New strategy for Pinus pinaster genomic library construction in bacterial artificial chromosomes

Abstract

To learn about the structure and organization of plant genomes, molecular cloning of large genomic fragments for representative gene libraries construction is required. The genome library construction on bacterial artificial chromosome (BAC) is one of the most frequently used tools for this purpose, and the chosen strategy in this work. But when working with species containing very large genomes, such as pine, construction of this kind of gene libraries is a laborious and expensive task. In this study we have developed a method that starts from P. pinaster cotyledons to construct a pooled BAC library that drastically reduces cost, space and time required to clone a genotype of interesThe current BAC library comprises 83 groups of cells with an average of 4000 clones per group and accounts for a 0.8 X genome content of P. pinaster. It is also shown that a BAC of interest can be quickly identified by PCR (polymerase chain reaction): a pool containing a BAC clone that carries a coding sequence similar to a Class I chitinase of Picea abies has been identified, as well as another BAC clone in a different pool containing a sequence with homology to an unknown P.taeda mRNA. This strategy will allow screening and storing gene libraries from large genome organisms and a quickly location of genes of interest, the method been described in detail.