- Poster presentation
- Open Access
Novel design and deployment of orthologous genic SSR markers in Eucalyptus camaldulensisDehnh
© Nagabhushana et al; licensee BioMed Central Ltd. 2011
- Published: 13 September 2011
- Repeat Motif
- Seed Orchard
- Eucalyptus Species
- Genome Scaffold
- Polymerase Chain Reaction Profile
Eucalyptus camaldulensis is a widely planted tree species in India, because of its rapid growth and adaptability to dry regions. Genetic improvement through informed breeding of E. camaldulensis largely depends on availability of molecular markers, linkage maps and genome information. Microsatellite markers, also called as simple sequence repeats (SSRs) have wide application due to their unique advantages over other marker systems. In Eucalyptus, SSR markers have been used in various breeding applications from DNA fingerprinting to QTL mapping [1, 4]. Despite of their advantages, the major drawback is the time required for their development . There are less than twenty five E. camaldulensis specific SSR markers available in the public database . Although, there are large number of SSRs available in other Eucalyptus species [2, 3], their species transferability in camaldulensis is questionable for practical use. Nevertheless, the existing camaldulensis specific SSRs are insufficient for developing linkage maps, QTL and comparative mapping studies. Besides, EST based markers are handicapped due to exclusion of introns, which sometimes lead to compromise on product sizing. Therefore we have modeled a novel strategy that targets highly conserved domain in the genic region using both publicly available ESTs as well as genome sequences.
Targeting conserved genic region
PCR, multiplexing and genotyping
We adopted an innovative two-tier polymerase chain reaction (PCR) system to reduce the cost 12 times on labeled oligo synthesis. The first PCR was performed with M13 tagged forward primer at 5’ end whereas the second PCR was performed with fluorescently labeled M13 as forward primer and SSR reverse primer. Multiplexing was carried out at post PCR stage. The first PCR consisted of template DNA (5 ng), primers (2 pM), 10X PCR buffer, dNTPs (1 µM), MgCl2 (1.5mM) and Amplitaq Gold Taq DNA polymerase (0.25 U). The PCR profile consisted of denaturing the template DNA at 94oC for 5 min, followed by 35 cycles, each at 94oC for 30 sec, 50-65oC for 30 sec and 72oC for 1 min, followed by 72oC for 8 min. The first PCR products were resolved on 2 % high resolution agarose. The second PCR consisted of template DNA (2 ng), 2 pM each of forward (labeled M13) and reverse (SSR) primer, 10X PCR buffer, dNTPs (1 µM), and 0.5U of Taq DNA polymerase. The PCR conditions remained same except reduced number of cycles to 20. The amplicons were resolved using ABI 3730 sequencer and each amplicon was manually validated for their allele size.
- Brondani RPV, Brondani C, Grattapaglia D: Towards a genus-wide reference linkage map for eucalyptus based exclusively on highly informative microsatellite markers. Mol Genet Genomics. 2002, 267: 338-347. 10.1007/s00438-002-0665-6.View ArticlePubMedGoogle Scholar
- Brondani RPV, Williams ER, Brondani C, Grattapaglia D: A microsatellite-based consensus linkage map for species of Eucalyptus and a novel set of 230 microsatellite markers for the genus. BMC Plant Biol. 2006, 6: 20-10.1186/1471-2229-6-20.PubMed CentralView ArticlePubMedGoogle Scholar
- Faria DA, Mamani EMC, Pappas MR, Pappas GJ, Grattapaglia D: A selected set of EST-derived microsatellites, polymorphic and transferable across 6 species of Eucalyptus. J Hered. 2010, 101: 512-520. 10.1093/jhered/esq024.View ArticlePubMedGoogle Scholar
- Kirst M, Cordeiro GD, Rezende SP, Grattapaglia D: Power of microsatellite markers for fingerprinting and parentage analysis in Eucalyptus grandis breeding populations. J Hered. 2005, 96: 1-6.View ArticleGoogle Scholar
- Silva JM, Souza ACB, Souza AP, Mori ES, Freitas MLM, Sebbenn AM, Moraes MLT: Development and characterization of 14 microsatellite loci from enriched genomic library of Eucalyptus camaldulensis Dehnh. Conservation Genet Resour. 2009, 1: 465-469. 10.1007/s12686-009-9107-7.View ArticleGoogle Scholar
- Zane L, Bargelloni L, Patarnello T: Strategies for microsatellite isolation: a review. Mol Ecol. 2002, 11: 1-16. 10.1046/j.0962-1083.2001.01418.x.View ArticlePubMedGoogle Scholar
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