A genome sequence for the threatened whitebark pine.

Whitebark pine (WBP, Pinus albicaulis) is a white pine of subalpine regions in the Western contiguous United States and Canada. WBP has become critically threatened throughout a significant part of its natural range due to mortality from the introduced fungal pathogen white pine blister rust (WPBR, Cronartium ribicola) and additional threats from mountain pine beetle (Dendroctonus ponderosae), wildfire, and maladaptation due to changing climate. Vast acreages of WBP have suffered nearly complete mortality. Genomic technologies can contribute to a faster, more cost-effective approach to the traditional practices of identifying disease-resistant, climate-adapted seed sources for restoration. With deep-coverage Illumina short reads of haploid megagametophyte tissue and Oxford Nanopore long reads of diploid needle tissue, followed by a hybrid, multistep assembly approach, we produced a final assembly containing 27.6 Gb of sequence in 92,740 contigs (N50 537,007 bp) and 34,716 scaffolds (N50 2.0 Gb). Approximately 87.2% (24.0 Gb) of total sequence was placed on the 12 WBP chromosomes. Annotation yielded 25,362 protein-coding genes, and over 77% of the genome was characterized as repeats. WBP has demonstrated the greatest variation in resistance to WPBR among the North American white pines. Candidate genes for quantitative resistance include disease resistance genes known as nucleotide-binding leucine-rich repeat receptors (NLRs). A combination of protein domain alignments and direct genome scanning was employed to fully describe the 3 subclasses of NLRs. Our high-quality reference sequence and annotation provide a marked improvement in NLR identification compared to previous assessments that leveraged de novo-assembled transcriptomes.

A Genome Sequence for the Threatened Whitebark Pine.

Whitebark pine (WBP, Pinus albicaulis ) is a white pine of subalpine regions in western contiguous US and Canada. WBP has become critically threatened throughout a significant part of its natural range due to mortality from the introduced fungal pathogen white pine blister rust (WPBR, Cronartium ribicola ) and additional threats from mountain pine beetle ( Dendroctonus ponderosae ), wildfire, and maladaptation due to changing climate. Vast acreages of WBP have suffered nearly complete mortality. Genomic technologies can contribute to a faster, more cost-effective approach to the traditional practices of identifying disease-resistant, climate-adapted seed sources for restoration. With deep-coverage Illumina short-reads of haploid megametophyte tissue and Oxford Nanopore long-reads of diploid needle tissue, followed by a hybrid, multistep assembly approach, we produced a final assembly containing 27.6 Gbp of sequence in 92,740 contigs (N50 537,007 bp) and 34,716 scaffolds (N50 2.0 Gbp). Approximately 87.2% (24.0 Gbp) of total sequence was placed on the twelve WBP chromosomes. Annotation yielded 25,362 protein-coding genes, and over 77% of the genome was characterized as repeats. WBP has demonstrated the greatest variation in resistance to WPBR among the North American white pines. Candidate genes for quantitative resistance include disease resistance genes known as nucleotide-binding leucine-rich-repeat receptors (NLRs). A combination of protein domain alignments and direct genome scanning was employed to fully describe the three subclasses of NLRs (TNL, CNL, RNL). Our high-quality reference sequence and annotation provide a marked improvement in NLR identification compared to previous assessments that leveraged de novo assembled transcriptomes.

Assembled and annotated 26.5 Gbp coast redwood genome: a resource for estimating evolutionary adaptive potential and investigating hexaploid origin.

Sequencing, assembly, and annotation of the 26.5 Gbp hexaploid genome of coast redwood (Sequoia sempervirens) was completed leading toward discovery of genes related to climate adaptation and investigation of the origin of the hexaploid genome. Deep-coverage short-read Illumina sequencing data from haploid tissue from a single seed were combined with long-read Oxford Nanopore Technologies sequencing data from diploid needle tissue to create an initial assembly, which was then scaffolded using proximity ligation data to produce a highly contiguous final assembly, SESE 2.1, with a scaffold N50 size of 44.9 Mbp. The assembly included several scaffolds that span entire chromosome arms, confirmed by the presence of telomere and centromere sequences on the ends of the scaffolds. The structural annotation produced 118,906 genes with 113 containing introns that exceed 500 Kbp in length and one reaching 2 Mb. Nearly 19 Gbp of the genome represented repetitive content with the vast majority characterized as long terminal repeats, with a 2.9:1 ratio of Copia to Gypsy elements that may aid in gene expression control. Comparison of coast redwood to other conifers revealed species-specific expansions for a plethora of abiotic and biotic stress response genes, including those involved in fungal disease resistance, detoxification, and physical injury/structural remodeling and others supporting flavonoid biosynthesis. Analysis of multiple genes that exist in triplicate in coast redwood but only once in its diploid relative, giant sequoia, supports a previous hypothesis that the hexaploidy is the result of autopolyploidy rather than any hybridizations with separate but closely related conifer species.

Aegilops tauschii genome assembly Aet v5.0 features greater sequence contiguity and improved annotation.

Aegilops tauschii is the donor of the D subgenome of hexaploid wheat and an important genetic resource. The reference-quality genome sequence Aet v4.0 for Ae. tauschii acc. AL8/78 was therefore an important milestone for wheat biology and breeding. Further advances in sequencing acc. AL8/78 and release of the Aet v5.0 sequence assembly are reported here. Two new optical maps were constructed and used in the revision of pseudomolecules. Gaps were closed with Pacific Biosciences long-read contigs, decreasing the gap number by 38,899. Transposable elements and protein-coding genes were reannotated. The number of annotated high-confidence genes was reduced from 39,635 in Aet v4.0 to 32,885 in Aet v5.0. A total of 2245 biologically important genes, including those affecting plant phenology, grain quality, and tolerance of abiotic stresses in wheat, was manually annotated and disease-resistance genes were annotated by a dedicated pipeline. Disease-resistance genes encoding nucleotide-binding site domains, receptor-like protein kinases, and receptor-like proteins were preferentially located in distal chromosome regions, whereas those encoding transmembrane coiled-coil proteins were dispersed more evenly along the chromosomes. Discovery, annotation, and expression analyses of microRNA (miRNA) precursors, mature miRNAs, and phasiRNAs are reported, including miRNA target genes. Other small RNAs, such as hc-siRNAs and tRFs, were characterized. These advances enhance the utility of the Ae. tauschii genome sequence for wheat genetics, biotechnology, and breeding.

A SNP-based genetic dissection of versatile traits in bread wheat (Triticum aestivum L.).

The continuous increase in global population prompts increased wheat production. Future wheat (Triticum aestivum L.) breeding will heavily rely on dissecting molecular and genetic bases of wheat yield and related traits which is possible through the discovery of quantitative trait loci (QTLs) in constructed populations, such as recombinant inbred lines (RILs). Here, we present an evaluation of 92 RILs in a bi-parental RIL mapping population (the International Triticeae Mapping Initiative Mapping Population [ITMI/MP]) using newly generated phenotypic data in 3-year experiments (2015), older phenotypic data (1997-2009), and newly created single nucleotide polymorphism (SNP) marker data based on 92 of the original RILs to search for novel and stable QTLs. Our analyses of more than 15 unique traits observed in multiple experiments included analyses of 46 traits in three environments in the USA, 69 traits in eight environments in Germany, 149 traits in 10 environments in Russia, and 28 traits in four environments in India (292 traits in 25 environments) with 7584 SNPs (292 × 7584 = 2 214 528 data points). A total of 874 QTLs were detected with limit of detection (LOD) scores of 2.01-3.0 and 432 QTLs were detected with LOD > 3.0. Moreover, 769 QTLs could be assigned to 183 clusters based on the common markers and relative proximity of related QTLs, indicating gene-rich regions throughout the A, B, and D genomes of common wheat. This upgraded genotype-phenotype information of ITMI/MP can assist breeders and geneticists who can make crosses with suitable RILs to improve or investigate traits of interest.

Introgression of perennial growth habit from Lophopyrum elongatum into wheat.

KEY MESSAGE: A locus for perennial growth was mapped on Lophopyrum elongatum chromosome arm 4ES and introgressed into the wheat genome. Evidence was obtained that in addition to chromosome 4E, other L. elongatum chromosomes control perennial growth. Monocarpy versus polycarpy is one of the fundamental developmental dichotomies in flowering plants. Advances in the understanding of the genetic basis of this dichotomy are important for basic biological reasons and practically for genetic manipulation of growth development in economically important plants. Nine wheat introgression lines (ILs) harboring germplasm of the Lophopyrum elongatum genome present in the octoploid amphiploid Triticum aestivum cv. Chinese Spring (subgenomes AABBDD) × L. elongatum (genomes EE) were selected from a population of ILs developed earlier. These ILs were employed here in genomic analyses of post-sexual cycle regrowth (PSCR), which is a component of polycarpy in caespitose L. elongatum. Analyses of disomic substitution (DS) lines confirmed that L. elongatum chromosome 4E confers PSCR on wheat. The gene was mapped into a short distal region of L. elongatum arm 4ES and was tentatively named Pscr1. ILs harboring recombined chromosomes with 4ES segments, including Pscr1, incorporated into the distal part of the 4DS chromosome arm were identified. Based on the location, Pscr1 is not orthologous with the rice rhizome-development gene Rhz2 located on rice chromosome Os3, which is homoeologous with chromosome 4E, but it may correspond to the Teosinte branched1 (TB1) gene, which is located in the introgressed region in the L. elongatum and Ae. tauschii genomes. A hexaploid IL harboring a large portion of the E-genome but devoid of chromosome 4E also expressed PSCR, which provided evidence that perennial growth is controlled by genes on other L. elongatum chromosomes in addition to 4E.

Genome-wide introgression from a bread wheat × Lophopyrum elongatum amphiploid into wheat.

KEY MESSAGE: We introgressed wheatgrass germplasm from the octoploid amphiploid Triticum aestivum× Lophopyrum elongatum into wheat by manipulating the wheat Ph1 gene and discovered and characterized 130 introgression lines harboring single or, in various combinations, complete and recombined L. elongatum chromosomes. Diploid wheatgrass Lophopyrum elongatum (genomes EE) possesses valuable traits for wheat genetics and breeding. We evaluated several strategies for introgression of this germplasm into wheat. To detect it, we developed and validated multiplexed sets of Sequenom MassARRAY single nucleotide polymorphism (SNP) markers, which differentiated disomic and monosomic L. elongatum chromosomes from wheat chromosomes. We identified 130 introgression lines (ILs), which harbored 108 complete and 89 recombined L. elongatum chromosomes. Of the latter, 59 chromosomes were recombined by one or more crossovers and 30 were involved in centromeric (Robertsonian) translocations or were telocentric. To identify wheat chromosomes substituted for or recombined with L. elongatum chromosomes, we genotyped the ILs with the wheat 90-K Infinium SNP array. We found that most of the wheat 90-K probes correctly detected their targets in the L. elongatum genome and showed that some wheat SNPs are ancient and had originated prior to the divergence of the wheat and L. elongatum lineages. Of the 130 ILs, 52% were homozygous for Ph1 deletion and thus are staged to be recombined further. We failed to detect in the L. elongatum genome the 4/5 reciprocal translocation that has been reported in Thinopyrum bessarabicum and several other Triticeae genomes.

Correction to: Recombination between homoeologous chromosomes induced in durum wheat by the Aegilops speltoides Su1-Ph1 suppressor.

Unfortunately, the 9th author name was incorrectly published in the original publication. The complete correct name is given below.

Recombination between homoeologous chromosomes induced in durum wheat by the Aegilops speltoides Su1-Ph1 suppressor.

KEY MESSAGE: Su1-Ph1, which we previously introgressed into wheat from Aegilops speltoides, is a potent suppressor of Ph1 and a valuable tool for gene introgression in tetraploid wheat. We previously introgressed Su1-Ph1, a suppressor of the wheat Ph1 gene, from Aegilops speltoides into durum wheat cv Langdon (LDN). Here, we evaluated the utility of the introgressed suppressor for inducing introgression of alien germplasm into durum wheat. We built LDN plants heterozygous for Su1-Ph1 that simultaneously contained a single LDN chromosome 5B and a single Ae. searsii chromosome 5Sse, which targeted them for recombination. We genotyped 28 BC1F1 and 84 F2 progeny with the wheat 90-K Illumina single-nucleotide polymorphism assay and detected extensive recombination between the two chromosomes, which we confirmed by non-denaturing fluorescence in situ hybridization (ND-FISH). We constructed BC1F1 and F2 genetic maps that were 65.31 and 63.71 cM long, respectively. Recombination rates between the 5B and 5Sse chromosomes were double the expected rate computed from their meiotic pairing, which we attributed to selection against aneuploid gametes. Recombination rate between 5B and 5Sse was depressed compared to that between 5B chromosomes in the proximal region of the long arm. We integrated ND-FISH signals into the genetic map and constructed a physical map, which we used to map a 172,188,453-bp Ph1 region. Despite the location of the region in a low-recombination region of the 5B chromosome, we detected three crossovers in it. Our data show that Su1-Ph1 is a valuable tool for gene introgression and gene mapping based on recombination between homoeologous chromosomes in wheat.

Sequencing a Juglans regia × J. microcarpa hybrid yields high-quality genome assemblies of parental species.

Members of the genus Juglans are monecious wind-pollinated trees in the family Juglandaceae with highly heterozygous genomes, which greatly complicates genome sequence assembly. The genomes of interspecific hybrids are usually comprised of haploid genomes of parental species. We exploited this attribute of interspecific hybrids to avoid heterozygosity and sequenced an interspecific hybrid Juglans microcarpa × J. regia using a novel combination of single-molecule sequencing and optical genome mapping technologies. The resulting assemblies of both genomes were remarkably complete including chromosome termini and centromere regions. Chromosome termini consisted of arrays of telomeric repeats about 8 kb long and heterochromatic subtelomeric regions about 10 kb long. The centromeres consisted of arrays of a centromere-specific Gypsy retrotransposon and most contained genes, many of them transcribed. Juglans genomes evolved by a whole-genome-duplication dating back to the Cretaceous-Paleogene boundary and consist of two subgenomes, which were fractionated by numerous short gene deletions evenly distributed along the length of the chromosomes. Fractionation was shown to be asymmetric with one subgenome exhibiting greater gene loss than the other. The asymmetry of the process is ongoing and mirrors an asymmetry in gene expression between the subgenomes. Given the importance of J. microcarpa × J. regia hybrids as potential walnut rootstocks, we catalogued disease resistance genes in the parental genomes and studied their chromosomal distribution. We also estimated the molecular clock rates for woody perennials and deployed them in estimating divergence times of Juglans genomes and those of other woody perennials.

Aegilops tauschii Genome Sequence: A Framework for Meta-analysis of Wheat QTLs.

Numerous quantitative trait loci (QTL) have been mapped in tetraploid and hexaploid wheat and wheat relatives, mostly with simple sequence repeat (SSR) or single nucleotide polymorphism (SNP) markers. To conduct meta-analysis of QTL requires projecting them onto a common genomic framework, either a consensus genetic map or genomic sequence. The latter strategy is pursued here. Of 774 QTL mapped in wheat and wheat relatives found in the literature, 585 (75.6%) were successfully projected onto the Aegilops tauschii pseudomolecules. QTL mapped with SNP markers were more successfully projected (92.2%) than those mapped with SSR markers (66.2%). The QTL were not distributed homogeneously along chromosome arms. Their frequencies increased in the proximal-to-distal direction but declined in the most distal regions and were weakly correlated with recombination rates along the chromosome arms. Databases for projected SSR markers and QTL were constructed and incorporated into the Ae. tauschii JBrowse. To facilitate meta-QTL analysis, eight clusters of QTL were used to estimate standard deviations ([Formula: see text]) of independently mapped QTL projected onto the Ae. tauschii genome sequence. The standard deviations [Formula: see text] were modeled as an exponential decay function of recombination rates along the Ae. tauschii chromosomes. We implemented four hypothesis tests for determining the membership of query QTL. The hypothesis tests and estimation procedure for [Formula: see text] were implemented in a web portal for meta-analysis of projected QTL. Twenty-one QTL for Fusarium head blight resistance mapped on wheat chromosomes 3A, 3B, and 3D were analyzed to illustrate the use of the portal for meta-QTL analyses.

Improved Genome Sequence of Wild Emmer Wheat Zavitan with the Aid of Optical Maps.

Wild emmer (Triticum turgidum ssp. dicoccoides) is the progenitor of all modern cultivated tetraploid wheat. Its genome is large (> 10 Gb) and contains over 80% repeated sequences. The successful whole-genome-shotgun assembly of the wild emmer (accession Zavitan) genome sequence (WEW_v1.0) was an important milestone for wheat genomics. In an effort to improve this assembly, an optical map of accession Zavitan was constructed using Bionano Direct Label and Stain (DLS) technology. The map spanned 10.4 Gb. This map and another map produced earlier by us with the Bionano's Nick Label Repair and Stain (NLRS) technology were used to improve the current wild emmer assembly. The WEW_v1.0 assembly consisted of 151,912 scaffolds. Of them, 3,102 could be confidently aligned on the optical maps. Forty-seven were chimeric. They were disjoined and new scaffolds were assembled with the aid of the optical maps. The total number of scaffolds was reduced from 151,912 to 149,252 and N50 increased from 6.96 Mb to 72.63 Mb. Of the 149,252 scaffolds, 485 scaffolds, which accounted for 97% of the total genome length, were aligned and oriented on genetic maps, and new WEW_v2.0 pseudomolecules were constructed. The new pseudomolecules included 333 scaffolds (68.51 Mb) which were originally unassigned, 226 scaffolds (554.84 Mb) were placed into new locations, and 332 scaffolds (394.83 Mb) were re-oriented. The improved wild emmer genome assembly is an important resource for understanding genomic modification that occurred by domestication.

Reassessment of the evolution of wheat chromosomes 4A, 5A, and 7B.

KEY MESSAGE: Comparison of genome sequences of wild emmer wheat and Aegilops tauschii suggests a novel scenario of the evolution of rearranged wheat chromosomes 4A, 5A, and 7B. Past research suggested that wheat chromosome 4A was subjected to a reciprocal translocation T(4AL;5AL)1 that occurred in the diploid progenitor of the wheat A subgenome and to three major rearrangements that occurred in polyploid wheat: pericentric inversion Inv(4AS;4AL)1, paracentric inversion Inv(4AL;4AL)1, and reciprocal translocation T(4AL;7BS)1. Gene collinearity along the pseudomolecules of tetraploid wild emmer wheat (Triticum turgidum ssp. dicoccoides, subgenomes AABB) and diploid Aegilops tauschii (genomes DD) was employed to confirm these rearrangements and to analyze the breakpoints. The exchange of distal regions of chromosome arms 4AS and 4AL due to pericentric inversion Inv(4AS;4AL)1 was detected, and breakpoints were validated with an optical Bionano genome map. Both breakpoints contained satellite DNA. The breakpoints of reciprocal translocation T(4AL;7BS)1 were also found. However, the breakpoints that generated paracentric inversion Inv(4AL;4AL)1 appeared to be collocated with the 4AL breakpoints that had produced Inv(4AS;4AL)1 and T(4AL;7BS)1. Inv(4AS;4AL)1, Inv(4AL;4AL)1, and T(4AL;7BS)1 either originated sequentially, and Inv(4AL;4AL)1 was produced by recurrent chromosome breaks at the same breakpoints that generated Inv(4AS;4AL)1 and T(4AL;7BS)1, or Inv(4AS;4AL)1, Inv(4AL;4AL)1, and T(4AL;7BS)1 originated simultaneously. We prefer the latter hypothesis since it makes fewer assumptions about the sequence of events that produced these chromosome rearrangements.

Structural variation and rates of genome evolution in the grass family seen through comparison of sequences of genomes greatly differing in size.

Homology was searched with genes annotated in the Aegilops tauschii pseudomolecules against genes annotated in the pseudomolecules of tetraploid wild emmer wheat, Brachypodium distachyon, sorghum and rice. Similar searches were performed with genes annotated in the rice pseudomolecules. Matrices of collinear genes and rearrangements in their order were constructed. Optical BioNano genome maps were constructed and used to validate rearrangements unique to the wild emmer and Ae. tauschii genomes. Most common rearrangements were short paracentric inversions and short intrachromosomal translocations. Intrachromosomal translocations outnumbered segmental intrachromosomal duplications. The densities of paracentric inversion lengths were approximated by exponential distributions in all six genomes. Densities of collinear genes along the Ae. tauschii chromosomes were highly correlated with meiotic recombination rates but those of rearrangements were not, suggesting different causes of the erosion of gene collinearity and evolution of major chromosome rearrangements. Frequent rearrangements sharing breakpoints suggested that chromosomes have been rearranged recurrently at some sites. The distal 4 Mb of the short arms of rice chromosomes Os11 and Os12 and corresponding regions in the sorghum, B. distachyon and Triticeae genomes contain clusters of interstitial translocations including from 1 to 7 collinear genes. The rates of acquisition of major rearrangements were greater in the large wild emmer wheat and Ae. tauschii genomes than in the lineage preceding their divergence or in the B. distachyon, rice and sorghum lineages. It is suggested that synergy between large quantities of dynamic transposable elements and annual growth habit have been the primary causes of the fast evolution of the Triticeae genomes.

Genome sequence of the progenitor of the wheat D genome Aegilops tauschii.

Aegilops tauschii is the diploid progenitor of the D genome of hexaploid wheat (Triticum aestivum, genomes AABBDD) and an important genetic resource for wheat. The large size and highly repetitive nature of the Ae. tauschii genome has until now precluded the development of a reference-quality genome sequence. Here we use an array of advanced technologies, including ordered-clone genome sequencing, whole-genome shotgun sequencing, and BioNano optical genome mapping, to generate a reference-quality genome sequence for Ae. tauschii ssp. strangulata accession AL8/78, which is closely related to the wheat D genome. We show that compared to other sequenced plant genomes, including a much larger conifer genome, the Ae. tauschii genome contains unprecedented amounts of very similar repeated sequences. Our genome comparisons reveal that the Ae. tauschii genome has a greater number of dispersed duplicated genes than other sequenced genomes and its chromosomes have been structurally evolving an order of magnitude faster than those of other grass genomes. The decay of colinearity with other grass genomes correlates with recombination rates along chromosomes. We propose that the vast amounts of very similar repeated sequences cause frequent errors in recombination and lead to gene duplications and structural chromosome changes that drive fast genome evolution.

The Douglas-Fir Genome Sequence Reveals Specialization of the Photosynthetic Apparatus in Pinaceae.

A reference genome sequence for Pseudotsuga menziesii var. menziesii (Mirb.) Franco (Coastal Douglas-fir) is reported, thus providing a reference sequence for a third genus of the family Pinaceae. The contiguity and quality of the genome assembly far exceeds that of other conifer reference genome sequences (contig N50 = 44,136 bp and scaffold N50 = 340,704 bp). Incremental improvements in sequencing and assembly technologies are in part responsible for the higher quality reference genome, but it may also be due to a slightly lower exact repeat content in Douglas-fir vs. pine and spruce. Comparative genome annotation with angiosperm species reveals gene-family expansion and contraction in Douglas-fir and other conifers which may account for some of the major morphological and physiological differences between the two major plant groups. Notable differences in the size of the NDH-complex gene family and genes underlying the functional basis of shade tolerance/intolerance were observed. This reference genome sequence not only provides an important resource for Douglas-fir breeders and geneticists but also sheds additional light on the evolutionary processes that have led to the divergence of modern angiosperms from the more ancient gymnosperms.

Sequencing and comparative analyses of Aegilops tauschii chromosome arm 3DS reveal rapid evolution of Triticeae genomes.

Bread wheat (Triticum aestivum, AABBDD) is an allohexaploid species derived from two rounds of interspecific hybridizations. A high-quality genome sequence assembly of diploid Aegilops tauschii, the donor of the wheat D genome, will provide a useful platform to study polyploid wheat evolution. A combined approach of BAC pooling and next-generation sequencing technology was employed to sequence the minimum tiling path (MTP) of 3176 BAC clones from the short arm of Ae. tauschii chromosome 3 (At3DS). The final assembly of 135 super-scaffolds with an N50 of 4.2 Mb was used to build a 247-Mb pseudomolecule with a total of 2222 predicted protein-coding genes. Compared with the orthologous regions of rice, Brachypodium, and sorghum, At3DS contains 38.67% more genes. In comparison to At3DS, the short arm sequence of wheat chromosome 3B (Ta3BS) is 95-Mb large in size, which is primarily due to the expansion of the non-centromeric region, suggesting that transposable element (TE) bursts in Ta3B likely occurred there. Also, the size increase is accompanied by a proportional increase in gene number in Ta3BS. We found that in the sequence of short arm of wheat chromosome 3D (Ta3DS), there was only less than 0.27% gene loss compared to At3DS. Our study reveals divergent evolution of grass genomes and provides new insights into sequence changes in the polyploid wheat genome.

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.

Synteny analysis in Rosids with a walnut physical map reveals slow genome evolution in long-lived woody perennials.

BACKGROUND: Mutations often accompany DNA replication. Since there may be fewer cell cycles per year in the germlines of long-lived than short-lived angiosperms, the genomes of long-lived angiosperms may be diverging more slowly than those of short-lived angiosperms. Here we test this hypothesis. RESULTS: We first constructed a genetic map for walnut, a woody perennial. All linkage groups were short, and recombination rates were greatly reduced in the centromeric regions. We then used the genetic map to construct a walnut bacterial artificial chromosome (BAC) clone-based physical map, which contained 15,203 exonic BAC-end sequences, and quantified with it synteny between the walnut genome and genomes of three long-lived woody perennials, Vitis vinifera, Populus trichocarpa, and Malus domestica, and three short-lived herbs, Cucumis sativus, Medicago truncatula, and Fragaria vesca. Each measure of synteny we used showed that the genomes of woody perennials were less diverged from the walnut genome than those of herbs. We also estimated the nucleotide substitution rate at silent codon positions in the walnut lineage. It was one-fifth and one-sixth of published nucleotide substitution rates in the Medicago and Arabidopsis lineages, respectively. We uncovered a whole-genome duplication in the walnut lineage, dated it to the neighborhood of the Cretaceous-Tertiary boundary, and allocated the 16 walnut chromosomes into eight homoeologous pairs. We pointed out that during polyploidy-dysploidy cycles, the dominant tendency is to reduce the chromosome number. CONCLUSION: Slow rates of nucleotide substitution are accompanied by slow rates of synteny erosion during genome divergence in woody perennials.

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.