Christopher J. Grassa

The genome of flax (Linum usitatissimum) assembled de novo from short shotgun sequence reads

Flax (Linum usitatissimum) is an ancient crop that is widely cultivated as a source of fiber, oil and medicinally relevant compounds. To accelerate crop improvement, we performed whole-genome shotgun sequencing of the nuclear genome of flax. Seven paired-end libraries ranging in size from 300 bp to 10 kb were sequenced using an Illumina genome analyzer. A de novo assembly, comprised exclusively of deep-coverage (approximately 94× raw, approximately 69× filtered) short-sequence reads (44-100 bp), produced a set of scaffolds with N(50) =694 kb, including contigs with N(50)=20.1 kb. The contig assembly contained 302 Mb of non-redundant sequence representing an estimated 81% genome coverage. Up to 96% of published flax ESTs aligned to the whole-genome shotgun scaffolds. However, comparisons with independently sequenced BACs and fosmids showed some mis-assembly of regions at the genome scale. A total of 43384 protein-coding genes were predicted in the whole-genome shotgun assembly, and up to 93% of published flax ESTs, and 86% of A. thaliana genes aligned to these predicted genes, indicating excellent coverage and accuracy at the gene level. Analysis of the synonymous substitution rates (K(s) ) observed within duplicate gene pairs was consistent with a recent (5-9 MYA) whole-genome duplication in flax. Within the predicted proteome, we observed enrichment of many conserved domains (Pfam-A) that may contribute to the unique properties of this crop, including agglutinin proteins. Together these results show that de novo assembly, based solely on whole-genome shotgun short-sequence reads, is an efficient means of obtaining nearly complete genome sequence information for some plant species.

A 34K SNP genotyping array for Populus trichocarpa: design, application to the study of natural populations and transferability to other Populus species.

Genetic mapping of quantitative traits requires genotypic data for large numbers of markers in many individuals. For such studies, the use of large single nucleotide polymorphism (SNP) genotyping arrays still offers the most cost‐effective solution. Herein we report on the design and performance of a SNP genotyping array for Populus trichocarpa (black cottonwood). This genotyping array was designed with SNPs pre‐ascertained in 34 wild accessions covering most of the species latitudinal range. We adopted a candidate gene approach to the array design that resulted in the selection of 34 131 SNPs, the majority of which are located in, or within 2 kb of, 3543 candidate genes. A subset of the SNPs on the array (539) was selected based on patterns of variation among the SNP discovery accessions. We show that more than 95% of the loci produce high quality genotypes and that the genotyping error rate for these is likely below 2%. We demonstrate that even among small numbers of samples (n = 10) from local populations over 84% of loci are polymorphic. We also tested the applicability of the array to other species in the genus and found that the number of polymorphic loci decreases rapidly with genetic distance, with the largest numbers detected in other species in section Tacamahaca. Finally, we provide evidence for the utility of the array to address evolutionary questions such as intraspecific studies of genetic differentiation, species assignment and the detection of natural hybrids.

LANDSCAPE GENOMICS OF POPULUS TRICHOCARPA: THE ROLE OF HYBRIDIZATION, LIMITED GENE FLOW, AND NATURAL SELECTION IN SHAPING PATTERNS OF POPULATION STRUCTURE

Populus trichocarpa is an ecologically important tree across western North America. We used a large population sample of 498 accessions over a wide geographical area genotyped with a 34K Populus SNP array to quantify geographical patterns of genetic variation in this species (landscape genomics). We present evidence that three processes contribute to the observed patterns: (1) introgression from the sister species P. balsamifera, (2) isolation by distance (IBD), and (3) natural selection. Introgression was detected only at the margins of the species’ distribution. IBD was significant across the sampled area as a whole, but no evidence of restricted gene flow was detected in a core of drainages from southern British Columbia (BC). We identified a large number of FST outliers. Gene Ontology analyses revealed that FST outliers are overrepresented in genes involved in circadian rhythm and response to red/far‐red light when the entire dataset is considered, whereas in southern BC heat response genes are overrepresented. We also identified strong correlations between geoclimate variables and allele frequencies at FST outlier loci that provide clues regarding the selective pressures acting at these loci.

Recent nonhybrid origin of sunflower ecotypes in a novel habitat.

The genomics of local adaptation is an increasingly active field, providing insights into the forces driving ecological speciation and the repeatability of evolution. Demography and gene flow play an important role in determining the paths by which parallel evolution occurs and the genomic signatures of adaptation. In the annual sunflowers, hybridization between species has repeatedly led to the colonization of extreme habitats, such as sand dunes. In a new case of adaptation to sand dunes that occurs in populations of H. petiolaris growing at Great Sand Dunes National Park and Preserve (Colorado), we wished to determine the age and long‐term migration patterns of the system, as well as its ancestry. We addressed these questions with restriction‐associated DNA (RAD) sequence data, aligned to a reference transcriptome. In an isolation with migration model using RAD sequences, coalescent analysis showed that the dune ecotype originated since the last ice age, which is very recent compared with the hybrid dune species, H. anomalus. Large effective population sizes and substantial numbers of gene migrants per generation between dune and nondune ecotypes explained the highly heterogeneous divergence observed among loci. Analysis of RAD‐derived SNPs identified heterogeneous divergence between the dune and nondune ecotypes, as well as identifying its nearest relative. Our results did not support the hypothesis that the dune ecotype has hybrid ancestry, suggesting that adaptation of sunflowers to dunes has occurred by multiple mechanisms. The ancestry and long‐term history of gene flow between incipient sunflower species provides valuable context for our understanding of ecological speciation and parallel adaptation.

The Population Genomics of Sunflowers and Genomic Determinants of Protein Evolution Revealed by RNAseq

Few studies have investigated the causes of evolutionary rate variation among plant nuclear genes, especially in recently diverged species still capable of hybridizing in the wild. The recent advent of Next Generation Sequencing (NGS) permits investigation of genome wide rates of protein evolution and the role of selection in generating and maintaining divergence. Here, we use individual whole-transcriptome sequencing (RNAseq) to refine our understanding of the population genomics of wild species of sunflowers (Helianthus spp.) and the factors that affect rates of protein evolution. We aligned 35 GB of transcriptome sequencing data and identified 433,257 polymorphic sites (SNPs) in a reference transcriptome comprising 16,312 genes. Using SNP markers, we identified strong population clustering largely corresponding to the three species analyzed here (Helianthus annuus, H. petiolaris, H. debilis), with one distinct early generation hybrid. Then, we calculated the proportions of adaptive substitution fixed by selection (alpha) and identified gene ontology categories with elevated values of alpha. The “response to biotic stimulus” category had the highest mean alpha across the three interspecific comparisons, implying that natural selection imposed by other organisms plays an important role in driving protein evolution in wild sunflowers. Finally, we examined the relationship between protein evolution (dN/dS ratio) and several genomic factors predicted to co-vary with protein evolution (gene expression level, divergence and specificity, genetic divergence [FST], and nucleotide diversity pi). We find that variation in rates of protein divergence was correlated with gene expression level and specificity, consistent with results from a broad range of taxa and timescales. This would in turn imply that these factors govern protein evolution both at a microevolutionary and macroevolutionary timescale. Our results contribute to a general understanding of the determinants of rates of protein evolution and the impact of selection on patterns of polymorphism and divergence.

Complete Genome Sequence of Haemophilus influenzae Strain 375 from the Middle Ear of a Pediatric Patient with Otitis Media

ABSTRACT Originally isolated from a pediatric patient with otitis media, Haemophilus influenzae strain 375 (Hi375) has been extensively studied as a model system for intracellular invasion of airway epithelial cells and other pathogenesis traits. Here, we report its complete genome sequence and methylome.

Draft Genome Sequence of Exiguobacterium pavilionensis Strain RW-2, with Wide Thermal, Salinity, and pH Tolerance, Isolated from Modern Freshwater Microbialites

ABSTRACT We report the draft genome sequence of Exiguobacterium pavilionensis strain RW-2, isolated from a cold thrombolytic microbialite. The isolate grows at temperatures from 4 to 50°C, at pH levels from 5 to 11, and in media without added NaCl or KCl or with 7% added NaCl.

Complete Mitochondrial Genome Sequence of Sunflower (Helianthus annuus L.)

ABSTRACT This is the first complete mitochondrial genome sequence for sunflower and the first complete mitochondrial genome for any member of Asteraceae, the largest plant family, which includes over 23,000 named species. The master circle is 300,945-bp long and includes 27 protein-coding sequences, 18 tRNAs, and the 26S, 5S, and 18S rRNAs.

First Draft Genome Sequence from a Member of the Genus Agrococcus, Isolated from Modern Microbialites

ABSTRACT We report the first draft genome sequence from a member of the genus Agrococcus, isolated from cold thrombolytic microbialites within Pavilion Lake, British Columbia, Canada. The draft genome assembly for Agrococcus pavilionensis strain RW-1 has a size of 2,878,403 bp with a G+C content of 72.56%.

Complete plastid genome assembly of invasive plant, Centaurea diffusa

Invasive plants present both problems and possibilities for discovery, which may be addressed utilizing new genomic tools. Here we present the completed plastome assembly for the problematic invasive weed, Centaurea diffusa. This new tool represents a significant contribution to future studies of the ecological genomics of invasive plants, particularly this weedy genus, and studies of the Asteraceae in general.