A comparative genomics examination of desiccation tolerance and sensitivity in two sister grass species.

Desiccation tolerance is an ancient and complex trait that spans all major lineages of life on earth. Although important in the evolution of land plants, the mechanisms that underlay this complex trait are poorly understood, especially for vegetative desiccation tolerance (VDT). The lack of suitable closely related plant models that offer a direct contrast between desiccation tolerance and sensitivity has hampered progress. We have assembled high-quality genomes for two closely related grasses, the desiccation-tolerant Sporobolus stapfianus and the desiccation-sensitive Sporobolus pyramidalis Both species are complex polyploids; S. stapfianus is primarily tetraploid, and S. pyramidalis is primarily hexaploid. S. pyramidalis undergoes a major transcriptome remodeling event during initial exposure to dehydration, while S. stapfianus has a muted early response, with peak remodeling during the transition between 1.5 and 1.0 grams of water (gH2O) g-1 dry weight (dw). Functionally, the dehydration transcriptome of S. stapfianus is unrelated to that for S. pyramidalis A comparative analysis of the transcriptomes of the hydrated controls for each species indicated that S. stapfianus is transcriptionally primed for desiccation. Cross-species comparative analyses indicated that VDT likely evolved from reprogramming of desiccation tolerance mechanisms that evolved in seeds and that the tolerance mechanism of S. stapfianus represents a recent evolution for VDT within the Chloridoideae. Orthogroup analyses of the significantly differentially abundant transcripts reconfirmed our present understanding of the response to dehydration, including the lack of an induction of senescence in resurrection angiosperms. The data also suggest that failure to maintain protein structure during dehydration is likely critical in rendering a plant desiccation sensitive.

A taxonomic, genetic and ecological data resource for the vascular plants of Britain and Ireland.

The vascular flora of Britain and Ireland is among the most extensively studied in the world, but the current knowledge base is fragmentary, with taxonomic, ecological and genetic information scattered across different resources. Here we present the first comprehensive data repository of native and alien species optimized for fast and easy online access for ecological, evolutionary and conservation analyses. The inventory is based on the most recent reference flora of Britain and Ireland, with taxon names linked to unique Kew taxon identifiers and DNA barcode data. Our data resource for 3,227 species and 26 traits includes existing and unpublished genome sizes, chromosome numbers and life strategy and life-form assessments, along with existing data on functional traits, species distribution metrics, hybrid propensity, associated biomes, realized niche description, native status and geographic origin of alien species. This resource will facilitate both fundamental and applied research and enhance our understanding of the flora's composition and temporal changes to inform conservation efforts in the face of ongoing climate change and biodiversity loss.

A highly contiguous, scaffold-level nuclear genome assembly for the fever tree (Cinchona pubescens Vahl) as a novel resource for Rubiaceae research.

The Andean fever tree (Cinchona L.; Rubiaceae) is a source of bioactive quinine alkaloids used to treat malaria. C. pubescens Vahl is a valuable cash crop within its native range in northwestern South America, however, genomic resources are lacking. Here we provide the first highly contiguous and annotated nuclear and plastid genome assemblies using Oxford Nanopore PromethION-derived long-read and Illumina short-read data. Our nuclear genome assembly comprises 603 scaffolds with a total length of 904 Mbp (∼82% of the full genome based on a genome size of 1.1 Gbp/1C). Using a combination of de novo and reference-based transcriptome assemblies we annotated 72,305 coding sequences comprising 83% of the BUSCO gene set and 4.6% fragmented sequences. Using additional plastid and nuclear datasets we place C. pubescens in the Gentianales order. This first genomic resource for C. pubescens opens new research avenues, including the analysis of alkaloid biosynthesis in the fever tree.

The nature of intraspecific and interspecific genome size variation in taxonomically complex eyebrights.

BACKGROUND AND AIMS: Genome size varies considerably across the diversity of plant life. Although genome size is, by definition, affected by genetic presence/absence variants, which are ubiquitous in population sequencing studies, genome size is often treated as an intrinsic property of a species. Here, we studied intra- and interspecific genome size variation in taxonomically complex British eyebrights (Euphrasia, Orobanchaceae). Our aim is to document genome size diversity and investigate underlying evolutionary processes shaping variation between individuals, populations and species. METHODS: We generated genome size data for 192 individuals of diploid and tetraploid Euphrasia and analysed genome size variation in relation to ploidy, taxonomy, population affiliation and geography. We further compared the genomic repeat content of 30 samples. KEY RESULTS: We found considerable intraspecific genome size variation, and observed isolation-by-distance for genome size in outcrossing diploids. Tetraploid Euphrasia showed contrasting patterns, with genome size increasing with latitude in outcrossing Euphrasia arctica, but with little genome size variation in the highly selfing Euphrasia micrantha. Interspecific differences in genome size and the genomic proportions of repeat sequences were small. CONCLUSIONS: We show the utility of treating genome size as the outcome of polygenic variation. Like other types of genetic variation, such as single nucleotide polymorphisms, genome size variation may be affected by ongoing hybridization and the extent of population subdivision. In addition to selection on associated traits, genome size is predicted to be affected indirectly by selection due to pleiotropy of the underlying presence/absence variants.

Low dispersal and ploidy differences in a grass maintain photosynthetic diversity despite gene flow and habitat overlap.

Geographical isolation facilitates the emergence of distinct phenotypes within a single species, but reproductive barriers or selection are needed to maintain the polymorphism after secondary contact. Here, we explore the processes that maintain intraspecific variation of C4 photosynthesis, a complex trait that results from the combined action of multiple genes. The grass Alloteropsis semialata includes C4 and non-C4 populations, which have coexisted as a polyploid series for more than 1 million years in the miombo woodlands of Africa. Using population genomics, we show that there is genome-wide divergence for the photosynthetic types, but the current geographical distribution does not reflect a simple habitat displacement scenario as the genetic clusters overlap, being occasionally mixed within a given habitat. Despite evidence of recurrent introgression between non-C4 and C4 groups, in both diploids and polyploids, the distinct genetic lineages retain their identity, potentially because of selection against hybrids. Coupled with strong isolation by distance within each genetic group, this selection created a geographical mosaic of photosynthetic types. Diploid C4 and non-C4 types never grew together, and the C4 type from mixed populations constantly belonged to the hexaploid lineage. By limiting reproductive interactions between photosynthetic types, the ploidy difference probably allows their co-occurrence, reinforcing the functional diversity within this species. Together, these factors enabled the persistence of divergent physiological traits of ecological importance within a single species despite gene flow and habitat overlap.

The Application of Flow Cytometry for Estimating Genome Size, Ploidy Level Endopolyploidy, and Reproductive Modes in Plants.

Over the years, the amount of DNA in a nucleus (genome size) has been estimated using a variety of methods, but increasingly, flow cytometry (FCM) has become the method of choice. The popularity of this technique lies in the ease of sample preparation and in the large number of particles (i.e., nuclei) that can be analyzed in a very short period of time. This chapter presents a step-by-step guide to estimating the nuclear DNA content of plant nuclei using FCM. Attempting to serve as a tool for daily laboratory practice, we list, in detail, the equipment required, specific reagents and buffers needed, as well as the most frequently used protocols to carry out nuclei isolation. In addition, solutions to the most common problems that users may encounter when working with plant material and troubleshooting advice are provided. Finally, information about the correct terminology to use and the importance of obtaining chromosome counts to avoid cytological misinterpretations of the FCM data are discussed.

Contrasted histories of organelle and nuclear genomes underlying physiological diversification in a grass species.

C4 photosynthesis evolved multiple times independently in angiosperms, but most origins are relatively old so that the early events linked to photosynthetic diversification are blurred. The grass Alloteropsis semialata is an exception, as this species encompasses C4 and non-C4 populations. Using phylogenomics and population genomics, we infer the history of dispersal and secondary gene flow before, during and after photosynthetic divergence in A. semialata. We further analyse the genome composition of individuals with varied ploidy levels to establish the origins of polyploids in this species. Detailed organelle phylogenies indicate limited seed dispersal within the mountainous region of origin and the emergence of a C4 lineage after dispersal to warmer areas of lower elevation. Nuclear genome analyses highlight repeated secondary gene flow. In particular, the nuclear genome associated with the C4 phenotype was swept into a distantly related maternal lineage probably via unidirectional pollen flow. Multiple intraspecific allopolyploidy events mediated additional secondary genetic exchanges between photosynthetic types. Overall, our results show that limited dispersal and isolation allowed lineage divergence, with photosynthetic innovation happening after migration to new environments, and pollen-mediated gene flow led to the rapid spread of the derived C4 physiology away from its region of origin.

Genome-wide association mapping of date palm fruit traits.

Date palms (Phoenix dactylifera) are an important fruit crop of arid regions of the Middle East and North Africa. Despite its importance, few genomic resources exist for date palms, hampering evolutionary genomic studies of this perennial species. Here we report an improved long-read genome assembly for P. dactylifera that is 772.3 Mb in length, with contig N50 of 897.2 Kb, and use this to perform genome-wide association studies (GWAS) of the sex determining region and 21 fruit traits. We find a fruit color GWAS at the R2R3-MYB transcription factor VIRESCENS gene and identify functional alleles that include a retrotransposon insertion and start codon mutation. We also find a GWAS peak for sugar composition spanning deletion polymorphisms in multiple linked invertase genes. MYB transcription factors and invertase are implicated in fruit color and sugar composition in other crops, demonstrating the importance of parallel evolution in the evolutionary diversification of domesticated species.

A Target Capture-Based Method to Estimate Ploidy From Herbarium Specimens.

Whole genome duplication (WGD) events are common in many plant lineages, but the ploidy status and possible occurrence of intraspecific ploidy variation are unknown for most species. Standard methods for ploidy determination are chromosome counting and flow cytometry approaches. While flow cytometry approaches typically use fresh tissue, an increasing number of studies have shown that recently dried specimens can be used to yield ploidy data. Recent studies have started to explore whether high-throughput sequencing (HTS) data can be used to assess ploidy levels by analyzing allelic frequencies from single copy nuclear genes. Here, we compare different approaches using a range of yam (Dioscorea) tissues of varying ages, drying methods and quality, including herbarium tissue. Our aims were to: (1) explore the limits of flow cytometry in estimating ploidy level from dried samples, including herbarium vouchers collected between 1831 and 2011, and (2) optimize a HTS-based method to estimate ploidy by considering allelic frequencies from nuclear genes obtained using a target-capture method. We show that, although flow cytometry can be used to estimate ploidy levels from herbarium specimens collected up to fifteen years ago, success rate is low (5.9%). We validated our HTS-based estimates of ploidy using 260 genes by benchmarking with dried samples of species of known ploidy (Dioscorea alata, D. communis, and D. sylvatica). Subsequently, we successfully applied the method to the 85 herbarium samples analyzed with flow cytometry, and successfully provided results for 91.7% of them, comprising species across the phylogenetic tree of Dioscorea. We also explored the limits of using this HTS-based approach for identifying high ploidy levels in herbarium material and the effects of heterozygosity and sequence coverage. Overall, we demonstrated that ploidy diversity within and between species may be ascertained from historical collections, allowing the determination of polyploidization events from samples collected up to two centuries ago. This approach has the potential to provide insights into the drivers and dynamics of ploidy level changes during plant evolution and crop domestication.

Decoding ice plants: challenges associated with barcoding and phylogenetics in the diverse succulent family Aizoaceae.

Aizoaceae is the largest succulent plant family in the world, including in excess of 1800 species. Despite its richness, a large proportion of its taxa are listed as data deficient and as such, has been identified as the top priority for taxonomic research in South Africa. Limitations to accurate taxonomic identification of taxa in the family may be partly attributed to the degree of technical knowledge required to identify taxa in the Aizoaceae. DNA barcoding may provide an alternative method of identification; however, the suitability of commonly used gene regions has not been tested in the family. Here, we analyse variable and parsimony informative characters (PIC), as well as the barcoding gap, in commonly used plastid regions (atpB-rbcL, matK, psbA-trnH, psbJ-petA, rpl16, rps16, trnD-trnT, trnL-trnF, trnQ-rps16, and trnS-trnG) and the nuclear region ITS (for Aizooideae only) across two subfamilies and two expanded clades within the Aizoaceae. The relative percentage of PIC was much greater in subfamilies Aizooideae and Mesembryanthemoideae than in Ruschioideae. Although nrITS had the highest percentage of PIC, barcoding gap analyses identified neither ITS nor any chloroplast region as suitable for barcoding of the family. From the results, it is evident that novel barcoding regions need to be explored within the Aizoaceae.