Unraveling phylogenetic relationships, reticulate evolution, and genome composition of polyploid plant complexes by RAD-Seq and Hyb-Seq

Complex genome evolution of young polyploid complexes is poorly understood. Besides challenges caused by hybridization, polyploidization, and incomplete lineage sorting, bioinformatic analyses are often exacerbated by missing information on progenitors, ploidy, and reproduction modes. By using a comprehensive, self-developed bioinformatic pipeline covering tree, structure, network, and SNP-origin analyses, we for the first time unraveled polyploid phylogenetic relationships and genome evolution within the large Eurasian Ranunculus auricomus species complex comprising more than 840 taxa. Our results rely on 97,312 genomic RADseq loci, target enrichment of 576 nuclear genes (48 phased), and 71 plastid regions (Hybseq; OMICS-data) derived from the 75 most widespread polyploid apomictic taxa and four di- and one tetraploid potential sexual progenitor species. Phylogenetic tree and structure analyses consistently showed 3-5 supported polyploid groups, each containing sexual progenitor species. In total, analyses revealed four diploid sexual progenitors and a one unknown, probably extinct progenitor, contributing to the genome composition of R. auricomus polyploids. Phylogenetic network, structure, and SNP-origin analyses based on RADseq loci and phased nuclear genes completed by plastid data demonstrated predominantly allopolyploid origins, each involving 2-3 different diploid sexual subgenomes. Allotetraploid genomes were characterized by subgenome dominance and large proportions of interspecific, non-hybrid SNPs, indicating an enormous degree of post-origin evolution (i.e., Mendelian segregation of the diploid hybrid generations, back-crossings, and gene flow due to facultative sexuality of apomicts), but only low proportions of lineage-specific SNPs. The R. auricomus model system is the first large European polyploid species complex studied with reduced representation OMICS data. Our bioinformatic pipeline underlines the importance of combining different approaches and datasets to successfully unveil how reticulate evolution and post-origin processes shape the diversity of polyploid plant complexes.

Colchicine induced polyploidy in coriander (Coriandrum sativum L.)

The aim of this study was to find a suitable treatment combination that would effectively induce polyploidy in Coriander. In this study, colchicine concentrations and treatment durations were examined for improving the induction of polyploidy. The combinations of three colchicine concentrations such as 0.1%, 0.2% and 0.3% for 3 hrs per day for two to three days were tested in coriander. Three tetraploids were obtained in 0.2% colchicine treated population. The treatment of colchicine (cotton swab method) seedling with 0.2% for 3 days was suitable for induction of chromosome doubling. The control plant showed eleven bivalents (2n=2x=22) and polyploid plant showed twenty two bivalents (2n=4x=44) at diakinesis/metaphase-I in most of the PMCs. Anaphase-I distribution of chromosomes was normal (11:11) in control and in tetraploids distribution of chromosomes was (22:22) at each poles. In contrast with the normal plants, those treated by colchicine treatment often showed changes in height and width, in thickness of branches, in size, shape, texture of leaves, flowers, size of fruits and seed setting.

Origin and Fates of TERT Gene Copies in Polyploid Plants

The gene coding for the telomerase reverse transcriptase (TERT) is essential for the maintenance of telomeres. Previously we described the presence of three TERT paralogs in the allotetraploid plant Nicotiana tabacum, while a single TERT copy was identified in the paleopolyploid model plant Arabidopsis thaliana. Here we examine the presence, origin and functional status of TERT variants in allotetraploid Nicotiana species of diverse evolutionary ages and their parental genome donors, as well as in other diploid and polyploid plant species. A combination of experimental and in silico bottom-up analyses of TERT gene copies in Nicotiana polyploids revealed various patterns of retention or loss of parental TERT variants and divergence in their functions. RT–qPCR results confirmed the expression of all the identified TERT variants. In representative plant and green algal genomes, our synteny analyses show that their TERT genes were located in a conserved locus that became advantageous after the divergence of eudicots, and the gene was later translocated in several plant groups. In various diploid and polyploid species, translocation of TERT became fixed in target loci that show ancient synapomorphy.

Proxies of CRISPR-Cas9 activity to aid in the identification of mutagenized Arabidopsis plants

CRISPR-Cas9 has become the preferred gene editing technology to obtain loss-of-function mutants in plants, and hence a valuable tool to study gene function. This is mainly due to the easy reprograming of Cas9 specificity using customizable small non-coding RNAs, and to the ability to target several independent genes simultaneously. Despite these advances, the identification of CRISPR-edited plants remains time and resource consuming. Here, based on the premise that one editing event in one locus is a good predictor of editing event/s in other locus/loci, we developed a CRISPR co-editing selection strategy that greatly facilitates the identification of CRISPR-mutagenized Arabidopsis plants. This strategy is based on targeting the gene/s of interest simultaneously with a proxy of CRISPR-Cas9-directed mutagenesis. The proxy is an endogenous gene whose loss-of-function mutation produces an easy-to-detect visible phenotype that is unrelated to the expected phenotype of the gene/s under study. We tested this strategy via assessing the frequency of co-editing of three functionally unrelated proxies. We found all three proxies predicted the occurrence of mutations in either or both of the other two proxies with efficiencies ranging from 40% to 100%, dramatically reducing the number of plants that need to be screened to identify CRISPR mutants. This selection strategy provides a framework to facilitate gene function studies of gene families as well as the function of single copy genes in polyploid plant species where the identification of multiplex mutants remains challenging.

Assigning confidence scores to homoeologs using fuzzy logic

In polyploid genomes, homoeologs are a specific subtype of homologs, and can be thought of as orthologs between subgenomes. In Orthologous MAtrix, we infer homoeologs in three polyploid plant species: upland cotton (Gossypium hirsutum), rapeseed (Brassica napus), and bread wheat (Triticum aestivum). While we can typically recognize the features of a “good” homoeolog prediction (a consistent evolutionary distance, high synteny, and a one-to-one relationship), none of them is a hard-fast criterion. We devised a novel fuzzy logic-based method to assign confidence scores to each pair of predicted homoeologs. We inferred homoeolog pairs and used the new and improved method to assign confidence scores, which ranged from 0 to 100. Most confidence scores were between 70 and 100, but the distribution varied between genomes. The new confidence scores show an improvement over our previous method and were manually evaluated using a subset from various confidence ranges.