Comprehensive Identification and Expression Analysis of CRY Gene Family in Gossypium

Background: The cryptochromes (CRY) comprise a specific blue light receptor for plants and animals, which play crucial roles in physiological processes of plant growth, development, and stress tolerance. Results: In the present work, a systematical analysis of CRY gene family from five allotetraploid cotton species, G. hirsutum, G. barbadense, G. tomentosum, G. mustelinum and G. darwinii together with seven diploid species. There were 18, 17, 17, 17, and 17 CRYs identified in G. hirsutum, G. barbadense, G. tomentosum, G. mustelinum and G. darwinii, respectively, whereas five to nine CRY genes were identified in the diploid species. Phylogenetic analysis of the protein-coding sequences revealed that CRY genes from the allotetraploids G. hirsutum and G. barbadense, three diploid cotton species (G. raimondii, G. herbaceum, and G. arboreum), and Arabidopsis thaliana could be classified into seven clades. Synteny analysis suggested that the homoeolog of G. hirsutum Gh_A02G0384 has undergone an evolutionary loss event in the other four allotetraploid cotton species. Cis-element analysis predicated the possible functions of CRY genes in G. hirsutum. Public RNA-seq data were investigated to analyze the expression patterns of G. hirsutum CRY genes in various tissues as well as gene expressions under abiotic stress treatments. Conclusion: These results indicated the possible functions of G. hirsutum CRY genes in differential tissues as well as in response to abiotic stress during the cotton plants life cycle.

The last missing piece of the Triangle of U: the evolution of the tetraploid Brassica carinata genome

Ethiopian mustard (Brassica carinata) is an ancient crop with significant potential for expanded cultivation as a biodiesel feedstock. The remarkable stress resilience of B. carinata and desirable seed fatty acid profile addresses the ongoing food vs. fuel debate as the crop is productive on marginal lands otherwise not suitable for even closely related species. B. carinata is one of six key Brassica spp. that share three major genomes: three diploid species (AA, BB, CC) that spontaneously hybridized in a pairwise manner, forming three allotetraploid species (AABB, AACC, and BBCC). Each of these genomes has been researched extensively, except for that of B. carinata. In the present study, we report a high-quality, 1.31 Gbp genome with 156.9-fold sequencing coverage for B. carinata var. Gomenzer, completing and confirming the classic Triangle of U, a theory of the evolutionary relationships among these six species that arose almost a century ago. Our assembly provides insights into the genomic features that give rise to B. carinata's superior agronomic traits for developing more climate-resilient Brassica crops with excellent oil production. Notably, we identified an expansion of transcription factor networks and agronomically-important gene families. Completing the Triangle of U comparative genomics platform allowed us to examine the dynamics of polyploid evolution and the role of subgenome dominance in domestication and agronomical improvement.

Smooth Descent: a Ploidy-Aware Algorithm To Improve Linkage Mapping In The Presence of Genotyping Errors

Linkage mapping is an approach to order markers based on recombination events. Mapping algorithms cannot easily handle genotyping errors, which are common in high-throughput genotyping data. To solve this issue, strategies have been developed, aimed mostly at identifying and eliminating these errors. One such strategy is SMOOTH (van Os et al. 2005), an iterative algorithm to detect genotyping errors. Unlike other approaches, SMOOTH can also be used to impute the most probable alternative genotypes, but its application is limited to diploid species and to markers heterozygous in only one of the parents. In this study we adapted SMOOTH to expand its use to any marker type and to autopolyploids with the use of identity-by-descent probabilities, naming the updated algorithm Smooth Descent (SD). We applied SD to real and simulated data, showing that in the presence of genotyping errors this method produces better genetic maps in terms of marker order and map length. SD is particularly useful for error rates between 5% and 20% and when error rates are not homogeneous among markers or individuals. Moreover, the simplicity of the algorithm allows thousands of markers to be efficiently processed, thus being particularly useful for error detection in high-density datasets. We have implemented this algorithm in the R package SmoothDescent.

Chloroplast phylogenomics in Camelina (Brassicaceae) reveals multiple origins of polyploid species and the maternal lineage of C. sativa

The genus Camelina (Brassicaceae) comprises 7–8 diploid, tetraploid, and hexaploid species. Of particular agricultural interest is the biofuel crop, C. sativa (gold-of-pleasure or false flax), an allohexaploid domesticated from the widespread weed, C. microcarpa. Recent cytogenetics and genomics work has uncovered the identity of the parental diploid species involved in ancient polyploidization events in Camelina. However, little is known about the maternal subgenome ancestry of contemporary polyploid species. To determine the diploid maternal contributors of polyploid Camelina lineages, we sequenced and assembled 84 Camelina chloroplast genomes for phylogenetic analysis. Divergence time estimation was used to infer the timing of polyploidization events. Chromosome counts were also determined for 82 individuals to assess ploidy and cytotypic variation. Chloroplast genomes showed minimal divergence across the genus, with no observed gene-loss or structural variation. Phylogenetic analyses revealed C. hispida as a maternal diploid parent to the allotetraploid Camelina rumelica, and C. neglecta as the closest extant diploid contributor to the allohexaploids C. microcarpa and C. sativa. The tetraploid C. rumelica appears to have evolved through multiple independent hybridization events. Divergence times for polyploid lineages closely related to C. sativa were all inferred to be very recent, at only ~65 thousand years ago. Chromosome counts confirm that there are two distinct cytotypes within C. microcarpa (2n = 38 and 2n = 40). Based on these findings and other recent research, we propose a model of Camelina subgenome relationships representing our current understanding of the hybridization and polyploidization history of this recently-diverged genus.

Karyotype and genome size variation in white-flowered Eranthis sect. Shibateranthis (Ranunculaceae)

Comparative karyomorphological analyses of six out of the eight white-flowered species of Eranthis sect. Shibateranthis have been carried out. All studied specimens of E. byunsanensis, E. lobulata, E. pinnatifida, and E. stellata had a somatic chromosome number 2n = 16 with basic chromosome number x = 8. On the contrary, E. tanhoensis and E. sibirica had a basic chromosome number x = 7. The specimens of E. tanhoensis were diploid with 2n = 14, while the specimens of E. sibirica were polyploid with 2n = 42. Monoploid chromosome sets of the investigated diploid species had 4–5 metacentric chromosomes and 2–4 submetacentric/subtelocentric/acrocentric chromosomes. The highest level of interchromosomal asymmetry, estimated via CVCL, was found in E. byunsanensis and E. pinnatifida. The highest levels of intrachromosomal asymmetry (MCA) and heterogeneity in centromere position (CVCI) were found in E. lobulata and E. byunsanensis, while E. sibirica had the most symmetric karyotype. A multivariate PCoA analysis of basic karyotype parameters (2n, x, THL, CVCL, MCA, and CVCI) highlighted no overlap among species accessions, which was also confirmed by LDA. The average absolute monoploid DNA content (1Cx) of the 23 investigated samples of six Eranthis species varied from 9.26 ± 0.25 pg in E. sibirica to 15.93 ± 0.32 pg in E. stellata. Overall karyological affinity was highlighted between E. lobulata and E. stellata, on one side, and between E. byunsanensis and E. pinnatifida, on the other side. Interestingly, there was no significant correlation between total haploid (monoploid) chromosome length (THL) and 1Cx values in these species.

Development of an SNP Marker Set for Marker-Assisted Backcrossing Using Genotyping-By-Sequencing in Tetraploid Perilla

High-quality molecular markers are essential for marker-assisted selection to accelerate breeding progress. Compared with diploid species, recently diverged polyploid crop species tend to have highly similar homeologous subgenomes, which is expected to limit the development of broadly applicable locus-specific single-nucleotide polymorphism (SNP) assays. Furthermore, it is particularly challenging to make genome-wide marker sets for species that lack a reference genome. Here, we report the development of a genome-wide set of kompetitive allele specific PCR (KASP) markers for marker-assisted recurrent selection (MARS) in the tetraploid minor crop perilla. To find locus-specific SNP markers across the perilla genome, we used genotyping-by-sequencing (GBS) to construct linkage maps of two F2 populations. The two resulting high-resolution linkage maps comprised 2,326 and 2,454 SNP markers that spanned a total genetic distance of 2,133 cM across 16 linkage groups and 2,169 cM across 21 linkage groups, respectively. We then obtained a final genetic map consisting of 22 linkage groups with 1,123 common markers from the two genetic maps. We selected 96 genome-wide markers for MARS and confirmed the accuracy of markers in the two F2 populations using a high-throughput Fluidigm system. We confirmed that 91.8% of the SNP genotyping results from the Fluidigm assay were the same as the results obtained through GBS. These results provide a foundation for marker-assisted backcrossing and the development of new varieties of perilla.

Chromosome Doubling in Genetically Diverse Bilberry (Vaccinium myrtillus L.) Accessions and Evaluation of Tetraploids in Terms of Phenotype and Ability to Cross with Highbush Blueberry (V. corymbosum L.)

To expand the gene pool and introduce new traits to the tetraploid cultivars of Vaccinium corymbosum from wild diploid species V. myrtillus, it is necessary to double the chromosome number in diploid species in order to overcome a post zygotic crossing barrier and a strong triploid block, existing within the genus Vaccinium. Five genetically diverse bilberry genotypes were selected from 21 accessions taken from the breeding collection of the National Institute of Horticultural Research (Skierniewice, Poland) for this study. The bilberry genotypes were derived from the Polish locations of Bolimów Landscape Park, Budy Grabskie and forest complex Zwierzyniec (Łódź Province), and habitats in Norway. The selection of genotypes was made based on the analysis of amplified fragment length polymorphism (AFLP-PCR). Analysis of the Jaccard similarity indexes and the UPGMA method revealed that the examined accessions formed two main groups on the dendrogram. The first group consisted of accessions from Norway, while the second group agglomerated Polish accessions. A further two classes were distinguished in the Polish group: the first included accessions from Budy Grabskie and the second from Zwierzyniec, located ca. 9 km from Budy Grabskie. In order to obtain plant material for in vitro polyploidisation, in vitro shoot cultures of the selected accessions were initiated and multiplied. Both antimitotics used, colchicine and APM, induced tetraploids for all of the accessions. The obtained tetraploids were multiplied, rooted ex vitro and grown in a greenhouse and then in a field. The first flowering was observed in 1.5-year-old plants, either diploid or tetraploid. Diploids bloomed slightly earlier and more profusely than tetraploid plants. Compared to diploids, autotetraploids had significantly larger flowers by ca. 64% and larger pollen tetrads by ca. 35%. The germination capacity of pollen tetrads was high in tetraploids (87.8%), although slightly lower than in diploids (94.3%). After pollinating the flowers of three highbush blueberry cultivars with pollen from the bilberry tetraploid accession, J-4-4x, the plants formed fruits, some of which contained properly formed seeds. The effectiveness of interspecific crossing between V. corymbosum and tetraploid V. myrtillus, defined as the percentage of obtained seedlings in relation to the number of pollinated flowers, was highest (53.3%) in the blueberry ‘Liberty’, and lower in ‘Bluecrop’ and ‘Northland’, 14.8% and 10.0%, respectively. Before using the seedlings for further breeding, their hybridity will be confirmed by molecular markers and the phenotype will be evaluated.

Precise editing using CRISPR-Cas9 to explore the contribution of clinically-derived mutations to antifungal resistance in the pathogenic yeast Candida parapsilosis

Introduction Candida parapsilosis is both a commensal/saprophytic yeast of the human skin and an opportunistic pathogen which can be responsible for life-threatening infections. The increasing reports of clonal outbreaks involving azole-resistant C. parapsilosis in the clinical setting is worrisome and urges for a better understanding of antifungal resistance in this species. Previous studies have identified mutations in key genes which can explain acquired fluconazole resistance. Reverse genetics approaches are now warranted to confirm their involvement and to determine whether they can affect other clinically-licensed antifungals. Here, we used a CRISPR-Cas9 technique to study the relative contributions of clinically-derived mutations to antifungal resistance and provide answers to these questions. Materials and Methods Six clinically-derived mutations were selected (ERG11Y132F, ERG11K143R,ERG11R398I, TAC1G650E, MRR1G583R, ERG3G111R) to be engineered in two C. parapsilosis fluconazole-susceptible backgrounds (ATCC22019, STZ5) using a previously described CRISPR-Cas9 method. In vitro susceptibility of the transformants to fluconazole, voriconazole, posaconazole, isavuconazole and micafungin was determined by Etest®. Results/Discussion The impact on fluconazole susceptibility was highly variable depending on the residue/gene involved, but roughly similar between the two genetic backgrounds. All but two(ERG11R398I, ERG3G111R) conferred fluconazole resistance, though the highest MIC increase was observed for MRR1G583R (≥650 fold). As expected in a diploid species, we noted an impact of allelic dosage. Some kind of cross-resistance to the other azoles was noted from some mutations, although the impact was lower for posaconazole and isavuconazole, except for MRR1G583R which led to multi-azole resistance. Finally, ERG3G111R increased tolerance to both azoles and echinocandins.

The genetic diversity of Asplenium viride (Aspleniaceae) fern colonizing heavy metal-polluted sites

Heavy metals can affect the morphology, physiology and evolution of plants. Asplenium viride is a diploid species, belonging to the largest genus of the cosmopolitan fern family Aspleniaceae, and occurring on various types of alkaline rocks. It is known to colonize sites with high concentrations of heavy metals, exhibiting changes in frond morphology. Microevolutionary processes, manifesting as changes in genome size and new genotype formation, can ultimately lead to the formation of new subspecies and speciation. This study aimed to evaluate the morphological and genetic diversity of A. viride, and to test for a potential correlation between variability and heavy metal concentration. Analysis of A. viride specimens, from one metalliferous site and five non-metalliferous localities, showed no apparent variation in genome size between plants from affected and non-affected sites. There was no significant correlation between genetic variability and heavy metal concentration. This was possibly due to intragametophytic selfing, caused by patchy habitats and subsequent founder effects, resulting from long-distance colonization by single spores.

Allele segregation analysis of F1 hybrids between independent Brassica allohexaploid lineages

In the Brassica genus we find both diploid species (one genome) and allotetraploid species (two different genomes) but no naturally occurring hexaploid species (three different genomes, AABBCC). Although hexaploids can be produced via human intervention, these neo-polyploids have quite unstable genomes and usually suffer from severe genome reshuffling. Whether these genome rearrangements continue in later generations and whether genomic arrangements follow similar, reproducible patterns between different lines is still unknown. We crossed Brassica hexaploids resulting from different species combinations to produce five F1 hybrids, and analyzed the karyotypes of the parents and the F1 hybrids, as well as allele segregation in a resulting test-cross population via molecular karyotyping using SNP array genotyping. Although some genomic regions were found to be more likely to be duplicated, deleted or rearranged, a consensus pattern was not shared between genotypes. Brassica hexaploids had a high tolerance for fixed structural rearrangements, but which rearrangements occur and become fixed over many generations does not seem to show either strong reproducibility or to indicate selection for stability. On average, we observed 10 de novo chromosome rearrangements contributed almost equally from both parents to the F1 hybrids. At the same time, the F1 hybrid meiosis produced on average 8.6 new rearrangements. Hence, the increased heterozygosity in the F1 hybrid did not significantly improve genome stability in our hexaploid hybrids, and might have had the opposite effect. However, hybridization between lineages was readily achieved and may be exploited for future genetics and breeding purposes.