Development of a BAC library for yellow-poplar (Liriodendron tulipifera) and the identification of genes associated with flower development and lignin biosynthesis
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Liriodendron tulipifera L., a member of the Magnoliaceae, occupies an important phylogenetic position as a basal angiosperm that has retained numerous putatively ancestral morphological characters, and thus has often been used in studies of the evolution of flowering plants and of specific gene families. However, genomic resources for these early branching angiosperm lineages are very limited. In this study, we describe the construction of a large-insert bacterial artificial chromosome (BAC) library from L. tulipifera. Flow cytometry estimates that this nuclear genome is approximately 1,802 Mbp per haploid genome (±16 SD). The BAC library contains 73,728 clones, a 4.8-fold genome coverage, with an average insert size of 117 kb, a chloroplast DNA content of 0.2%, and little to no bacterial sequences nor empty vector content clones. As a test of the utility of this BAC library, we screened the library with six single/low-copy genic probes. We obtained at least two positive clones for each gene and confirmed the clones by DNA sequencing. A total of 182 paired end sequences were obtained from 96 of the BAC clones. Using BLAST searches, we found that 25% of the BAC end sequences were similar to DNA sequences in GenBank. Of these, 68% shared sequence with transposable elements and 25% with genes from other taxa. This result closely reflected the content of random sequences obtained from a small insert genomic library for L. tulipifera, indicating that the BAC library construction process was not biased. The first genomic DNA sequences for Liriodendron genes are also reported. All the Liriodendron genomic sequences described in this paper have been deposited in the GenBank data library. The end sequences from shotgun genomic clones and BAC clones are under accession DU169330–DU169684. Partial sequences of Gigantea, Frigida, LEAFY, cinnamyl alcohol dehydrogenase, 4-coumarate:CoA ligase, and phenylalanine ammonia-lyase genes are under accession DQ223429–DQ223434.
KeywordsYellow-poplar BAC library
This study was supported by the National Science Foundation grant numbers 0207202 (dePamphilis and Carlson), 0211611 (Wing), and 0208502 (Mandoli) to The Green Plant BAC Library Project, and by the Schatz Center for Tree Molecular Genetics at Pennsylvania State University. We thank Jayson Talag, Sheila Plock, Kerr Wall, and Deb Grove for their assistance in developing and characterizing the BAC resource. We greatly acknowledge The Floral Genome Project for the prepublication access to cDNA probes and EST information on yellow-poplar, without which the library characterization might not have been possible. We are indebted to Abdelali Barakat for the many helpful discussions during the project and for the insightful comments on the manuscript.
CWD, DFM, JEC, JAB, JPT, and RAD participated in the design of the study and were co-PIs on the project. SES provided the biological samples from which the libraries were made. KA performed the cell flow cytometry genome-size determination. EGF and JPT constructed the BAC library. ML and DK constructed the plasmid shotgun library. DK, EGF, HL, HRK, ML, and SZ participated in library characterization. HL was the primary author of the manuscript. All authors have read and approved this report.
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