Abstract
BackgroundEucalyptus species are among the most planted hardwoods in the world because of their rapid growth, adaptability and valuable wood properties. The development and integration of genomic resources into breeding practice will be increasingly important in the decades to come. Bacterial artificial chromosome (BAC) libraries are key genomic tools that enable positional cloning of important traits, synteny evaluation, and the development of genome framework physical maps for genetic linkage and genome sequencing.ResultsWe describe the construction and characterization of two deep-coverage BAC libraries EG_Ba and EG_Bb obtained from nuclear DNA fragments of E. grandis (clone BRASUZ1) digested with HindIII and BstYI, respectively. Genome coverages of 17 and 15 haploid genome equivalents were estimated for EG_Ba and EG_Bb, respectively. Both libraries contained large inserts, with average sizes ranging from 135 Kb (Eg_Bb) to 157 Kb (Eg_Ba), very low extra-nuclear genome contamination providing a probability of finding a single copy gene ≥ 99.99%. Libraries were screened for the presence of several genes of interest via hybridizations to high-density BAC filters followed by PCR validation. Five selected BAC clones were sequenced and assembled using the Roche GS FLX technology providing the whole sequence of the E. grandis chloroplast genome, and complete genomic sequences of important lignin biosynthesis genes.ConclusionsThe two E. grandis BAC libraries described in this study represent an important milestone for the advancement of Eucalyptus genomics and forest tree research. These BAC resources have a highly redundant genome coverage (> 15×), contain large average inserts and have a very low percentage of clones with organellar DNA or empty vectors. These publicly available BAC libraries are thus suitable for a broad range of applications in genetic and genomic research in Eucalyptus and possibly in related species of Myrtaceae, including genome sequencing, gene isolation, functional and comparative genomics. Because they have been constructed using the same tree (E. grandis BRASUZ1) whose full genome is being sequenced, they should prove instrumental for assembly and gap filling of the upcoming Eucalyptus reference genome sequence.
Highlights
Eucalyptus species are among the most planted hardwoods in the world because of their rapid growth, adaptability and valuable wood properties
Bacterial artificial chromosome (BAC) library characterization Two genomic BAC libraries (EG_Ba and EG_Bb) were constructed using partially digested (HindIII or BstYI) and size-selected nuclear DNA isolated from E. grandis and the pAGIBAC1 cloning vector, as described in Materials and Methods
To evaluate the average insert size of each library, BAC DNA was isolated about 384 randomly selected clones from each library, restriction enzyme digested with the rare cutter NotI, and analyzed by Pulsed-Field Gel Electrophoresis (PFGE)
Summary
Eucalyptus species are among the most planted hardwoods in the world because of their rapid growth, adaptability and valuable wood properties. Bacterial artificial chromosome (BAC) libraries are key genomic tools that enable positional cloning of important traits, synteny evaluation, and the development of genome framework physical maps for genetic linkage and genome sequencing Renowned for their fast growth, valuable wood properties and wide adaptability, Eucalyptus species are among the most planted hardwoods in the world. The genus Eucalyptus includes over 700 species [1], including the most planted species E. grandis and E. urophylla (section Transversaria), E. globulus (section Maidenaria) hybrids are among the most widely used for industrial plantations because of their rapid growth rate, their adaptability to diverse ecological conditions and their good quality wood fiber. The complete nucleotide sequence of the chloroplast genome from E. globulus is available [17], and is similar to other angiosperms, with an inverted repeat (IR) separated by a large single copy (LCS) region, and a small single copy (SSC) region
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