Breviario, Diego , Hilu, Khidir W. , Blumetti, Pietro , Gianě, Silvia , Sangoi, Shail , Baird, Wm. Vance .
Tubulin intron-based polymorphisms as a new DNA fingerprinting tool with application to systematics, breeding and variety protection.
Tubulin-Based-Polymorphism (TBP) is a novel method for assaying genetic diversity in plants. Tubulin structural proteins, alpha and beta, form heterodimers that are the primary building blocks of microtubules. TBP was based initially on polymorphism resulting from the PCR amplification of the first intron in the coding region of members of the b-tubulin gene family. Although this method was successful in genetic assessment of a number of plant species, it suffered from a low number of molecular markers caused by limited variation in first intron size. Inclusion of the second intron of the b-tubulin genes as an additional source of molecular variation rectified this limitation. Degenerate primer pairs for PCR amplification were designed for conserved coding sequences flanking each intron. The targeted introns were amplified from purified total genomic DNA, and the products were run on acrylamide gels and silver-stained for visualization. Representative examples of the polymorphic bands were cloned to confirm their origin and identity. Only reproducible and unambiguously resolved bands were scored. A genetic similarity matrix with simple matching coefficients was generated using NTSVSpc2.1. A dendrogram was computed using the UPGMA clustering method. The mean Polymorphism Information Content value for the TBP markers was calculated. Combined use of the two introns substantially increases the number of molecular markers. After preliminary work with five cultivars of Brassica napus (Brassicaceae) to validate the approach, this new combinatorial method was tested on nine species each of Arachis (Fabaceae) and Eleusine (Poaceae). Patterns of species relationship obtained with the combinatorial TBP approach were consistent with recently published studies resulting from more elaborated methods, which included DNA sequencing. Combinatorial TBP is a simple, fast, reliable, reproducible and easy method to assess genetic variation that is very useful for breeding programs, and species and variety assessments.
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1 - Istituto Biologia e Biotecnologia Agraria, ibba-cnr, Via Bassini 15, Milano, 20133, Italy
2 - Virginia Tech, Biological Sciences, Blacksburg, Virginia, 24061, USA
3 - Istituto Biologia e Biotecnologia Agraria, ibba - cnr, Via Bassini 15, Milano, 20133, Italy
4 - Clemson University, Horticulture Department, Clemson, SC, 29634-0319, USA
5 - Clemson University, Horticulture Department, Poole Agr Cntr, Box 340319, Clemson, South Carolina, 29634-0319, USA
Presentation Type: Oral Paper:Papers for Sections
Location: Lake Huron/Hilton
Date: Monday, July 9th, 2007
Time: 2:15 PM