Application of CRISPR/Cas9 to Tragopogon (Asteraceae), an evolutionary model for the study of polyploidy.

Tragopogon (Asteraceae) is an excellent natural system for studies of recent polyploidy. Development of an efficient CRISPR/Cas9-based genome editing platform in Tragopogon will facilitate novel studies of the genetic consequences of polyploidy. Here, we report our initial results of developing CRISPR/Cas9 in Tragopogon. We have established a feasible tissue culture and transformation protocol for Tragopogon. Through protoplast transient assays, use of the TragCRISPR system (i.e. the CRISPR/Cas9 system adapted for Tragopogon) was capable of introducing site-specific mutations in Tragopogon protoplasts. Agrobacterium-mediated transformation with Cas9-sgRNA constructs targeting the phytoene desaturase gene (TraPDS) was implemented in this model polyploid system. Sequencing of PCR amplicons from the target regions indicated simultaneous mutations of two alleles and four alleles of TraPDS in albino shoots from Tragopogon porrifolius (2x) and Tragopogon mirus (4x), respectively. The average proportions of successfully transformed calli with the albino phenotype were 87% and 78% in the diploid and polyploid, respectively. This appears to be the first demonstration of CRISPR/Cas9-based genome editing in any naturally formed neopolyploid system. Although a more efficient tissue culture system should be developed in Tragopogon, application of a robust CRISPR/Cas9 system will permit unique studies of biased fractionation, the gene-balance hypothesis and cytonuclear interactions in polyploids. In addition, the CRISPR/Cas9 platform enables investigations of those genes involved in phenotypic changes in polyploids and will also facilitate novel functional biology studies in Asteraceae. Our workflow provides a guide for applying CRISPR/Cas9 to other nongenetic model plant systems.

What is Atraphaxis L. (Polygonaceae, Polygoneae): cryptic taxa and resolved taxonomic complexity instead of the formal lumping and the lack of morphological synapomorphies.

BACKGROUND: The recently proposed recircumscription of the genus Atraphaxis (incl. Atraphaxis section Ovczinnikovia O.V. Yurtseva ex. S. Tavakkoli and Polygonum sect. Spinescentia Boissier (=A. sect. Polygonoides S. Tavakkoli, Kaz. Osaloo & Mozaff.) makes this genus fairly heterogeneous and therefore almost undefinable based on morphology. A critical comprehensive reappraisal of the group is necessary. METHODS: Using the DNA sequence data (ITS1&2 regions of nrDNA and combined trnL intron + trnL-F IGS and rpl32-trnL((UAG)) IGS regions of plastid genome), Maximum Likelihood (ML) and Bayesian analyses (BI) were applied for phylogenetic reconstructions of the tribe Polygoneae with special attention to Atraphaxis, and related taxa. Maximum parsimony reconstructions of the evolution of perianth morphology and sporoderm ornamentation in the tribe Polygoneae were also performed. Life history, morphology of shoots, leaf blades, ocreas, perianth and achene morphology, ultrasculpture of achene surface, and pollen morphology were compared, and SEM and LM images were provided. PRINCIPAL FINDINGS: The genera Atraphaxis and Polygonum were found to be widely polyphyletic. The rarest and morphologically remarkable endemic of Tian-Shan and Pamir Atraphaxis ovczinnikovii (Atraphaxis sect. Ovczinnikovia O.V. Yurtseva ex. S. Tavakkoli) was confirmed to be a sister of the clade (Atraphaxis + Polygonum sect. Spinescentia) in plastid topology. The genus Bactria (=Atraphaxis sect. Ovczinnikovia), which circumscribes two species, is newly established as a result of this analyses. Morphological data confirm the originality of the taxon. DISCUSSION: We are arguing for a narrow delimitation of Atraphaxis with petalloid segments and striato-perforate sporoderm ornamentation as morphological synapomorphies. The recently proposed inclusion of Polygonum sect. Spinescentia in Atraphaxis is fairly questionable from a morphological standpoint. The rank of Polygonum sect. Spinescentia requires further clarification. The generic composition of the tribe Polygoneae also requires future reappraisals.

250 years of hybridization between two biennial herb species without speciation.

Hybridization between plant species can generate novel morphological diversity and lead to speciation at homoploid or polyploid levels. Hybrids between biennial herbs Tragopogon pratensis and T. porrifolius have been studied in experimental and natural populations for over 250 years. Here we examine their current status in natural populations in southeast England. All hybrids found were diploid; they tended to grow taller and with more buds than their parental species; many showed partial fertility; a few showed evidence of backcrossing. However, we found no evidence to suggest that the hybrids are establishing as a new species, nor can we find literature documenting speciation of these hybrids elsewhere. This lack of speciation despite at least 250 years of hybridization contrasts with the fact that both parental species have formed new allopolyploid species through hybridization with another diploid, T. dubius. Understanding why hybrids often do not speciate, despite repeated opportunities, would enhance our understanding of both the evolutionary process and risk assessments of invasive species.

Multiple origins and chromosomal novelty in the allotetraploid Tragopogon castellanus (Asteraceae).

Tragopogon includes two classic examples of recently formed allopolyploid species in North America: T. mirus and T. miscellus. Older Tragopogon allotetraploids from Eurasia offer ideal taxa for comparing the longer term outcomes of allopolyploidy. To help resolve the ancestry of one of these older polyploids, phylogenetic analyses of multiple populations of the allotetraploid T. castellanus (2n = 24) and its putative diploid parents, T. crocifolius and T. lamottei, were conducted using sequences from nuclear (internal transcribed spacer, ITS; and alcohol dehydrogenase 1A, Adh) and plastid (trnT-trnL spacer, trnL intron, trnL-trnF spacer and rpl16 intron) loci. Fluorescence in situ hybridization (FISH) and genomic in situ hybridization (GISH) were used to investigate the chromosomal constitution of T. castellanus. Our data confirm that the widely distributed T. crocifolius and the Iberian endemic, T. lamottei, are the diploid parents of T. castellanus, and that this polyploid formed at least three times. One group of populations of T. castellanus is distinct in exhibiting two pairs of rearranged chromosomes. These data suggest that some of the chromosomal variants that originate in young polyploids (here, an intergenomic translocation) may become fixed in populations, contributing to novelty in older polyploid lineages. The geographical distributions of the allopolyploids and parents are also complex, with allotetraploid populations being disjunct from one or both of the most closely related diploid parental populations.

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.