Adventures in the Enormous: A 1.8 Million Clone BAC Library for the 21.7 Gb Genome of Loblolly Pine

Zenaida V Magbanua,C Dana Nelson,Richard C Cronn,Daniel G Peterson,Seval Ozkan,Philippe Chouvarine,Christopher A Saski,Aaron Liston,Benjamin D Bartlett

doi:10.1371/journal.pone.0016214

Abstract

Loblolly pine (LP; Pinus taeda L.) is the most economically important tree in the U.S. and a cornerstone species in southeastern forests. However, genomics research on LP and other conifers has lagged behind studies on flowering plants due, in part, to the large size of conifer genomes. As a means to accelerate conifer genome research, we constructed a BAC library for the LP genotype 7-56. The LP BAC library consists of 1,824,768 individually-archived clones making it the largest single BAC library constructed to date, has a mean insert size of 96 kb, and affords 7.6X coverage of the 21.7 Gb LP genome. To demonstrate the efficacy of the library in gene isolation, we screened macroarrays with overgos designed from a pine EST anchored on LP chromosome 10. A positive BAC was sequenced and found to contain the expected full-length target gene, several gene-like regions, and both known and novel repeats. Macroarray analysis using the retrotransposon IFG-7 (the most abundant repeat in the sequenced BAC) as a probe indicates that IFG-7 is found in roughly 210,557 copies and constitutes about 5.8% or 1.26 Gb of LP nuclear DNA; this DNA quantity is eight times the Arabidopsis genome. In addition to its use in genome characterization and gene isolation as demonstrated herein, the BAC library should hasten whole genome sequencing of LP via next-generation sequencing strategies/technologies and facilitate improvement of trees through molecular breeding and genetic engineering. The library and associated products are distributed by the Clemson University Genomics Institute (www.genome.clemson.edu).

Highlights

Pine (LP; Pinus taeda L.) is an organism of tremendous economic and ecological importance and a key representative of the conifers, an ancient lineage of plants that dominates many of the world’s temperate and boreal ecosystems [1]
Freezer banks at the Mississippi Genome Exploration Laboratory (MGEL; www.mgel.msstate.edu) while the original was sent to the Clemson University Genomics Institute (CUGI; www.genome. clemson.edu) for distribution and remote storage
Unlike other plant and animal species for which we typically obtain clones with mean insert sizes in excess of 100 kb with only modest optimization, application of standard bacterial artificial chromosome (BAC) library construction protocols resulted in Loblolly pine (LP) clones with mean insert sizes,75 kb

Summary

Introduction

Pine (LP; Pinus taeda L.) is an organism of tremendous economic and ecological importance and a key representative of the conifers, an ancient lineage of plants that dominates many of the world’s temperate and boreal ecosystems [1]. Approximately eight years to sexual maturity, poses an obstacle to tree improvement through traditional breeding techniques. Though molecular resources such as genetic maps [6,7,8], a FISH-based karyotype [9], EST sequences [10,11,12,13], and QTL maps are available [6,7,8] for LP, efficient tree improvement will require integration of EST, sequence polymorphism, gene expression, and genetic data with actual genomic sequence including noncoding regulatory regions missed by EST approaches. The 7-56 BAC library is a high quality resource that will expedite research on pine and conifers in general

Results and Discussion

Conclusion

Materials and Methods