Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress

Verona, Italy - 24/27 September, 2003

ISBN 88-900622-4-X

Poster Abstract - 5.55

CONSTRUCTION OF A BACTERIAL ARTIFICIAL CHROMOSOME LIBRARY OF A SEEDLESS CLONE OF CITRUS SINENSIS CV “VANIGLIA”

S. FERRANTE, G. GERACI, D. GRAZIANO. M.-T. SCARANO

C.N.R. IGV-sez di Palermo, Corso Calatafimi 414, 90129 Palermo, Italy

BAC, genomics, sweet orange, DNA cloning

Isolation of genes controlling expression of important agronomical traits is at heart of plant genetic improvement. For this purpose, cloning of large DNA fragments within proper vectors represents an outstanding tool for genome analysis. BAC vectors (Bacterial Artificial Chromosome), due to high cloning efficiency, clone stability and ease of use, represent, currently the system of choice for the construction of large insert genomic DNA libraries. These vectors utilize the E. coli single-copy fertility (F plasmid) and can maintain genomic DNA fragments up to 350 kb. A prerequisite for the constitution of large fragment DNA libraries is the isolation of DNA of high molecular weight (megabase-size) and high quality. We followed the protocols previously proposed by Zhang et al. (1995) isolating nuclei from fresh leaves and subsequently embedding them in low melting agarose plugs. To prepare high molecular weight restriction fragments we applied the procedure from D.G. Peterson and the vector pIndigoBAC-5 (Epicentre) has been used because purchase dephosphorylated and linearized with HindIII restriction enzyme. Pulsed Field Gel Electrophoresis (PFGE) has been carried out with Gene Navigator System (Amersham Pharmacia Biotech) provided with and hexagonal electrode to generate homogeneous electric field (CHEF). Size-selected DNA fragments have been isolated from agarose via electroelution with Electroelution System Model 422 (BioRad). After ligation of insert DNA with the chosen vector, to form BACs, transformation of competent cells (TransforMax EC100 Electrocompetent E. coli, Epicentre) has been performed with BioRad Gene Pulser II apparatus. The most common form of insertional inactivation (alpha-complementation) has been utilized to select recombinant clones. Because the mean DNA length in the plugs resulted to be greater than 600 kb, the plugs have been evaluated adequate for the library construction. In order to obtain an high number of transformants, in the ligation the optimal molar ratio of size-selected DNA/vector was determined to be 6/1. The high number of colonies observed per plate (430 cfu/plate, 8.6 cfu/microl), the reduced number of blue-colored colonies (non-recombinant clones) and false positives detected, confirms that the tested procedure resulted to be efficient and, therefore, useful for the subsequent phases of clone collection. The described methodology has been conceived to generate clones with inserts of exogenous DNA between 100-350 kb. In this project, the mean insert length, estimated on a random sample of white colonies, was equal to 135 kb. It is possible that screening of a greater number of clones could determine a reduction of the estimated mean insert length, without nevertheless compromize the efficacy of the library under construction.

Peterson D.G., Tomkins J.P., Frisch D.A., Wing R.A., Paterson A.H. 2000. Construction of plant bacterial artificial chromosome (BAC) libraries: An illustrated guide. J. Agric. Genomics 5 (http://www.ncgr.org/research/jag).

Zhang H.B. , Zhao X., Ding X., Paterson A.H., Wing R.A. 1995. Preparation of megabase-size DNA from plant nuclei. The Plant Journal 7(1): 175-184.