Lenka Havlickova

Genome sequence and genetic diversity of European ash trees

Ash trees (genus Fraxinus, family Oleaceae) are widespread throughout the Northern Hemisphere, but are being devastated in Europe by the fungus Hymenoscyphus fraxineus, causing ash dieback, and in North America by the herbivorous beetle Agrilus planipennis. Here we sequence the genome of a low-heterozygosity Fraxinus excelsior tree from Gloucestershire, UK, annotating 38,852 protein-coding genes of which 25% appear ash specific when compared with the genomes of ten other plant species. Analyses of paralogous genes suggest a whole-genome duplication shared with olive (Olea europaea, Oleaceae). We also re-sequence 37 F. excelsior trees from Europe, finding evidence for apparent long-term decline in effective population size. Using our reference sequence, we re-analyse association transcriptomic data, yielding improved markers for reduced susceptibility to ash dieback. Surveys of these markers in British populations suggest that reduced susceptibility to ash dieback may be more widespread in Great Britain than in Denmark. We also present evidence that susceptibility of trees to H. fraxineus is associated with their iridoid glycoside levels. This rapid, integrated, multidisciplinary research response to an emerging health threat in a non-model organism opens the way for mitigation of the epidemic.

Different stress responsive strategies to drought and heat in two durum wheat cultivars with contrasting water use efficiency

BackgroundDurum wheat often faces water scarcity and high temperatures, two events that usually occur simultaneously in the fields. Here we report on the stress responsive strategy of two durum wheat cultivars, characterized by different water use efficiency, subjected to drought, heat and a combination of both stresses.ResultsThe cv Ofanto (lower water use efficiency) activated a large set of well-known drought-related genes after drought treatment, while Cappelli (higher water use efficiency) showed the constitutive expression of several genes induced by drought in Ofanto and a modulation of a limited number of genes in response to stress. At molecular level the two cvs differed for the activation of molecular messengers, genes involved in the regulation of chromatin condensation, nuclear speckles and stomatal closure. Noteworthy, the heat response in Cappelli involved also the up-regulation of genes belonging to fatty acid β-oxidation pathway, glyoxylate cycle and senescence, suggesting an early activation of senescence in this cv. A gene of unknown function having the greatest expression difference between the two cultivars was selected and used for expression QTL analysis, the corresponding QTL was mapped on chromosome 6B.ConclusionOfanto and Cappelli are characterized by two opposite stress-responsive strategies. In Ofanto the combination of drought and heat stress led to an increased number of modulated genes, exceeding the simple cumulative effects of the two single stresses, whereas in Cappelli the same treatment triggered a number of differentially expressed genes lower than those altered in response to heat stress alone. This work provides clear evidences that the genetic system based on Cappelli and Ofanto represents an ideal tool for the genetic dissection of the molecular response to drought and other abiotic stresses.

Molecular markers for tolerance of European ash (Fraxinus excelsior) to dieback disease identified using Associative Transcriptomics.

Tree disease epidemics are a global problem, impacting food security, biodiversity and national economies. The potential for conservation and breeding in trees is hampered by complex genomes and long lifecycles, with most species lacking genomic resources. The European Ash tree Fraxinus excelsior is being devastated by the fungal pathogen Hymenoscyphus fraxineus, which causes ash dieback disease. Taking this system as an example and utilizing Associative Transcriptomics for the first time in a plant pathology study, we discovered gene sequence and gene expression variants across a genetic diversity panel scored for disease symptoms and identified markers strongly associated with canopy damage in infected trees. Using these markers we predicted phenotypes in a test panel of unrelated trees, successfully identifying individuals with a low level of susceptibility to the disease. Co-expression analysis suggested that pre-priming of defence responses may underlie reduced susceptibility to ash dieback.

Extensive homoeologous genome exchanges in allopolyploid crops revealed by mRNAseq‐based visualization

Summary Polyploidy, the possession of multiple sets of chromosomes, has been a predominant factor in the evolution and success of the angiosperms. Although artificially formed allopolyploids show a high rate of genome rearrangement, the genomes of cultivars and germplasm used for crop breeding were assumed stable and genome structural variation under the artificial selection process of commercial breeding has remained little studied. Here, we show, using a repurposed visualization method based on transcriptome sequence data, that genome structural rearrangement occurs frequently in varieties of three polyploid crops (oilseed rape, mustard rape and bread wheat), meaning that the extent of genome structural variation present in commercial crops is much higher than expected. Exchanges were found to occur most frequently where homoeologous chromosome segments are collinear to telomeres and in material produced as doubled haploids. The new insights into genome structural evolution enable us to reinterpret the results of recent studies and implicate homoeologous exchanges, not deletions, as being responsible for variation controlling important seed quality traits in rapeseed. Having begun to identify the extent of genome structural variation in polyploid crops, we can envisage new strategies for the global challenge of broadening crop genetic diversity and accelerating adaptation, such as the molecular identification and selection of genome deletions or duplications encompassing genes with trait‐controlling dosage effects.

Validation of an updated Associative Transcriptomics platform for the polyploid crop species Brassica napus by dissection of the genetic architecture of erucic acid and tocopherol isoform variation in seeds

Summary An updated platform was developed to underpin association genetics studies in the polyploid crop species Brassica napus (oilseed rape). Based on 1.92 × 1012 bases of leaf mRNAseq data, functional genotypes, comprising 355 536 single‐nucleotide polymorphism markers and transcript abundance were scored across a genetic diversity panel of 383 accessions using a transcriptome reference comprising 116 098 ordered coding DNA sequence (CDS) gene models. The use of the platform for Associative Transcriptomics was first tested by analysing the genetic architecture of variation in seed erucic acid content, as high‐erucic rapeseed oil is highly valued for a variety of applications in industry. Known loci were identified, along with a previously undetected minor‐effect locus. The platform was then used to analyse variation for the relative proportions of tocopherol (vitamin E) forms in seeds, and the validity of the most significant markers was assessed using a take‐one‐out approach. Furthermore, the analysis implicated expression variation of the gene Bo2g050970.1, an orthologue of VTE4 (which encodes a γ‐tocopherol methyl transferase converting γ‐tocopherol into α‐tocopherol) associated with the observed trait variation. The establishment of the first full‐scale Associative Transcriptomics platform for B. napus enables rapid progress to be made towards an understanding of the genetic architecture of trait variation in this important species, and provides an exemplar for other crops.

Carbohydrate microarrays and their use for the identification of molecular markers for plant cell wall composition

Significance Plant cell wall (PCW) composition determines the nature and quality of many biologically derived products and, therefore, is a major target for genetic improvement. However, the identities and functions of many genes involved in PCW synthesis are still not known. Genome-wide association mapping studies (GWAS) are one of the few ways to identify these genes. However, collecting precise and quantitative PCW phenotype data at the scale required for GWAS is a significant challenge. Here, we demonstrate that high-density carbohydrate microarrays can be used as a PCW phenotyping strategy suitable for GWAS. Results presented in this study will aid in the understanding of PCW genetics and crop breeding for improved PCW composition. Genetic improvement of the plant cell wall has enormous potential to increase the quality of food, fibers, and fuels. However, the identification and characterization of genes involved in plant cell wall synthesis is far from complete. Association mapping is one of the few techniques that can help identify candidate genes without relying on our currently incomplete knowledge of cell wall synthesis. However, few cell wall phenotyping methodologies have proven sufficiently precise, robust, or scalable for association mapping to be conducted for specific cell wall polymers. Here, we created high-density carbohydrate microarrays containing chemically extracted cell wall polysaccharides collected from 331 genetically diverse Brassica napus cultivars and used them to obtain detailed, quantitative information describing the relative abundance of selected noncellulosic polysaccharide linkages and primary structures. We undertook genome-wide association analysis of data collected from 57 carbohydrate microarrays and identified molecular markers reflecting a diversity of specific xylan, xyloglucan, pectin, and arabinogalactan moieties. These datasets provide a detailed insight into the natural variations in cell wall carbohydrate moieties between B. napus genotypes and identify associated markers that could be exploited by marker-assisted breeding. The identified markers also have value beyond B. napus for functional genomics, facilitated by the close genetic relatedness to the model plant Arabidopsis. Together, our findings provide a unique dissection of the genetic architecture that underpins plant cell wall biosynthesis and restructuring.

Detection of self-incompatible oilseed rape plants (Brassica napus L.) based on molecular markers for identification of the class I S haplotype.

The selection of desirable genotypes with recessive characteristics, such as self-incompatible plants, is often difficult or even impossible and represents a crucial barrier in accelerating the breeding process. Molecular approaches and selection based on molecular markers can allow breeders to overcome this limitation. The use of self-incompatibility is an alternative in hybrid breeding of oilseed rape. Unfortunately, stable self-incompatibility is recessive and phenotype-based selection is very difficult and time-consuming. The development of reliable molecular markers for detecting desirable plants with functional self-incompatible genes is of great importance for breeders and allows selection at early stages of plant growth. Because most of these reliable molecular markers are based on discrimination of class I S-locus genes that are present in self-compatible plants, there is a need to use an internal control in order to detect possible PCR inhibition that gives false results during genotyping. In this study, 269 double haploid F2 oilseed rape plants obtained by microspore embryogenesis were used to verify the applicability of an improved PCR assay based on the detection of the class I SLG gene along with an internal control. Comparative analysis of the PCR genotyping results vs. S phenotype analysis confirmed the applicability of this molecular approach in hybrid breeding programs. This approach allows accurate detection of self-incompatible plants via a different amplification profile.

Molecular genetics and functional genomics of abiotic stress-responsive genes in oilseed rape (Brassica napus L.): a review of recent advances and future

Abiotic stresses are the key factors which negatively influence plant development and productivity and are the main cause of extensive agricultural production losses worldwide. Brassica napus is an oilseed crop of global economic significance and major contributor to the total oilseed production, quite often encounters abiotic stresses, resulting in reduced agricultural productivity. Hence, there is an immediate need being felt to raise B. napus cultivars which would be more suitable for various abiotic stress conditions presently and in the years to come. Biotechnology and molecular plant breeding has emerged as an important tool for molecular understanding of plant response to various abiotic stresses. Currently, various stress-responsive genes and mechanisms have been identified and functionally characterized in model plant Arabidopsis and other major crop plants such as Oryza sativa and Zea mays. However, very inadequate success has been achieved in this direction in a major oilseed crop such as B. napus. In this review, we present the latest methods and approaches of studying abiotic stress in B. napus. In this review, we describe the genes functioning as markers for crop breeding and discuss the recent progress and advances in genome editing by break through CRISPR/Cas9 multigene–multiplex approaches for developing multiple abiotic stress tolerance with our on-going research as a scheme. We also throw some light on molecular genetics, plant breeding and abiotic stress biotechnology of B. napus which offer a new prospective on the research directions for the practical plant breeding and functional genomics of B. napus in response to different abiotic stress conditions.

Identification of candidate genes for calcium and magnesium accumulation in Brassica napus L. by association genetics

Calcium (Ca) and magnesium (Mg) are essential plant nutrients and vital for human and animal nutrition. Biofortification of crops has previously been suggested to alleviate widespread human Ca and Mg deficiencies. In this study, new candidate genes influencing the leaf accumulation of Ca and Mg were identified in young Brassica napus plants using associative transcriptomics of ionomics datasets. A total of 247 and 166 SNP markers were associated with leaf Ca and Mg concentration, respectively, after false discovery rate correction and removal of SNPs with low second allele frequency. Gene expression markers at similar positions were also associated with leaf Ca and Mg concentration, including loci on chromosomes A10 and C2, within which lie previously identified transporter genes ACA8 and MGT7. Further candidate genes were selected from seven loci and the mineral composition of whole Arabidopsis thaliana shoots were characterized from lines mutated in orthologous genes. Four and two mutant lines had reduced shoot Ca and Mg concentration, respectively, compared to wild type plants. Three of these mutations were found to have tissue specific effects; notably reduced silique Ca in all three such mutant lines. This knowledge could be applied in targeted breeding, with the possibility of increasing Ca and Mg in plant tissue for improving human and livestock nutrition.

Species-wide variation in shoot nitrate concentration, and genetic loci controlling nitrate, phosphorus and potassium accumulation in Brassica napus L.

Large nitrogen, phosphorus and potassium fertilizer inputs are used in many crop systems. Identifying genetic loci controlling nutrient accumulation may be useful in crop breeding strategies to increase fertilizer use efficiency and reduce financial and environmental costs. Here, variation in leaf nitrate concentration across a diversity population of 383 genotypes of Brassica napus was characterized. Genetic loci controlling variation in leaf nitrate, phosphorus and potassium concentration were then identified through Associative Transcriptomics using single nucleotide polymorphism (SNP) markers and gene expression markers (GEMs). Leaf nitrate concentration varied over 8-fold across the diversity population. A total of 455 SNP markers were associated with leaf nitrate concentration after false-discovery-rate (FDR) correction. In linkage disequilibrium of highly associated markers are a number of known nitrate transporters and sensors, including a gene thought to mediate expression of the major nitrate transporter NRT1.1. Several genes influencing root and root-hair development co-localize with chromosomal regions associated with leaf P concentration. Orthologs of three ABC-transporters involved in suberin synthesis in roots also co-localize with association peaks for both leaf nitrate and phosphorus. Allelic variation at nearby, highly associated SNPs confers large variation in leaf nitrate and phosphorus concentration. A total of five GEMs associated with leaf K concentration after FDR correction including a GEM that corresponds to an auxin-response family protein. Candidate loci, genes and favorable alleles identified here may prove useful in marker-assisted selection strategies to improve fertilizer use efficiency in B. napus.