DoMY-Seq: A yeast two-hybrid-based technique for precision mapping of protein-protein interaction motifs.

Interactions between proteins are fundamental for every biological process and especially important in cell signaling pathways. Biochemical techniques that evaluate these protein-protein interactions (PPIs), such as in vitro pull downs and coimmunoprecipitations, have become popular in most laboratories and are essential to identify and validate novel protein binding partners. Most PPIs occur through small domains or motifs, which are challenging and laborious to map by using standard biochemical approaches because they generally require the cloning of several truncation mutants. Moreover, these classical methodologies provide limited resolution of the interacting interface. Here, we describe the development of an alternative technique to overcome these limitations termed "Protein Domain mapping using Yeast 2 Hybrid-Next Generation Sequencing" (DoMY-Seq), which leverages both yeast two-hybrid and next-generation sequencing techniques. In brief, our approach involves creating a library of fragments derived from an open reading frame of interest and enriching for the interacting fragments using a yeast two-hybrid reporter system. Next-generation sequencing is then subsequently employed to read and map the sequence of the interacting fragment, yielding a high-resolution plot of the binding interface. We optimized DoMY-Seq by taking advantage of the well-described and high-affinity interaction between KRAS and CRAF, and we provide high-resolution domain mapping on this and other protein-interacting pairs, including CRAF-MEK1, RIT1-RGL3, and p53-MDM2. Thus, DoMY-Seq provides an unbiased alternative method to rapidly identify the domains involved in PPIs by advancing the use of yeast two-hybrid technology.

Sequence of the Sugar Pine Megagenome.

Until very recently, complete characterization of the megagenomes of conifers has remained elusive. The diploid genome of sugar pine (Pinus lambertiana Dougl.) has a highly repetitive, 31 billion bp genome. It is the largest genome sequenced and assembled to date, and the first from the subgenus Strobus, or white pines, a group that is notable for having the largest genomes among the pines. The genome represents a unique opportunity to investigate genome "obesity" in conifers and white pines. Comparative analysis of P. lambertiana and P. taeda L. reveals new insights on the conservation, age, and diversity of the highly abundant transposable elements, the primary factor determining genome size. Like most North American white pines, the principal pathogen of P. lambertiana is white pine blister rust (Cronartium ribicola J.C. Fischer ex Raben.). Identification of candidate genes for resistance to this pathogen is of great ecological importance. The genome sequence afforded us the opportunity to make substantial progress on locating the major dominant gene for simple resistance hypersensitive response, Cr1 We describe new markers and gene annotation that are both tightly linked to Cr1 in a mapping population, and associated with Cr1 in unrelated sugar pine individuals sampled throughout the species' range, creating a solid foundation for future mapping. This genomic variation and annotated candidate genes characterized in our study of the Cr1 region are resources for future marker-assisted breeding efforts as well as for investigations of fundamental mechanisms of invasive disease and evolutionary response.

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.

Sequencing and assembly of the 22-gb loblolly pine genome.

Conifers are the predominant gymnosperm. The size and complexity of their genomes has presented formidable technical challenges for whole-genome shotgun sequencing and assembly. We employed novel strategies that allowed us to determine the loblolly pine (Pinus taeda) reference genome sequence, the largest genome assembled to date. Most of the sequence data were derived from whole-genome shotgun sequencing of a single megagametophyte, the haploid tissue of a single pine seed. Although that constrained the quantity of available DNA, the resulting haploid sequence data were well-suited for assembly. The haploid sequence was augmented with multiple linking long-fragment mate pair libraries from the parental diploid DNA. For the longest fragments, we used novel fosmid DiTag libraries. Sequences from the linking libraries that did not match the megagametophyte were identified and removed. Assembly of the sequence data were aided by condensing the enormous number of paired-end reads into a much smaller set of longer "super-reads," rendering subsequent assembly with an overlap-based assembly algorithm computationally feasible. To further improve the contiguity and biological utility of the genome sequence, additional scaffolding methods utilizing independent genome and transcriptome assemblies were implemented. The combination of these strategies resulted in a draft genome sequence of 20.15 billion bases, with an N50 scaffold size of 66.9 kbp.

Insights into the loblolly pine genome: characterization of BAC and fosmid sequences.

Despite their prevalence and importance, the genome sequences of loblolly pine, Norway spruce, and white spruce, three ecologically and economically important conifer species, are just becoming available to the research community. Following the completion of these large assemblies, annotation efforts will be undertaken to characterize the reference sequences. Accurate annotation of these ancient genomes would be aided by a comprehensive repeat library; however, few studies have generated enough sequence to fully evaluate and catalog their non-genic content. In this paper, two sets of loblolly pine genomic sequence, 103 previously assembled BACs and 90,954 newly sequenced and assembled fosmid scaffolds, were analyzed. Together, this sequence represents 280 Mbp (roughly 1% of the loblolly pine genome) and one of the most comprehensive studies of repetitive elements and genes in a gymnosperm species. A combination of homology and de novo methodologies were applied to identify both conserved and novel repeats. Similarity analysis estimated a repetitive content of 27% that included both full and partial elements. When combined with the de novo investigation, the estimate increased to almost 86%. Over 60% of the repetitive sequence consists of full or partial LTR (long terminal repeat) retrotransposons. Through de novo approaches, 6,270 novel, full-length transposable element families and 9,415 sub-families were identified. Among those 6,270 families, 82% were annotated as single-copy. Several of the novel, high-copy families are described here, with the largest, PtPiedmont, comprising 133 full-length copies. In addition to repeats, analysis of the coding region reported 23 full-length eukaryotic orthologous proteins (KOGS) and another 29 novel or orthologous genes. These discoveries, along with other genomic resources, will be used to annotate conifer genomes and address long-standing questions about gymnosperm evolution.