36th International Carrot Conference Abstract

β-tubulin intron-length-polymorphism marker (cTBP) – a useful tool to assist species/subspecies discrimination and genetic diversity analysis in carrot

Hélia G. Cardoso, Alexandre Ferreira, Maria D. Campos, A. M. Frederico and Birgit Arnholdt-Schmitt

EU Marie Curie Chair, ICAAM - Instituto de Ciências Agrárias e Ambientais Mediterrânicas, Universidade de Évora, Núcleo da Mitra, Ap. 94, 7002-554 Évora, Portugal

The availability of informative, polymorphic, and robust PCR-based markers are valuable resources to assist breeding, genetic diversity, and genomic studies in carrot. Different marker systems have been applied, including anonymous dominant AFLP markers [1] or laborious time-consuming codominant marker such as RFLPs [2] and SSRs [3]. The development of a method which involves low cost and being less time consuming will represent high advantages. Among the classes of available DNA markers, intron polymorphism can be exploited as a valuable molecular tool [4], being the Intron Length Polymorphism (ILPs) the most convenient molecular marker [5]. ILP can be detected by polymerase chain reaction (PCR) with a pair of primers designed on the flanking exons (exon-primed intron-crossing PCR or EPIC-PCR) [6]. The plant β-tubulin gene family provides the basis for one of the most successful and versatile form of ILPs, named TBP for Tubulin-Based Polymorphism [7]. An upgrade of the TBP methodology (cTBP) and is based in the combined analysis of intron 1 and 2 of β-tubulin [8,9]. cTBP allows the identification of different alleles providing stable and specific, neutral co-dominant marker DNA fragments. Combination of the genetic variability in both introns results in a more reliable assessment of species/cultivars/ecotypes relationships [8,9]. The discrimination power, of both TBP and cTBP methods, among species, varieties and ecotypes has been largely assessed and discussed by many authors [7-11]. Applicability of the marker in breeding application as a tool to evaluate the inbred level was also pointed out. In a first approach we confirm the applicability of the cTBP method for species/subspecies discrimination in Daucus. Genetic diversity and phylogenetic reconstructions could be accessed among several genotypes from wild and cultivated material and inbreed lines showing the highest level of genetic diversity within the wild material.

References:

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[2] Vivek and Simon (1999) Euphytica, 105:183-189.

[3] Cavagnaro et al. (2011) BMC Genomics, 12:386.

[4] Poczai et al. (2013) Plant Methods, doi:10.1186/1746-4811-9-6.

[5] Chen et al. (2011) New Forests, 41:379-388.

[6] Bierne et al. (2000) Mol Ecol, 9:233-235.

[7] Bardini et al. (2004) Genome, 247:281-291.

[8] Breviario et al. (2007) Mol Breeding, 20:249–259.

[9] Braglia et al. (2010) Diversity, 2:572-585.

[10] Casazza et al. (2011) Food Chemistry, 124:685–691.

[11] Gavazzi et al. (2012) Electrophoresis, 33: 2840–2851.

Acknowledgements:

This work is funded by FEDER Funds through the Operational Programme for Competitiveness Factors - COMPETE and National Funds through FCT - Foundation for Science and Technology under the Strategic Project PEst-C/AGR/UI0115/2011 and the European Project FEED-CODE. The authors would like to thank to FCT for the support given under the program POPH – Programa Operacional Potencial Humano (Ciência 2007 and 2008).

Last updated Thursday, 25-Jul-2013 12:30:47 CDT