Michael Chester

Extensive chromosomal variation in a recently formed natural allopolyploid species, Tragopogon miscellus (Asteraceae).

Polyploidy, or whole genome duplication, has played a major role in the evolution of many eukaryotic lineages. Although the prevalence of polyploidy in plants is well documented, the molecular and cytological consequences are understood largely from newly formed polyploids (neopolyploids) that have been grown experimentally. Classical cytological and molecular cytogenetic studies both have shown that experimental neoallopolyploids often have meiotic irregularities, producing chromosomally variable gametes and progeny; however, little is known about the extent or duration of chromosomal variation in natural neoallopolyploid populations. We report the results of a molecular cytogenetic study on natural populations of a neoallopolyploid, Tragopogon miscellus, which formed multiple times in the past 80 y. Using genomic and fluorescence in situ hybridization, we uncovered massive and repeated patterns of chromosomal variation in all populations. No population was fixed for a particular karyotype; 76% of the individuals showed intergenomic translocations, and 69% were aneuploid for one or more chromosomes. Importantly, 85% of plants exhibiting aneuploidy still had the expected chromosome number, mostly through reciprocal monosomy-trisomy of homeologous chromosomes (1:3 copies) or nullisomy-tetrasomy (0:4 copies). The extensive chromosomal variation still present after ca. 40 generations in this biennial species suggests that substantial and prolonged chromosomal instability might be common in natural populations after whole genome duplication. A protracted period of genome instability in neoallopolyploids may increase opportunities for alterations to genome structure, losses of coding and noncoding DNA, and changes in gene expression.

Next Generation Sequencing Reveals Genome Downsizing in Allotetraploid Nicotiana tabacum, Predominantly through the Elimination of Paternally Derived Repetitive DNAs

We used next generation sequencing to characterize and compare the genomes of the recently derived allotetraploid, Nicotiana tabacum (<200,000 years old), with its diploid progenitors, Nicotiana sylvestris (maternal, S-genome donor), and Nicotiana tomentosiformis (paternal, T-genome donor). Analysis of 14,634 repetitive DNA sequences in the genomes of the progenitor species and N. tabacum reveal all major types of retroelements found in angiosperms (genome proportions range between 17-22.5% and 2.3-3.5% for Ty3-gypsy elements and Ty1-copia elements, respectively). The diploid N. sylvestris genome exhibits evidence of recent bursts of sequence amplification and/or homogenization, whereas the genome of N. tomentosiformis lacks this signature and has considerably fewer homogenous repeats. In the derived allotetraploid N. tabacum, there is evidence of genome downsizing and sequences loss across most repeat types. This is particularly evident amongst the Ty3-gypsy retroelements in which all families identified are underrepresented in N. tabacum, as is 35S ribosomal DNA. Analysis of all repetitive DNA sequences indicates the T-genome of N. tabacum has experienced greater sequence loss than the S-genome, revealing preferential loss of paternally derived repetitive DNAs at a genome-wide level. Thus, the three genomes of N. sylvestris, N. tomentosiformis, and N. tabacum have experienced different evolutionary trajectories, with genomes that are dynamic, stable, and downsized, respectively.

Searching for the relatives of Coffea (Rubiaceae, Ixoroideae): the circumscription and phylogeny of Coffeeae based on plastid sequence data and morphology.

The circumscription of Coffeeae (Rubiaceae) and phylogenetic relationships within the tribe were evaluated using sequence data from four plastid regions (trnL-F intron, trnL-F intergenic spacer [IGS], rpl16 intron, and accD-psa1 IGS) and a morphological data set. Eleven candidates for inclusion in Coffeeae were examined using plastid data, and a further three were investigated using morphology alone. Based on previous phylogenetic analysis of the subfamily Ixoroideae, nine genera representing five tribes were used as outgroups. Our results support an enlarged circumscription for Coffeeae, containing 11 genera, viz. Argocoffeopsis, Belonophora, Calycosiphonia, Coffea, Diplospora, Discospermum, Nostolachma, Psilanthus, Tricalysia, Sericanthe, and Xantonnea. The inclusion of Diplospora and Tricalysia within Coffeeae, based on published molecular data, and the inclusion of Argocoffeopsis, Belonophora, Calycosiphonia, Discospermum, and Sericanthe, based on morphological evidence, are well supported. Nostolachma is newly transferred from Gardenieae subtribe Diplosporinae to Coffeeae, and Xantonnea from Octotropideae to Coffeeae. The exclusion of Bertiera from Coffeeae and placement in tribe Bertiereae is supported on the basis of molecular and morphological data. The removal of Diplospora and all other genera from Gardenieae subtribe Diplosporinae to Coffeeae and Octotropideae renders Diplosporinae superfluous. It is proposed that Xantonneopsis be transferred to Octotropideae; Petitiocodon is tentatively placed in Gardenieae. The monophyly of seven genera is supported, but Coffea is identified as paraphyletic in relation to Psilanthus on the basis of molecular and combined molecular and morphological data.

Rapid Y degeneration and dosage compensation in plant sex chromosomes

Significance Sex chromosomes have repeatedly evolved in animals and plants, but the evolutionary forces driving this process are not entirely understood. Nonrecombining Y chromosomes undergo rapid loss of functional genes in animals; however, it remains unclear whether this holds true in plants. We report the first genome sequence-based analysis of sex chromosomes in white campion, to our knowledge, which evolved large sex chromosomes only 10 million years ago. We demonstrate that the Y chromosome has lost nearly half its functional genes, at a rate of Y degeneration comparable to that of animal Y chromosomes. This degeneration is accommodated for by highly variable dosage compensation. Our results resolve the puzzling discrepancy in evolutionary trajectories of sex chromosomes between the plant and animal kingdoms. The nonrecombining regions of animal Y chromosomes are known to undergo genetic degeneration, but previous work has failed to reveal large-scale gene degeneration on plant Y chromosomes. Here, we uncover rapid and extensive degeneration of Y-linked genes in a plant species, Silene latifolia, that evolved sex chromosomes de novo in the last 10 million years. Previous transcriptome-based studies of this species missed unexpressed, degenerate Y-linked genes. To identify sex-linked genes, regardless of their expression, we sequenced male and female genomes of S. latifolia and integrated the genomic contigs with a high-density genetic map. This revealed that 45% of Y-linked genes are not expressed, and 23% are interrupted by premature stop codons. This contrasts with X-linked genes, in which only 1.3% of genes contained stop codons and 4.3% of genes were not expressed in males. Loss of functional Y-linked genes is partly compensated for by gene-specific up-regulation of X-linked genes. Our results demonstrate that the rate of genetic degeneration of Y-linked genes in S. latifolia is as fast as in animals, and that the evolutionary trajectories of sex chromosomes are similar in the two kingdoms.

Independent, rapid and targeted loss of highly repetitive DNA in natural and synthetic allopolyploids of Nicotiana tabacum.

Allopolyploidy (interspecific hybridisation and polyploidy) has played a significant role in the evolutionary history of angiosperms and can result in genomic, epigenetic and transcriptomic perturbations. We examine the immediate effects of allopolyploidy on repetitive DNA by comparing the genomes of synthetic and natural Nicotiana tabacum with diploid progenitors N. tomentosiformis (paternal progenitor) and N. sylvestris (maternal progenitor). Using next generation sequencing, a recently developed graph-based repeat identification pipeline, Southern blot and fluorescence in situ hybridisation (FISH) we characterise two highly repetitive DNA sequences (NicCL3 and NicCL7/30). Analysis of two independent high-throughput DNA sequencing datasets indicates NicCL3 forms 1.6–1.9% of the genome in N. tomentosiformis, sequences that occur in multiple, discontinuous tandem arrays scattered over several chromosomes. Abundance estimates, based on sequencing depth, indicate NicCL3 is almost absent in N. sylvestris and has been dramatically reduced in copy number in the allopolyploid N. tabacum. Surprisingly elimination of NicCL3 is repeated in some synthetic lines of N. tabacum in their forth generation. The retroelement NicCL7/30, which occurs interspersed with NicCL3, is also under-represented but to a much lesser degree, revealing targeted elimination of the latter. Analysis of paired-end sequencing data indicates the tandem component of NicCL3 has been preferentially removed in natural N. tabacum, increasing the proportion of the dispersed component. This occurs across multiple blocks of discontinuous repeats and based on the distribution of nucleotide similarity among NicCL3 units, was concurrent with rounds of sequence homogenisation.

Review of the Application of Modern Cytogenetic Methods (FISH/GISH) to the Study of Reticulation (Polyploidy/Hybridisation)

The convergence of distinct lineages upon interspecific hybridisation, including when accompanied by increases in ploidy (allopolyploidy), is a driving force in the origin of many plant species. In plant breeding too, both interspecific hybridisation and allopolyploidy are important because they facilitate introgression of alien DNA into breeding lines enabling the introduction of novel characters. Here we review how fluorescence in situ hybridisation (FISH) and genomic in situ hybridisation (GISH) have been applied to: 1) studies of interspecific hybridisation and polyploidy in nature, 2) analyses of phylogenetic relationships between species, 3) genetic mapping and 4) analysis of plant breeding materials. We also review how FISH is poised to take advantage of nextgeneration sequencing (NGS) technologies, helping the rapid characterisation of the repetitive fractions of a genome in natural populations and agricultural plants.

Patterns of chromosomal variation in natural populations of the neoallotetraploid Tragopogon mirus (Asteraceae)

Cytological studies have shown many newly formed allopolyploids (neoallopolyploids) exhibit chromosomal variation as a result of meiotic irregularities, but few naturally occurring neoallopolyploids have been examined. Little is known about how long chromosomal variation may persist and how it might influence the establishment and evolution of allopolyploids in nature. In this study we assess chromosomal composition in a natural neoallotetraploid, Tragopogon mirus, and compare it with T. miscellus, which is an allotetraploid of similar age (~40 generations old). We also assess whether parental gene losses in T. mirus correlate with entire or partial chromosome losses. Of 37 T. mirus individuals that were karyotyped, 23 (62%) were chromosomally additive of the parents, whereas the remaining 14 individuals (38%) had aneuploid compositions. The proportion of additive versus aneuploid individuals differed from that found previously in T. miscellus, in which aneuploidy was more common (69%; Fisher’s exact test, P=0.0033). Deviations from parental chromosome additivity within T. mirus individuals also did not reach the levels observed in T. miscellus, but similar compensated changes were observed. The loss of T. dubius-derived genes in two T. mirus individuals did not correlate with any chromosomal changes, indicating a role for smaller-scale genetic alterations. Overall, these data for T. mirus provide a second example of prolonged chromosomal instability in natural neoallopolyploid populations.

The Early Stages of Polyploidy: Rapid and Repeated Evolution in Tragopogon

Elucidating the causes and consequences of polyploidy (whole-genome duplication; WGD) is arguably central to understanding the evolution of most eukaryotic lineages. However, much of what we know about these processes is derived from the study of crops and synthetic polyploids. Tragopogon provides the unique opportunity to investigate the genetic and genomic changes that occur across an evolutionary series from F1 hybrids, synthetic allopolyploids, independently formed natural populations of T. mirus and T. miscellus that are 60–80 years post-formation, to older Eurasian polyploids that are dated by molecular clocks at several million years old, and finally to a putative ancient polyploidization thought to have occurred prior to or early in the history of the Asteraceae (40–43 mya). Tragopogon joins other well-studied natural polyploid systems (e.g., Glycine, Nicotiana, Gossypium, Spartina, Senecio), but presents a range of research possibilities that is not available in any other system. We have shown in T. mirus and T. miscellus that upon allopolyploidization, massive gene loss occurs in patterns that are repeated across populations of independent origin and with a bias against genes derived from T. dubius, the diploid parent shared by both new allotetraploids. We have also shown significant changes in gene expression (transcriptomic shock) in the early generations of allopolyploidy in these species. Massive and repeated patterns of chromosomal variation (intergenomic translocations and aneuploidy) have been revealed by fluorescence in situ hybridization. Aneuploidy results in substitutions between homeologous chromosomes, through reciprocal monosomy-trisomy (1:3 copies) or nullisomy-tetrasomy (0:4 copies). We propose that substantial chromosomal instability results in karyotype restructuring, a likely common process following WGD and a driver of allopolyploid speciation, which has largely unexplored implications for gene losses, gains, and expression patterns. But gene loss and expression changes as well as karyotypic changes are ongoing in T. mirus and T. miscellus, in that no population is fixed for any of these events; thus, we have literally caught evolution in the act.

An assessment of karyotype restructuring in the neoallotetraploid Tragopogon miscellus (Asteraceae).

Tragopogon miscellus and Tragopogon mirus are two rare examples of allopolyploids that have formed recently in nature. Molecular cytogenetic studies have revealed chromosome copy number variation and intergenomic translocations in both allotetraploids. Due to a lack of interstitial chromosome markers, there remained the possibility of additional karyotype restructuring in these neopolyploids, via intrachromosomal and intragenomic rearrangements. To address this issue, we searched for additional high-copy tandem repeats in genomic sequences of the diploid progenitor species—Tragopogon dubius, Tragopogon pratensis and Tragopogon porrifolius—for application to the chromosomes of the allotetraploids. Eight novel repeats were localised by fluorescence in situ hybridisation (FISH) in the diploids; one of these repeats, TTR3, provided interstitial coverage. TTR3 was included in a cocktail with other previously characterised probes, producing better-resolved karyotypes for the three diploids. The cocktail was then used to test a hypothesis of karyotype restructuring in the recent allotetraploid T. miscellus by comparing repeat distributions to its diploid progenitors, T. dubius and T. pratensis. Five individuals of T. miscellus were selected from across the range of karyotypic variation previously observed in natural populations. FISH signal distributions mostly matched those observed in the diploid progenitors, with the exception of several losses or gains of signal at chromosomal subtermini and previously noted intergenomic translocations. Thus, in T. miscellus, we find most changes restricted to the subterminal regions, and although some were recurrent, none of the changes were common to all individuals analysed. We consider these findings in relation to the gene loss reported previously for T. miscellus.

Natural hybrids between Tragopogon mirus and T. miscellus (Asteraceae): A new perspective on karyotypic changes following hybridization at the polyploid level

UNLABELLED PREMISE OF THE STUDY Natural hybrids have formed in Pullman, Washington, United States between the recently formed allotetraploids Tragopogon miscellus and T. mirus. In addition to forming spontaneously, these hybrids are semifertile, propagating via achenes. Previous work indicated that the tetraploid hybrids have genetic contributions from three progenitor diploids: T. dubius, T. pratensis, and T. porrifolius. Because the hybrids contain genomes from three species, they should be karyotypically variable and have very low fertility. To better understand how these hybrids are semifertile, we applied fluorescent probes to determine chromosome composition. • METHODS We sequentially conducted fluorescence and genomic in situ hybridization to generate karyotypes for five hybrid individuals grown from field-collected achenes. • KEY RESULTS All plants had the expected somatic chromosome number (2n = 24), but none showed an additive F1 chromosome complement, i.e., two sets of chromosomes from T. dubius and one set of chromosomes each from T. porrifolius and T. pratensis. No individuals shared an identical karyotype, but chromosomal variation followed a compensatory pattern of substitutions, with all groups of putatively homeologous chromosomes consistently totaling four. • CONCLUSIONS The hybrids appear to be shifting away from a parentally additive F1 karyotype to chromosomal compositions that are mostly, or entirely, disomic. We hypothesize that this process may eventually lead to the elimination of chromosomes from a population and produce a stabilized karyotype distinct from both allotetraploid parents. This work has implications for other hybrids formed between polyploids, in that they may be hard to detect using sequence data alone due to multilateral patterns of chromosome elimination.