Jaume Pellicer

Genome sequence of dwarf birch (Betula nana) and cross‐species RAD markers

New sequencing technologies allow development of genome‐wide markers for any genus of ecological interest, including plant genera such as Betula (birch) that have previously proved difficult to study due to widespread polyploidy and hybridization. We present a de novo reference genome sequence assembly, from 66× short read coverage, of Betula nana (dwarf birch) – a diploid that is the keystone woody species of subarctic scrub communities but of conservation concern in Britain. We also present 100 bp PstI RAD markers for B. nana and closely related Betula tree species. Assembly of RAD markers in 15 individuals by alignment to the reference B. nana genome yielded 44–86k RAD loci per individual, whereas de novo RAD assembly yielded 64–121k loci per individual. Of the loci assembled by the de novo method, 3k homologous loci were found in all 15 individuals studied, and 35k in 10 or more individuals. Matching of RAD loci to RAD locus catalogues from the B. nana individual used for the reference genome showed similar numbers of matches from both methods of RAD locus assembly but indicated that the de novo RAD assembly method may overassemble some paralogous loci. In 12 individuals hetero‐specific to B. nana 37–47k RAD loci matched a catalogue of RAD loci from the B. nana individual used for the reference genome, whereas 44–60k RAD loci aligned to the B. nana reference genome itself. We present a preliminary study of allele sharing among species, demonstrating the utility of the data for introgression studies and for the identification of species‐specific alleles.

Analysis of the giant genomes of Fritillaria (Liliaceae) indicates that a lack of DNA removal characterizes extreme expansions in genome size.

Summary Plants exhibit an extraordinary range of genome sizes, varying by > 2000‐fold between the smallest and largest recorded values. In the absence of polyploidy, changes in the amount of repetitive DNA (transposable elements and tandem repeats) are primarily responsible for genome size differences between species. However, there is ongoing debate regarding the relative importance of amplification of repetitive DNA versus its deletion in governing genome size. Using data from 454 sequencing, we analysed the most repetitive fraction of some of the largest known genomes for diploid plant species, from members of Fritillaria. We revealed that genomic expansion has not resulted from the recent massive amplification of just a handful of repeat families, as shown in species with smaller genomes. Instead, the bulk of these immense genomes is composed of highly heterogeneous, relatively low‐abundance repeat‐derived DNA, supporting a scenario where amplified repeats continually accumulate due to infrequent DNA removal. Our results indicate that a lack of deletion and low turnover of repetitive DNA are major contributors to the evolution of extremely large genomes and show that their size cannot simply be accounted for by the activity of a small number of high‐abundance repeat families.

A universe of dwarfs and giants: genome size and chromosome evolution in the monocot family Melanthiaceae

• Since the occurrence of giant genomes in angiosperms is restricted to just a few lineages, identifying where shifts towards genome obesity have occurred is essential for understanding the evolutionary mechanisms triggering this process. • Genome sizes were assessed using flow cytometry in 79 species and new chromosome numbers were obtained. Phylogenetically based statistical methods were applied to infer ancestral character reconstructions of chromosome numbers and nuclear DNA contents. • Melanthiaceae are the most diverse family in terms of genome size, with C-values ranging more than 230-fold. Our data confirmed that giant genomes are restricted to tribe Parideae, with most extant species in the family characterized by small genomes. Ancestral genome size reconstruction revealed that the most recent common ancestor (MRCA) for the family had a relatively small genome (1C = 5.37 pg). Chromosome losses and polyploidy are recovered as the main evolutionary mechanisms generating chromosome number change. • Genome evolution in Melanthiaceae has been characterized by a trend towards genome size reduction, with just one episode of dramatic DNA accumulation in Parideae. Such extreme contrasting profiles of genome size evolution illustrate the key role of transposable elements and chromosome rearrangements in driving the evolution of plant genomes.

Recent updates and developments to plant genome size databases

Two plant genome size databases have been recently updated and/or extended: the Plant DNA C-values database (http://data.kew.org/cvalues), and GSAD, the Genome Size in Asteraceae database (http://www.asteraceaegenomesize.com). While the first provides information on nuclear DNA contents across land plants and some algal groups, the second is focused on one of the largest and most economically important angiosperm families, Asteraceae. Genome size data have numerous applications: they can be used in comparative studies on genome evolution, or as a tool to appraise the cost of whole-genome sequencing programs. The growing interest in genome size and increasing rate of data accumulation has necessitated the continued update of these databases. Currently, the Plant DNA C-values database (Release 6.0, Dec. 2012) contains data for 8510 species, while GSAD has 1219 species (Release 2.0, June 2013), representing increases of 17 and 51%, respectively, in the number of species with genome size data, compared with previous releases. Here we provide overviews of the most recent releases of each database, and outline new features of GSAD. The latter include (i) a tool to visually compare genome size data between species, (ii) the option to export data and (iii) a webpage containing information about flow cytometry protocols.

Cytotype diversity in the Sorbus complex (Rosaceae) in Britain: sorting out the puzzle.

BACKGROUND AND AIMS Large-scale ploidy surveys using flow cytometry have become an essential tool to study plant genome dynamics and to gain insight into the mechanisms and genetic barriers framing ploidy diversity. As an ideal complement to traditional techniques such as chromosome counting, the analysis of cytotype diversity in plant systems such as Sorbus provides primary investigation into the potential patterns and evolutionary implications of hybrid speciation. METHODS Ploidy was assessed by means of relative nuclear DNA content using propidium iodide flow cytometry in 474 Sorbus samples collected from 65 populations in southern Wales and South-West England. Statistical tests were applied to evaluate the utility of this technique to confidently discriminate ploidy in the genus. KEY RESULTS Flow cytometric profiles revealed the presence of four cytotypes (2x, 3x, 4x and 5x), confirming in many cases chromosome counts previously reported and demonstrating cytotype heterogeneity within specific Sorbus aggregates. Diploid cytotypes were restricted to the potential parental species and homoploid hybrids. Most of the samples processed were polyploid. The occurrence of the pentaploid cytotype had previously only been reported from a single specimen; it is now confirmed for two taxa occurring at different sites. CONCLUSIONS Flow cytometry results obtained have proved useful in shedding light on the taxonomy of several controversial taxa and in confirming the presence of cytoypes which occur at very low frequencies. Notably, the coexistence of several cytotypes in Sorbus populations has probably been facilitated by the overlapping distribution of many of the species studied, which might also explain the high incidence of potential hybrid apomictic polyploids. These results will provide a solid baseline for molecular research aiming to better understand the genetic pathways controlling the formation and establishment of polyploid Sorbus.

A molecular phylogenetic approach to western North America endemic Artemisia and allies (Asteraceae): Untangling the sagebrushes

PREMISE OF THE STUDY Artemisia subgenus Tridentatae plants characterize the North American Intermountain West. These are landscape-dominant constituents of important ecological communities and habitats for endemic wildlife. Together with allied species and genera (Picrothamnus and Sphaeromeria), they make up an intricate series of taxa whose limits are uncertain, likely the result of reticulate evolution. The objectives of this study were to resolve relations among Tridentatae species and their near relatives by delimiting the phylogenetic positions of subgenus Tridentatae species with particular reference to its New World geographic placement and to provide explanations for the relations of allied species and genera with the subgenus with an assessment of their current taxonomic placement. METHODS Bayesian inference and maximum parsimony analysis were based on 168 newly generated sequences (including the nuclear ITS and ETS and the plastid trnS(UGA)-trnfM(CAU) and trnS(GCU)-trnC(GCA)) and 338 previously published sequences (ITS and ETS). Genome size by flow cytometry of species from Sphaeromeria was also determined. KEY RESULTS The results support an expanded concept and reconfiguration of Tridentatae to accommodate additional endemic North American Artemisia species. The monotypic Picrothamnus and all Sphaeromeria species appear nested within subgenus Tridentatae clade. CONCLUSIONS A redefinition of subgenus Tridentatae to include other western North American endemics is supported. We propose a new circumscription of the subgenus and divide it into three sections: Tridentatae, Filifoliae, and Nebulosae. The position of the circumboreal and other North American species suggests that subgenus Artemisia is the ancestral stock for the New World endemics, including those native to South America.

Biology, Genome Evolution, Biotechnological Issues and Research Including Applied Perspectives in Artemisia (Asteraceae)

Abstract Artemisia is one of the largest genera of the family Asteraceae or Compositae, itself the biggest flowering plant family. It comprises around 600 taxa at specific and subspecific levels, present in all continents but Antarctica, mostly distributed in the Northern Hemisphere, with no more than 25 taxa in the Southern Hemisphere. The genus displays a huge ecological plasticity, with species occurring from sea level to high mountains and from arid zones to wetlands. Some species are cosmopolitan, including landscape-dominating plants over large areas, and others are endemics with a quite restricted distribution area. Many species of the genus have economic uses at both folk and industrial levels, and some of them are widely cultivated and submitted to breeding programmes as crops. In this review, we will set out the state of art of Artemisia systematics and phylogeny, as well as all the biological aspects of the genus, with particular attention paid to those of genome organization, and of applied questions related to its useful taxa. In the first part of this chapter, we will review all the systematic points in the genus and in some closely related genera that constitute, with the core genus Artemisia, a pool with controversial structuring. Besides, the infrageneric classification will be addressed. All these questions will be treated in the light of recent molecular phylogenetic studies, which have had an important impact on its systematics and taxonomy. A second part will be devoted to genome organization and evolution in Artemisia, with special attention to cytogenetic data, including genome size, and genetic variability. These points are relevant for understanding the evolutionary pathways in the genus and for applied purposes. The third and fourth parts of the chapter will review, respectively, the uses of Artemisia species in different domains and the biotechnological issues linked to their productivity. Finally, the perspectives of the knowledge and applied aspects of the genus will be addressed.

Ribosomal DNA, heterochromatin, and correlation with genome size in diploid and polyploid North American endemic sagebrushes (Artemisia, Asteraceae)

Subgenus Tridentatae (Artemisia, Asteraceae) can be considered a polyploid complex. Both polyploidy and hybridization have been documented in the Tridentatae. Fluorescent in situ hybridization (FISH) and fluorochrome banding were used to detect and analyze ribosomal DNA changes linked to polyploidization in this group by studying four diploid-polyploid species pairs. In addition, genome sizes and heterochromatin patterns were compared between these populations. The linked 5S and 35S rRNA genes are confirmed as characteristic for Artemisia, and a pattern at the diploid level of three rDNA loci located at telomeric positions proved to be typical. Loss of rDNA loci was observed in some polyploids, whereas others showed additivity with respect to their diploid relatives. Genome downsizing was observed in all polyploids. Banding patterns differed depending on the pair of species analysed, but some polyploid populations showed an increased number of heterochromatic bands. FISH and fluorochrome banding were useful in determining the systematic position of Artemisia bigelovii, for which a differential pattern was found as compared with the rest of the group. Additionally, FISH was used to detect the presence of the Arabidopsis-type telomere repeat for the first time in Artemisia.

Genome size variation and evolution in the family Asteraceae

The nuclear DNA content data available in “A genome size database in the Asteraceae” (GSAD: www.asteraceaegenomesize.com) have been analyzed, together with other parameters (i.e. ecological, karyological, cytogenetic), in order to establish hypotheses on the systematic, phylogenetic and evolutionary aspects of genome size in one of the largest angiosperm families. The novelty of this work is a comprehensive analysis of the whole family with the following aims: (1) to update the knowledge of genome size values in the Asteraceae; (2) to infer evolutionary trends of genome size, compared with other plant groups; and (3) to detect gaps in this field in the family and outline further research priorities. The analysis of this dataset shows that most Asteraceae genomes (57.23%) range from very small (1C ⩽ 1.4 pg) to small (1C ⩽ 3.5 pg). Gains and losses of DNA occur throughout the phylogeny of the family but although ancestral values for the basal nodes remain mostly equivocal, often small and very small ancestral genome sizes are reconstructed. Most genome size data (96.74%) are concentrated in five tribes, which broadly reflect their species richness. The relationships between genome size and other cytogenetic and ecological features have been analyzed and discussed, highlighting several general patterns. Further studies are needed to fill the gaps in genome size knowledge in the Asteraceae and more detailed research in some groups could provide information about mechanisms regulating genome expansions and contractions.

Why size really matters when sequencing plant genomes

Genome sequencing has been restricted to species with a small genome size. With the advent of second- and third-generation sequencing technologies, the potential to sequence genomes of all sizes is becoming a reality. As the field of whole genome sequencing has developed, there has been a growing appreciation of the need to better represent the major lineages of the plant tree of life, rather than just those that contain economically important taxa. We argue that as well as accounting for phylogenetic diversity when selecting species to analyse, in order to gain a comprehensive understanding of genome evolution, large-scale sequencing projects also need to reflect the diversity of genome sizes in plants. In this article we briefly outline evidence from the literature to support this view.