Aaron Liston

The genome of woodland strawberry ( Fragaria vesca )

The woodland strawberry, Fragaria vesca (2n = 2x = 14), is a versatile experimental plant system. This diminutive herbaceous perennial has a small genome (240 Mb), is amenable to genetic transformation and shares substantial sequence identity with the cultivated strawberry (Fragaria × ananassa) and other economically important rosaceous plants. Here we report the draft F. vesca genome, which was sequenced to ×39 coverage using second-generation technology, assembled de novo and then anchored to the genetic linkage map into seven pseudochromosomes. This diploid strawberry sequence lacks the large genome duplications seen in other rosids. Gene prediction modeling identified 34,809 genes, with most being supported by transcriptome mapping. Genes critical to valuable horticultural traits including flavor, nutritional value and flowering time were identified. Macrosyntenic relationships between Fragaria and Prunus predict a hypothetical ancestral Rosaceae genome that had nine chromosomes. New phylogenetic analysis of 154 protein-coding genes suggests that assignment of Populus to Malvidae, rather than Fabidae, is warranted.

The genome of Eucalyptus grandis

Eucalypts are the world’s most widely planted hardwood trees. Their outstanding diversity, adaptability and growth have made them a global renewable resource of fibre and energy. We sequenced and assembled >94% of the 640-megabase genome of Eucalyptus grandis. Of 36,376 predicted protein-coding genes, 34% occur in tandem duplications, the largest proportion thus far in plant genomes. Eucalyptus also shows the highest diversity of genes for specialized metabolites such as terpenes that act as chemical defence and provide unique pharmaceutical oils. Genome sequencing of the E. grandis sister species E. globulus and a set of inbred E. grandis tree genomes reveals dynamic genome evolution and hotspots of inbreeding depression. The E. grandis genome is the first reference for the eudicot order Myrtales and is placed here sister to the eurosids. This resource expands our understanding of the unique biology of large woody perennials and provides a powerful tool to accelerate comparative biology, breeding and biotechnology.

Phylogeny and classification of Pinus

We used chloroplast DNA sequences from matK and rbcL to infer the phylogeny for 101 of the approximately 111 species of Pinus (Pinaceae). At the level of subsection and above, the cpDNA tree is congruent with phylogenies based on nuclear DNA with one notable exception: cpDNA sequences from subsect. Contortae are sister to all other North American hard pines rather than occupying a more derived position in the same clade. We used the cpDNA tree plus evidence from nuclear ribosomal DNA and morphology to propose a new classification for the genus. The molecular phylogenies are symmetrical at the deepest branches of the genus, allowing for the delineation of two subgenera, each with two sections that form sister groups. Within sections, clades were slightly asymmetric and sometimes ambiguously resolved. To accomodate ambiguity in some interrelationships, avoid the creation of new ranks, and retain traditional names, we recognised up to three monophyletic subsections per section. Subgenus Pinus (the diploxylon, or hard pines) is divided into the predominantly Eurasian and Mediterranean section Pinus, composed of subsections Pinus and Pinaster, and the strictly North American section Trifoliae, composed of subsections Australes, Ponderosae, and Contortae. Subgenus Strobus (the haploxylon, or soft pines) is divided into the strictly North American section Parrya, composed of subsections Cembroides, Nelsoniae, and Balfourianae, and the Eurasian and North American section Quinquefoliae, composed of subsections Gerardianae, Krempfianae, and Strobus. Mapping of ten morphological and distributional characters indicates that two were diagnostic for infrageneric taxa: the number of vascular bundles per leaf distinguishes subgenus Pinus from subgenus Strobus, and a terminal-positioned umbo on the ovulate cone scale is diagnostic of subsect. Strobus.

Increasing phylogenetic resolution at low taxonomic levels using massively parallel sequencing of chloroplast genomes

BackgroundMolecular evolutionary studies share the common goal of elucidating historical relationships, and the common challenge of adequately sampling taxa and characters. Particularly at low taxonomic levels, recent divergence, rapid radiations, and conservative genome evolution yield limited sequence variation, and dense taxon sampling is often desirable. Recent advances in massively parallel sequencing make it possible to rapidly obtain large amounts of sequence data, and multiplexing makes extensive sampling of megabase sequences feasible. Is it possible to efficiently apply massively parallel sequencing to increase phylogenetic resolution at low taxonomic levels?ResultsWe reconstruct the infrageneric phylogeny of Pinus from 37 nearly-complete chloroplast genomes (average 109 kilobases each of an approximately 120 kilobase genome) generated using multiplexed massively parallel sequencing. 30/33 ingroup nodes resolved with ≥ 95% bootstrap support; this is a substantial improvement relative to prior studies, and shows massively parallel sequencing-based strategies can produce sufficient high quality sequence to reach support levels originally proposed for the phylogenetic bootstrap. Resampling simulations show that at least the entire plastome is necessary to fully resolve Pinus, particularly in rapidly radiating clades. Meta-analysis of 99 published infrageneric phylogenies shows that whole plastome analysis should provide similar gains across a range of plant genera. A disproportionate amount of phylogenetic information resides in two loci (ycf1, ycf2), highlighting their unusual evolutionary properties.ConclusionPlastome sequencing is now an efficient option for increasing phylogenetic resolution at lower taxonomic levels in plant phylogenetic and population genetic analyses. With continuing improvements in sequencing capacity, the strategies herein should revolutionize efforts requiring dense taxon and character sampling, such as phylogeographic analyses and species-level DNA barcoding.

Navigating the tip of the genomic iceberg: Next‐generation sequencing for plant systematics

PREMISE OF THE STUDY Just as Sanger sequencing did more than 20 years ago, next-generation sequencing (NGS) is poised to revolutionize plant systematics. By combining multiplexing approaches with NGS throughput, systematists may no longer need to choose between more taxa or more characters. Here we describe a genome skimming (shallow sequencing) approach for plant systematics. METHODS Through simulations, we evaluated optimal sequencing depth and performance of single-end and paired-end short read sequences for assembly of nuclear ribosomal DNA (rDNA) and plastomes and addressed the effect of divergence on reference-guided plastome assembly. We also used simulations to identify potential phylogenetic markers from low-copy nuclear loci at different sequencing depths. We demonstrated the utility of genome skimming through phylogenetic analysis of the Sonoran Desert clade (SDC) of Asclepias (Apocynaceae). KEY RESULTS Paired-end reads performed better than single-end reads. Minimum sequencing depths for high quality rDNA and plastome assemblies were 40× and 30×, respectively. Divergence from the reference significantly affected plastome assembly, but relatively similar references are available for most seed plants. Deeper rDNA sequencing is necessary to characterize intragenomic polymorphism. The low-copy fraction of the nuclear genome was readily surveyed, even at low sequencing depths. Nearly 160000 bp of sequence from three organelles provided evidence of phylogenetic incongruence in the SDC. CONCLUSIONS Adoption of NGS will facilitate progress in plant systematics, as whole plastome and rDNA cistrons, partial mitochondrial genomes, and low-copy nuclear markers can now be efficiently obtained for molecular phylogenetics studies.

Phylogenetic and Systematic Inferences from Chloroplast DNA and Isozyme Variation in Helianthus sect. Helianthus (Asteraceae)

Chloroplast DNA (cpDNA) and isozyme data were used to reconstruct the evolu- tionary history of the 21 taxa comprising Helianthus sect Helianthus. Low levels of cpDNA and isozyme divergence suggest a recent origin, perhaps within the last 1-2 million years, with subsequent rapid evolution and diversification throughout North America. This, combined with evolutionary phe- nomena such as phylogenetic sorting and introgression, makes phylogenetic reconstruction difficult. A strict consensus tree derived from Wagner parsimony analysis of the isozyme data set showed almost complete lack of resolution. The three clades resolved were, however, largely consistent with previous phylogenetic hypotheses. The most parsimonious cpDNA-based Wagner tree was poorly resolved, but showed that sect. Helianthus as presently circumscribed is monophyletic. Sur- prisingly, the cpDNA-based phylogeny does not resemble any of the previous phylogenetic hy- potheses for this group based on evidence from morphology, crossability, sesquiterpene lactone chemistry, and inferred chromosomal end arrangements. In general, species geographically most proximal are most closely related in terms of cpDNA, maternal inheritance of which is demonstrated herein. All four polytypic species in sect. Helianthus are polyphyletic in terms of cpDNA. Morpho- logical classification and cpDNA genotype were discordant for populations or species of H. annuus, H. debilis subsp. cucumerifolius, H. neglectus, H. petiolaris subsp. fallax, H. petiolaris subsp. petiolaris, and H. anomalus. Cytoplasmic introgression appears to be the most parsimonius explanation for these discrepancies. The edaphic differentiation and generally allopatric distribution of most taxa in sect. Helianthus are consistent with an allopatric mode of speciation. Molecular and chromosomal evi- dence, however, suggest that allopatric, parapatric, and quantum speciation all operate. In addition, at least one geographic race may have been derived through introgression and three species appear to be derived via selection or random fixation of recombinant genotypes following interspecific hybridization. We also tested the hypothesis that levels of genetic polymorphism will be lower in geographically restricted species than in more widespre-ad species. Eight of the 11 narrow endemics analyzed had lower levels of genetic diversity than any of their more widespread congeners.

Targeted enrichment strategies for next-generation plant biology

PREMISE OF THE STUDY The dramatic advances offered by modern DNA sequencers continue to redefine the limits of what can be accomplished in comparative plant biology. Even with recent achievements, however, plant genomes present obstacles that can make it difficult to execute large-scale population and phylogenetic studies on next-generation sequencing platforms. Factors like large genome size, extensive variation in the proportion of organellar DNA in total DNA, polyploidy, and gene number/redundancy contribute to these challenges, and they demand flexible targeted enrichment strategies to achieve the desired goals. METHODS In this article, we summarize the many available targeted enrichment strategies that can be used to target partial-to-complete organellar genomes, as well as known and anonymous nuclear targets. These methods fall under four categories: PCR-based enrichment, hybridization-based enrichment, restriction enzyme-based enrichment, and enrichment of expressed gene sequences. KEY RESULTS Examples of plant-specific applications exist for nearly all methods described. While some methods are well established (e.g., transcriptome sequencing), other promising methods are in their infancy (hybridization enrichment). A direct comparison of methods shows that PCR-based enrichment may be a reasonable strategy for accessing small genomic targets (e.g., ≤50 kbp), but that hybridization and transcriptome sequencing scale more efficiently if larger targets are desired. CONCLUSIONS While the benefits of targeted sequencing are greatest in plants with large genomes, nearly all comparative projects can benefit from the improved throughput offered by targeted multiplex DNA sequencing, particularly as the amount of data produced from a single instrument approaches a trillion bases per run.

Widespread Genealogical Nonmonophyly in Species of Pinus Subgenus Strobus

Phylogenetic relationships among Pinus species from subgenus Strobus remain unresolved despite combined efforts based on nrITS and cpDNA. To provide greater resolution among these taxa, a 900-bp intron from a late embryogenesis abundant (LEA)-like gene (IFG8612)was sequenced from 39 pine species, with two or more alleles representing 33 species. Nineteen of 33 species exhibited allelic nonmonphyly in the strict consensus tree, and 10 deviated significantly from allelic monophyly based on topology incongruence tests. Intraspecific nucleotide diversity ranged from 0.0 to 0.0211, and analysis of variance shows that nucleotide diversity was strongly associated (P < 0.0001)with the degree of species monophyly. Although species nonmonophyly complicates phylogenetic interpretations, this nuclear locus offers greater topological support than previously observed for cpDNA or nrITS. Lacking evidence for hybridization, recombination, or imperfect taxonomy, we feel that incomplete lineage sorting remains the best explanation for the polymorphisms shared among species. Depending on the species, coalescent expectations indicate that reciprocal monophyly will be more likely than paraphyly in 1.71 to 24.0 x 10(6) years, and that complete genome-wide coalescence in these species may require up to 76.3 x 10(6) years. The absence of allelic coalescence is a severe constraint in the application of phylogenetic methods in Pinus, and taxa sharing similar life history traits with Pinus are likely to show species nonmonophyly using nuclear markers.

POPULATION STRUCTURE AND GENETIC DIVERSITY OF BOTRYCHIUM PUMICOLA (OPHIOGLOSSACEAE) BASED ON INTER-SIMPLE SEQUENCE REPEATS (ISSR)'

Species of Botrychium reproduce by spores that form subterranean gametophytes and a few, like B. pumicola, also reproduce asexually with subterranean sporophytic gemmae. The goal of this study was to examine the genetic diversity of B. pumicola populations and to better understand the role of gemmae. Ninety-nine individuals from three monitored populations were sampled. The technique of inter-simple sequence repeats (ISSR) produced 15 polymorphic loci and identified 71 ISSR genotypes. Sixteen of the ISSR genotypes were shared by more that one individual in a population, representing potential clones. Ten of the 16 shared genotypes were not limited to clusters of plants (groups of plants growing from the same point). The ten potential clones were disjunct (separated by other genotypes) and not in patches as might be expected for an underground propagule. There is a high probability that these shared genotypes arose from independent sexual events suggesting they were not clones. These results suggest that the long-distance dispersal of gemmae is at best a rare event.

Length Variation in the Nuclear Ribosomal DNA Internal Transcribed Spacer Region of Non-flowering Seed Plants

The nuclear ribosomal DNA (rDNA) internal transcribed spacer (ITS) region was PCR- amplified in 32 genera of non-flowering seed plants. Length of the ITS region was determined by restriction site mapping of PCR products and nucleotide sequences were obtained from the ITS-2 and 5.8S rDNA of selected genera. In contrast to the relatively narrow range of ITS region lengths reported from angiosperms (565-700 base pairs (bp)), substantial length variation (975-3125 bp) is observed in the ITS region of Coniferales, Cycadales, Ginkgoales, and Gnetales. Restriction site analyses indicate that the 5.8S rDNA + ITS-2 ranges from 375-450 bp, while the ITS-1 is responsible for most of the length variation found in gymnosperm ITS regions. The representatives of Pinaceae exhibit the greatest variation in ITS region length (1550-3125 bp), while those of sampled members of Cupressaceae, Taxodiaceae, Cephalotaxaceae, and Taxaceae are relatively stable (975-1125 bp). The observed ITS region lengths in Sciadopityaceae (1250 bp) and Araucariaceae (1325-1350 bp) are somewhat larger than those of Cupressaceae, Taxodiaceae, Cephalotaxa- ceae, and Taxaceae, while those of sampled Podocarpaceae (2000-2100 bp) fall into the range observed in the Pinaceae. Outside of the Coniferales, ITS region length is similar among Cycadales (1150- 1450 bp), Ginkgoales (1200 bp), and two of the three members of Gnetales, Ephedra (1500 bp) and Gnetum (1200 bp). In contrast, the ITS region of Welwitschia is only 750 bp long, ca. 50 bp longer than the longest known angiosperm ITS region. Levels of nucleotide sequence variation as estimated by restriction site mapping suggest that phylogenetic analysis of the ITS region will be informative at the intrageneric level in most non-flowering seed plants. In Cupressaceae, intergeneric comparisons may also be feasible. Because the ITS region is relatively long, it may also permit population-level phylogenetic analysis in many non-flowering seed plants.