N, Jain S, Rana P, Singh RK, Jain RK Genetic analysis of a CSR10 (indica) x Taraori Basmati F3 population segregating for salt tolerance using ISSR markers Euphytica 2003;134:231–8 Kelly JD, Miklas PN The role of RAPD markers in breeding for disease resistance in common bean Mol Breed 1998;4:1–11 Kwok S, Kellogg DE, McKinney N, Spasic D, Goda D, Levenson C, et al Effects of primer-template mismatches on the polymerase chain reaction: human immunodeficiency virus type model studies Nucleic Acid Res 1990;18:999–1005 Li G, Quiros CF Sequence-related amplified polymorphism (SRAP), a new marker system based on a simple PCR reaction: its application to mapping and gene tagging in Brassica Theor Appl Genet 2001;103:455–61 Mackill DJ Classifying japonica rice cultivars with RAPD markers Crop Sci 1995;35:889–94 Monna L, Miyao A, Inoue T, Fukuoka S, Yamazaki M, Zhong HS, et al Determination of RAPD markers in rice and their conversion into sequence tagged sites (STSs) and STS-specific primers DNA Res 1994;1:138–48 Mueller UG, Wolfenbarger LL AFLP genotyping and fingerprinting Trends Ecol Evol 1999;14:389–94 Page RDM TREEVIEW: an application to display phylogenetic trees on personal computers Comput Appl Biosci 1996;12: 357–8 Paran I, Michelmore R Development of reliable PCR-based markers linked to downy mildew resistance genes in lettuce Theor Appl Genet 1993;85:985–93 Parsons BJ, Newbury HJ, Jackson MT, Ford-Lloyd BV Contrasting genetic diversity relationships are revealed in rice (Oryza sativa L.) using different marker types Mol Breed 1997;3:115–25 Pavlicek A, Hrda S, Flegr J FreeTree—freeware program for construction of phylogenetic trees on the basis of distance data and bootstrap/jackknife analysis of the tree robustness Application in the RAPD analysis of the genus Frenkelia Folia Biol (Praha) 1999;45:97–9 Penner G RAPD analysis of plant genomes In: Jauhar PP, editor Methods of genome analysis in plants Boca Raton: CRC; 1996 p 251–268 Perry AL, Worthington T, Hilton AC, Lambert PA, Stirling AJ, Elliott TSJ Analysis of clinical isolates of Propionibacterium acnes by optimised RAPD FEMS Microbiol Lett 2003;228:51–5 Rao KK, Lakshminarasu M, Jena KK DNA markers and markerassisted breeding for durable resistance to bacterial blight disease in rice Biotechnol Adv 2002;20:33–47 Sawant SV, Singh PK, Gupta SK, Madnala R, Tuli R Conserved nucleotide sequences in highly expressed genes in plants J Genet 1999;78:123–31 Semagn K, Bjornstad A, Ndjiondjop MN An overview of molecular marker methods for plants Afr J Biotechnol 2006;5:2540–68 Sommer R, Tautz D Minimal homology requirements for PCR primers Nucleic Acid Res 1989;17:6749 Tanaka J, Taniguchi F Emphasized-RAPD (e-RAPD): a simple and efficient technique to make RAPD bands clearer Breed Sci 2002;52:225–9 Tyler KD, Wang G, Tyler SD, Johnson WM Factors affecting reliability and reproducibility of amplification-based DNA fingerprinting of representative bacterial pathogens J Clin Microbiol 1997;35:339–46 Plant Mol Biol Rep (2009) 27:86–93 Vos P, Hogers R, Bleeker M, Reijans M, van de Lee T, Hoernes M, et al AFLP: a new technique for DNA fingerprinting Nucleic Acids Res 1995;23:4407–14 Welsh J, McClelland M Fingerprinting genomes using PCR with arbitrary primers Nucleic Acids Res 1990;18:7213–8 Williams J, Kubelik A, Livak K, Rafalski J, Tingey S DNA Polymorphisms amplified by arbitrary primers are useful as genetic markers Nucleic Acids Res 1990;18:6531–5 93 Winter P, Kahl G Molecular marker technologies for plant improvement World J Microbiol Biotechnol 1995;11:438–48 Ye G-N, Hemmat M, Lodhi MA, Weeden NF, Reisch BI Long primers for RAPD mapping and fingerprinting of grape and pear BioTechniques 1996;20:368–71 Zheng K, Subudhi PK, Domingo J, Maopantay G, Huang N Rapid DNA isolation for marker assisted selection in rice breeding Rice Genet Newsl 1995;12:48  ... 250 3 1500 1000 250 7 10 11 10 11 8 10 11 SCoT 34 SCoT 31 SCoT 30 1500 4 SCoT 29 SCoT 22 SCoT 20 SCoT 19 1500 10 11 10 11 1500 1000 250 Fig Examples of SCoT marker profiles Lane IR64, FL478, Swarna,... with the exception of SCoT 28 This emphasizes the need to carefully test all markers generated by using this technique, 250 SCoT 12 SCoT 13 10 11 1500 1500 1000 1000 250 250 SCoT 18 10 11 1500 1000... example, SCoT primers 29, 30, and 31 differ only in the last nucleotide at the 3′ end yet produced different profiles SCoT primers 18 and 20 differed only in the second last nucleotide and SCoT primers