Decoding the massive genome of loblolly pine using haploid DNA and novel assembly strategies.

BACKGROUND: The size and complexity of conifer genomes has, until now, prevented full genome sequencing and assembly. The large research community and economic importance of loblolly pine, Pinus taeda L., made it an early candidate for reference sequence determination. RESULTS: We develop a novel strategy to sequence the genome of loblolly pine that combines unique aspects of pine reproductive biology and genome assembly methodology. We use a whole genome shotgun approach relying primarily on next generation sequence generated from a single haploid seed megagametophyte from a loblolly pine tree, 20-1010, that has been used in industrial forest tree breeding. The resulting sequence and assembly was used to generate a draft genome spanning 23.2 Gbp and containing 20.1 Gbp with an N50 scaffold size of 66.9 kbp, making it a significant improvement over available conifer genomes. The long scaffold lengths allow the annotation of 50,172 gene models with intron lengths averaging over 2.7 kbp and sometimes exceeding 100 kbp in length. Analysis of orthologous gene sets identifies gene families that may be unique to conifers. We further characterize and expand the existing repeat library based on the de novo analysis of the repetitive content, estimated to encompass 82% of the genome. CONCLUSIONS: In addition to its value as a resource for researchers and breeders, the loblolly pine genome sequence and assembly reported here demonstrates a novel approach to sequencing the large and complex genomes of this important group of plants that can now be widely applied.

Unique features of the loblolly pine (Pinus taeda L.) megagenome revealed through sequence annotation.

The largest genus in the conifer family Pinaceae is Pinus, with over 100 species. The size and complexity of their genomes (∼20-40 Gb, 2n = 24) have delayed the arrival of a well-annotated reference sequence. In this study, we present the annotation of the first whole-genome shotgun assembly of loblolly pine (Pinus taeda L.), which comprises 20.1 Gb of sequence. The MAKER-P annotation pipeline combined evidence-based alignments and ab initio predictions to generate 50,172 gene models, of which 15,653 are classified as high confidence. Clustering these gene models with 13 other plant species resulted in 20,646 gene families, of which 1554 are predicted to be unique to conifers. Among the conifer gene families, 159 are composed exclusively of loblolly pine members. The gene models for loblolly pine have the highest median and mean intron lengths of 24 fully sequenced plant genomes. Conifer genomes are full of repetitive DNA, with the most significant contributions from long-terminal-repeat retrotransposons. In depth analysis of the tandem and interspersed repetitive content yielded a combined estimate of 82%.

Marking embryonic stem cells with a 2A self-cleaving peptide: a NKX2-5 emerald GFP BAC reporter.

BACKGROUND: Fluorescent reporters are useful for assaying gene expression in living cells and for identifying and isolating pure cell populations from heterogeneous cultures, including embryonic stem (ES) cells. Multiple fluorophores and genetic selection markers exist; however, a system for creating reporter constructs that preserve the regulatory sequences near a gene's native ATG start site has not been widely available. METHODOLOGY: Here, we describe a series of modular marker plasmids containing independent reporter, bacterial selection, and eukaryotic selection components, compatible with both Gateway recombination and lambda prophage bacterial artificial chromosome (BAC) recombineering techniques. A 2A self-cleaving peptide links the reporter to the native open reading frame. We use an emerald GFP marker cassette to create a human BAC reporter and ES cell reporter line for the early cardiac marker NKX2-5. NKX2-5 expression was detected in differentiating mouse ES cells and ES cell-derived mice. CONCLUSIONS: Our results describe a NKX2-5 ES cell reporter line for studying early events in cardiomyocyte formation. The results also demonstrate that our modular marker plasmids could be used for generating reporters from unmodified BACs, potentially as part of an ES cell reporter library.

Genome sequence, comparative analysis and haplotype structure of the domestic dog.

Here we report a high-quality draft genome sequence of the domestic dog (Canis familiaris), together with a dense map of single nucleotide polymorphisms (SNPs) across breeds. The dog is of particular interest because it provides important evolutionary information and because existing breeds show great phenotypic diversity for morphological, physiological and behavioural traits. We use sequence comparison with the primate and rodent lineages to shed light on the structure and evolution of genomes and genes. Notably, the majority of the most highly conserved non-coding sequences in mammalian genomes are clustered near a small subset of genes with important roles in development. Analysis of SNPs reveals long-range haplotypes across the entire dog genome, and defines the nature of genetic diversity within and across breeds. The current SNP map now makes it possible for genome-wide association studies to identify genes responsible for diseases and traits, with important consequences for human and companion animal health.

1-Mb resolution array-based comparative genomic hybridization using a BAC clone set optimized for cancer gene analysis.

Array-based comparative genomic hybridization (aCGH) is a recently developed tool for genome-wide determination of DNA copy number alterations. This technology has tremendous potential for disease-gene discovery in cancer and developmental disorders as well as numerous other applications. However, widespread utilization of a CGH has been limited by the lack of well characterized, high-resolution clone sets optimized for consistent performance in aCGH assays and specifically designed analytic software. We have assembled a set of approximately 4100 publicly available human bacterial artificial chromosome (BAC) clones evenly spaced at approximately 1-Mb resolution across the genome, which includes direct coverage of approximately 400 known cancer genes. This aCGH-optimized clone set was compiled from five existing sets, experimentally refined, and supplemented for higher resolution and enhancing mapping capabilities. This clone set is associated with a public online resource containing detailed clone mapping data, protocols for the construction and use of arrays, and a suite of analytical software tools designed specifically for aCGH analysis. These resources should greatly facilitate the use of aCGH in gene discovery.

Biomedical applications and studies of molecular evolution: a proposal for a primate genomic library resource.

The anticipated completion of two of the most biomedically relevant genomes, mouse and human, within the next three years provides an unparalleled opportunity for the large-scale exploration of genome evolution. Targeted sequencing of genomic regions in a panel of primate species and comparison to reference genomes will provide critical insight into the nature of single-base pair variation, mechanisms of chromosomal rearrangement, patterns of selection, and species adaptation. Although not recognized as model "genetic organisms" because of their longevity and low fecundity, 30 of the approximately 300 primate species are targets of biomedical research. The existence of a human reference sequence and genomic primate BAC libraries greatly facilitates the recovery of genes/genomic regions of high biological interest because of an estimated maximum neutral nucleotide sequence divergence of 25%. Primate species, therefore, may be regarded as the ideal model "genomic organisms". Based on existing BAC library resources, we propose the construction of a panel of primate BAC libraries from phylogenetic anchor species for the purpose of comparative medicine as well as studies of genome evolution.