Target capture and genome skimming for plant diversity studies.

Recent technological advances in long-read high-throughput sequencing and assembly methods have facilitated the generation of annotated chromosome-scale whole-genome sequence data for evolutionary studies; however, generating such data can still be difficult for many plant species. For example, obtaining high-molecular-weight DNA is typically impossible for samples in historical herbarium collections, which often have degraded DNA. The need to fast-freeze newly collected living samples to conserve high-quality DNA can be complicated when plants are only found in remote areas. Therefore, short-read reduced-genome representations, such as target capture and genome skimming, remain important for evolutionary studies. Here, we review the pros and cons of each technique for non-model plant taxa. We provide guidance related to logistics, budget, the genomic resources previously available for the target clade, and the nature of the study. Furthermore, we assess the available bioinformatic analyses, detailing best practices and pitfalls, and suggest pathways to combine newly generated data with legacy data. Finally, we explore the possible downstream analyses allowed by the type of data generated using each technique. We provide a practical guide to help researchers make the best-informed choice regarding reduced genome representation for evolutionary studies of non-model plants in cases where whole-genome sequencing remains impractical.

A high quality, high molecular weight DNA extraction method for PacBio HiFi genome sequencing of recalcitrant plants.

BACKGROUND: PacBio HiFi sequencing provides highly accurate long-read sequencing datasets which are of great advantage for whole genome sequencing projects. One limitation of the method is the requirement for high quality, high molecular weight input DNA. This can be particularly challenging for plants that frequently contain common and species-specific secondary metabolites, which often interfere with downstream processes. Cape Primroses (genus Streptocarpus), are some of these recalcitrant plants and are selected here as material to develop a high quality, high molecular weight DNA extraction protocol for long read genome sequencing. RESULTS: We developed a DNA extraction method for PacBio HiFi sequencing for Streptocarpus grandis and Streptocarpus kentaniensis. A CTAB lysis buffer was employed to avoid guanidine, and the traditional chloroform and phenol purification steps were replaced with pre-lysis sample washes. Best cells/nucleus lysis was achieved with 4 h at 58 °C. The obtained high quality and high molecular weight DNAs were tested in PacBio SMRTBell™ library preparations, which resulted in circular consensus sequencing (CCS) reads from 17 to 27 Gb per cell, and a read length N50 from 14 to 17 kbp. To evaluate the quality of the reads for whole genome sequencing, they were assembled with HiFiasm into draft genomes, with N50 = 49 Mb and 23 Mb, and L50 = 10 and 11. The longest contigs were 95 Mb and 57 Mb respectively, showing good contiguity as these are longer than the theoretical chromosome length (genome size/chromosome number) of 78 Mb and 55 Mb, for S. grandis and S. kentaniensis respectively. CONCLUSIONS: DNA extraction is a critical step towards obtaining a complete genome assembly. Our DNA extraction method here provided the required high quality, high molecular weight DNA for successful standard-input PacBio HiFi library preparation. The contigs from those reads showed a high contiguity, providing a good starting draft assembly towards obtaining a complete genome. The results obtained here were highly promising, and demonstrated that the DNA extraction method developed here is compatible with PacBio HiFi sequencing and suitable for de novo whole genome sequencing projects of plants.

The first genome for the Cape Primrose Streptocarpus rexii (Gesneriaceae), a model plant for studying meristem-driven shoot diversity.

Cape Primroses (Streptocarpus, Gesneriaceae) are an ideal study system for investigating the genetics underlying species diversity in angiosperms. Streptocarpus rexii has served as a model species for plant developmental research for over five decades due to its unusual extended meristem activity present in the leaves. In this study, we sequenced and assembled the complete nuclear, chloroplast, and mitochondrial genomes of S. rexii using Oxford Nanopore Technologies long read sequencing. Two flow cells of PromethION sequencing resulted in 32 billion reads and were sufficient to generate a draft assembly including the chloroplast, mitochondrial and nuclear genomes, spanning 776 Mbp. The final nuclear genome assembly contained 5,855 contigs, spanning 766 Mbp of the 929-Mbp haploid genome with an N50 of 3.7 Mbp and an L50 of 57 contigs. Over 70% of the draft genome was identified as repeats. A genome repeat library of Gesneriaceae was generated and used for genome annotation, with a total of 45,045 genes annotated in the S. rexii genome. Ks plots of the paranomes suggested a recent whole genome duplication event, shared between S. rexii and Primulina huaijiensis. A new chloroplast and mitochondrial genome assembly method, based on contig coverage and identification, was developed, and successfully used to assemble both organellar genomes of S. rexii. This method was developed into a pipeline and proved widely applicable. The nuclear genome of S. rexii and other datasets generated and reported here will be invaluable resources for further research to aid in the identification of genes involved in morphological variation underpinning plant diversification.

Mix and match: Patchwork domain evolution of the land plant-specific Ca2+-permeable mechanosensitive channel MCA.

Multidomain proteins can have a complex evolutionary history that may involve de novo domain evolution, recruitment and / or recombination of existing domains and domain losses. Here, the domain evolution of the plant-specific Ca2+-permeable mechanosensitive channel protein, MID1-COMPLEMENTING ACTIVITY (MCA), was investigated. MCA, a multidomain protein, possesses a Ca2+-influx-MCAfunc domain and a PLAC8 domain. Profile Hidden Markov Models (HMMs) of domains were assessed in 25 viridiplantae proteomes. While PLAC8 was detected in plants, animals, and fungi, MCAfunc was found in streptophytes but not in chlorophytes. Full MCA proteins were only found in embryophytes. We identified the MCAfunc domain in all streptophytes including charophytes where it appeared in E3 ubiquitin ligase-like proteins. Our Maximum Likelihood (ML) analyses suggested that the MCAfunc domain evolved early in the history of streptophytes. The PLAC8 domain showed similarity to Plant Cadmium Resistance (PCR) genes, and the coupling of MCAfunc and PLAC8 seemed to represent a single evolutionary event. This combination is unique in MCA, and does not exist in other plant mechanosensitive channels. Within angiosperms, gene duplications increased the number of MCAs. Considering their role in mechanosensing in roots, MCA might be instrumental for the rise of land plants. This study provides a textbook example of de novo domain emergence, recombination, duplication, and losses, leading to the convergence of function of proteins in plants.

Phylogenomics of Gesneriaceae using targeted capture of nuclear genes.

Gesneriaceae (ca. 3400 species) is a pantropical plant family with a wide range of growth form and floral morphology that are associated with repeated adaptations to different environments and pollinators. Although Gesneriaceae systematics has been largely improved by the use of Sanger sequencing data, our understanding of the evolutionary history of the group is still far from complete due to the limited number of informative characters provided by this type of data. To overcome this limitation, we developed here a Gesneriaceae-specific gene capture kit targeting 830 single-copy loci (776,754 bp in total), including 279 genes from the Universal Angiosperms-353 kit. With an average of 557,600 reads and 87.8% gene recovery, our target capture was successful across the family Gesneriaceae and also in other families of Lamiales. From our bait set, we selected the most informative 418 loci to resolve phylogenetic relationships across the entire Gesneriaceae family using maximum likelihood and coalescent-based methods. Upon testing the phylogenetic performance of our baits on 78 taxa representing 20 out of 24 subtribes within the family, we showed that our data provided high support for the phylogenetic relationships among the major lineages, and were able to provide high resolution within more recent radiations. Overall, the molecular resources we developed here open new perspectives for the study of Gesneriaceae phylogeny at different taxonomical levels and the identification of the factors underlying the diversification of this plant group.

Virus-induced Gene Silencing in Streptocarpus rexii (Gesneriaceae).

Many members of the family Gesneriaceae are cultivated as ornamental plants, including Cape primrose (Streptocarpus) species. The range of plant architecture found in this genus has also made it a model to study leaf and meristem development and their evolution. However, the lack of tools to study gene functions through reverse genetics in Streptocarpus has limited the exploitation of its genetic potential. To aid functional genomic studies in Streptocarpus rexii, we sought to investigate virus-induced gene silencing (VIGS). Using the broad host range Tobacco Rattle Virus (TRV) to target the PHYTOENE DESATURASE (PDS) gene of S. rexii, we show that infection with sap from Nicotiana benthamiana triggered VIGS efficiently. VIGS was most effective in the seedling leaves 8 weeks after sowing, but was limited in duration and systemic spread. This study reports the first successful use of VIGS in Streptocarpus and in the family Gesneriaceae. The inoculation of viral sap derived from N. benthamiana was able to overcome the difficulties of standard Agrobacterium-mediated transformation in this genus. Irrespective of its transient effect, this VIGS system will be useful to assess gene function at the cellular level and represent an important tool for further understanding molecular mechanisms in Streptocarpus.

Tangled history of a multigene family: The evolution of ISOPENTENYLTRANSFERASE genes.

ISOPENTENYLTRANSFERASE (IPT) genes play important roles in the initial steps of cytokinin synthesis, exist in plant and pathogenic bacteria, and form a multigene family in plants. Protein domain searches revealed that bacteria and plant IPT proteins were to assigned to different protein domains families in the Pfam database, namely Pfam IPT (IPTPfam) and Pfam IPPT (IPPTPfam) families, both are closely related in the P-loop NTPase clan. To understand the origin and evolution of the genes, a species matrix was assembled across the tree of life and intensively in plant lineages. The IPTPfam domain was only found in few bacteria lineages, whereas IPPTPfam is common except in Archaea and Mycoplasma bacteria. The bacterial IPPTPfam domain miaA genes were shown as ancestral of eukaryotic IPPTPfam domain genes. Plant IPTs diversified into class I, class II tRNA-IPTs, and Adenosine-phosphate IPTs; the class I tRNA-IPTs appeared to represent direct successors of miaA genes were found in all plant genomes, whereas class II tRNA-IPTs originated from eukaryotic genes, and were found in prasinophyte algae and in euphyllophytes. Adenosine-phosphate IPTs were only found in angiosperms. Gene duplications resulted in gene redundancies with ubiquitous expression or diversification in expression. In conclusion, it is shown that IPT genes have a complex history prior to the protein family split, and might have experienced losses or HGTs, and gene duplications that are to be likely correlated with the rise in morphological complexity involved in fine tuning cytokinin production.

From shoot to leaf: step-wise shifts in meristem and KNOX1 activity correlate with the evolution of a unifoliate body plan in Gesneriaceae.

Typical dicots possess equal-sized cotyledons and leaf-bearing shoots topped with a shoot apical meristem (SAM), the source of lateral organs, and where KNOX1 homeobox genes act as key regulators. New World Gesneriaceae show typical cotyledons, whereas Old World Gesneriaceae show anisocotyly, the unequal post-germination growth of cotyledons, and include unifoliate (one-leaf) plants. One-leaf plants show an extremely reduced body plan: the adult above-ground photosynthetic tissue consisting of a single cotyledon, a macrocotyledon enlarged by the basal meristem (BM), but lacking a SAM. To investigate the origin and evolution of the BM and one-leaf plants, the meristem activity and KNOX1 SHOOTMERISTEMLESS (STM) expression in cotyledons and leaves were systematically studied by RT-PCR and in situ hybridization across the family Gesneriaceae, Jovellana in Calceolariaceae (sister family to Gesneriaceae), and Antirrhinum in Plantaginaceae, all families of order Lamiales (asterids), in comparison to Arabidopsis (Brassicales, rosids). In all examined Lamiales samples, unlike Arabidopsis, BM activity accompanied by STM expression was found in both cotyledons in early stages. Foliage leaves of Gesneriaceae and Jovellana also showed the correlation of BM and STM expression. An extension of BM activity was found following a phylogenetic trajectory towards one-leaf plants where it is active throughout the lifetime of the macrocotyledon. Our results suggest that KNOX1 involvement in early cotyledon expansion originated early on in the diversification of Lamiales and is proposed as the prerequisite for the evolution of vegetative diversity in Gesneriaceae. Step-wise morphological shifts, driven by transfers of meristematic activity, as evidenced by shifts in KNOX1 expression, may be one mechanism by which morphological diversity evolves in plants.

Taxonomic status, phylogenetic affinities and genetic diversity of a presumed extinct genus, Paraisometrum W.T. Wang (gesneriaceae) from the karst regions of Southwest China.

BACKGROUND: The karst regions in South China have an abundance of endemic plants that face high extinction risks. The Chinese Gesneriaceae endemic Paraisometrum mileense ( = Oreocharis mileensis), was presumed extinct for 100 years. After its re-discovery, the species has become one of five key plants selected by the Chinese forestry government to establish a new conservation category for plants with extremely small populations. For conservation purposes, we studied the phylogenetic and population genetic status of P. mileense at the three only known localities in Guangxi, Guizhou and Yunnan. METHODOLOGY/PRINCIPAL FINDINGS: We collected 64 samples (52 species) of Oreocharis and 8 samples from three provinces of P. mileense and generated molecular phylogenies, and inferred that P. mileense represents a relatively isolated and derived taxonomic unit within Oreocharis. Phylogeographic results of 104 samples of 12 populations of P. mileense indicated that the populations in Yunnan have derived from those in Guangxi and Guizhou. Based on AFLP data, the populations were found to harbor low levels of genetic diversity (He = 0.118), with no apparent gradient across the species' range, a restricted gene flow and significant isolation-by-distance with limited genetic differentiation among the populations across the three provinces (FST = 0.207, P<0.001). The 10 populations in Yunnan were found to represent two distinct lineages residing at different altitudes and distances from villages. CONCLUSION/SIGNIFICANCE: The low levels of genetic diversity found in P. mileense are perhaps a consequence of severe bottlenecks in the recent past. The distribution of the genetic diversity suggests that all populations are significant for conservation. Current in situ and ex situ measures are discussed. Further conservation actions are apparently needed to fully safeguard this conservation flagship species. Our work provides a model of an integrated study for the numerous endemic species in the karst regions with extremely small populations.

A complex case of simple leaves: indeterminate leaves co-express ARP and KNOX1 genes.

The mutually exclusive relationship between ARP and KNOX1 genes in the shoot apical meristem and leaf primordia in simple leaved plants such as Arabidopsis has been well characterized. Overlapping expression domains of these genes in leaf primordia have been described for many compound leaved plants such as Solanum lycopersicum and Cardamine hirsuta and are regarded as a characteristic of compound leaved plants. Here, we present several datasets illustrating the co-expression of ARP and KNOX1 genes in the shoot apical meristem, leaf primordia, and developing leaves in plants with simple leaves and simple primordia. Streptocarpus plants produce unequal cotyledons due to the continued activity of a basal meristem and produce foliar leaves termed "phyllomorphs" from the groove meristem in the acaulescent species Streptocarpus rexii and leaves from a shoot apical meristem in the caulescent Streptocarpus glandulosissimus. We demonstrate that the simple leaves in both species possess a greatly extended basal meristematic activity that persists over most of the leaf's growth. The area of basal meristem activity coincides with the co-expression domain of ARP and KNOX1 genes. We suggest that the co-expression of ARP and KNOX1 genes is not exclusive to compound leaved plants but is associated with foci of meristematic activity in leaves.

Temperature-dependent changes of cell shape during heterophyllous leaf formation in Ludwigia arcuata (Onagraceae).

Although elongation of epidermal cells in submerged leaves is thought to be a common feature of heterophyllous aquatic plants, such elongation has not been observed in Ludwigia arcuata Walt. (Onagraceae). In this study we found that reduced culture temperature induced the elongation of epidermal cells of submerged leaves in L. arcuata. Since such submerged leaves also showed a reduction in the number of epidermal cells aligned across the leaf transverse axis, these data indicate that heterophyllous leaf formation in L. arcuata is partially temperature sensitive, i.e., the elongation of epidermal cells was temperature sensitive while the reduction in the number of epidermal cells did not show such temperature sensitivity. To clarify the mechanisms that cause such temperature sensitivity, we examined the effects of ethylene, which induced the formation of submerged-type leaves on aerial shoots at the relatively high culture-temperature of 28 degrees C. At 23 degrees C, ethylene induced both cell elongation and reduction in the number of epidermal cells across the leaf transverse axis, while cell elongation was not observed at 28 degrees C. Moreover, both submergence and ethylene treatment induced a change in the arrangement of cortical microtubules (MTs) in epidermal cells of developing leaves at 23 degrees C. Such changes in the arrangement of MTs was not induced at 28 degrees C. Factors involved in the temperature-sensitive response to ethylene would be critical for temperature-sensitive heterophyllous leaf formation in L. arcuata.

Characterization of anisocotylous leaf formation in Streptocarpus wendlandii (Gesneriaceae): significance of plant growth regulators.

BACKGROUND AND AIMS: Unifoliate species of Gesneriaceae are unique, as they bear only one leaf throughout their life history. The development of this leaf (termed a macrocotyledon) derived from one of two cotyledons is intriguing. The other cotyledon does not develop further and is termed a microcotyledon. This process of unequal cotyledon development is termed anisocotyly. In this study the process of macrocotyeldon formation was studied and the effects of plant hormones on the macrocotyledon development were investigated. METHODS: Streptocarpus wendlandii was chosen as the main subject material, as it was found to be suitable for experimental studies in laboratory conditions. Morphological analyses were carried out with light and scanning electron microscopy. Plant hormones were applied exogenously. KEY RESULTS: The macrocotyledon of S. wendlandii is produced through cell division activity in the basal meristem of the enlarging cotyledon. The newly developed region in the macrocotyledon displayed distinct morphological changes, including the formation of long, needle-shaped trichomes. The newly formed region was surrounded by lateral veins. No such change was observed in the microcotyledon. Furthermore, it was shown that development of anisocotyly is suppressed by the application of cytokinin, resulting in the formation of two nearly equal-sized cotyledons. Both cotyledons displayed macrocotyledon characteristics. This observation in S. wendlandii was confirmed using Monophyllaea glabra, another unifoliate species in the same family. CONCLUSIONS: It is proposed that developmental changes of the macrocotyledon have characteristics of a developmental phase-change, and cytokinins may be involved in its formation. These results are discussed in the light of current knowledge of phase-change transitions in plant vegetative development.