Sequencing and Assembly of the 22-Gb Loblolly Pine Genome

Abstract

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.

Genome sequence and genetic diversity of European ash trees

“…Analysis of gene families. The OrthoMCL pipeline (version 2.0.9) 73 was used to identify clusters of orthologous and paralogous genes from F. excelsior and the following: Amborella 74 , Arabidopsis 75 , barrel medic 76 , bladderwort 64 , coffee 65 , grape 66 , loblolly pine 77 , monkey flower 8 , poplar 78 and tomato 4 (Supplementary Table 10). Input proteomes contained a single transcript per gene and were filtered with orthomclFilterFasta to remove any sequences of fewer than ten amino acids in length and/or > 20% stop codons.…”

Genomic innovation for crop improvement

“…Another important advance has been the increase in the length of sequence reads. Single molecule, real-time sequencing has dramatically improved the contiguity and repeat representation of mammalian 29 and large pine 30 and grass 31 genomes. This has proved useful for the production of higher-quality plant-genome assemblies and has been applied to the de novo assembly of smaller plant genomes such as Arabidopsis and the grape vine Vitis vinifera cv.…”

Nitrogen Economy and Nitrogen Environmental Interactions in Conifers

“…However, only limited knowledge is currently available on the expression of key genes involved in arginine biosynthesis and the transcriptional regulation of this process in conifers. In last few years, the assembly of pine and spruce transcriptomes [36,37] and genomes have been reported [38,39] and these new resources will facilitate the identification and functional characterization of genes involved in arginine metabolism. development [36].…”