Transcriptomic analyses to summarize gene expression patterns that occur during leaf initiation of Chinese cabbage.

In Chinese cabbage, rosette leaves expose their adaxial side to the light converting light energy into chemical energy, acting as a source for the growth of the leafy head. In the leafy head, the outer heading leaves expose their abaxial side to the light while the inner leaves are shielded from the light and have become a sink organ of the growing Chinese cabbage plant. Interestingly, variation in several ad/abaxial polarity genes is associated with the typical leafy head morphotype. The initiation of leaf primordia and the establishment of leaf ad/abaxial polarity are essential steps in the initiation of marginal meristem activity leading to leaf formation. Understanding the molecular genetic mechanisms of leaf primordia formation, polar differentiation, and leaf expansion is thus relevant to understand leafy head formation. As Brassica's are mesa-hexaploids, many genes have multiple paralogues, complicating analysis of the genetic regulation of leaf development. In this study, we used laser dissection of Chinese cabbage leaf primordia and the shoot apical meristem (SAM) to compare gene expression profiles between both adaxial and abaxial sides and the SAM aiming to capture transcriptome changes underlying leaf primordia development. We highlight genes with roles in hormone pathways and transcription factors. We also assessed gene expression gradients along expanded leaf blades from the same plants to analyze regulatory links between SAM, leaf primordia and the expanding rosette leaf. The catalogue of differentially expressed genes provides insights in gene expression patterns involved in leaf development and form a starting point to unravel leafy head formation.

Cabbage (Brassica oleracea var. capitata) Development in Time: How Differential Parenchyma Tissue Growth Affects Leafy Head Formation.

This study aims to categorize the morphological changes during cabbage (B. oleracea ssp. capitata) development, seedling, rosette, folding, and heading, and to elucidate the cellular mechanisms of the leaf curvature, essential for the formation of the leafy head. We followed the growth of two cabbage cultivars with distinct head shapes (round and pointed) and one non-heading collard cultivar; we phenotyped the size and volume of the whole plant as well as the size, shape, and curvature of the leaves during growth. By integrating these phenotypic data, we determined the four vegetative stages for both cabbages. The histological phenotypes of microtome sections from five distinct leaf positions of the rosette, folding, and heading leaves at two timepoints during leaf growth were quantified and revealed variations in cellular parameters among leaf types, between leaf positions, and between the adaxial and abaxial sides. We identified two synergistic cellular mechanisms contributing to the curvature of heading leaves: differential growth across the leaf blade, with increased growth at the leaf's center relative to the margins; and the increased expansion of the spongy parenchyma layer compared to the palisade parenchyma layer, resulting in the direction of the curvature, which is inwards. These two processes together contribute to the typical leafy heads of cabbages.

Large-scale gene expression alterations introduced by structural variation drive morphotype diversification in Brassica oleracea.

Brassica oleracea, globally cultivated for its vegetable crops, consists of very diverse morphotypes, characterized by specialized enlarged organs as harvested products. This makes B. oleracea an ideal model for studying rapid evolution and domestication. We constructed a B. oleracea pan-genome from 27 high-quality genomes representing all morphotypes and their wild relatives. We identified structural variations (SVs) among these genomes and characterized these in 704 B. oleracea accessions using graph-based genome tools. We show that SVs exert bidirectional effects on the expression of numerous genes, either suppressing through DNA methylation or promoting probably by harboring transcription factor-binding elements. The following examples illustrate the role of SVs modulating gene expression: SVs promoting BoPNY and suppressing BoCKX3 in cauliflower/broccoli, suppressing BoKAN1 and BoACS4 in cabbage and promoting BoMYBtf in ornamental kale. These results provide solid evidence for the role of SVs as dosage regulators of gene expression, driving B. oleracea domestication and diversification.

A butterfly egg-killing hypersensitive response in Brassica nigra is controlled by a single locus, PEK, containing a cluster of TIR-NBS-LRR receptor genes.

Knowledge of plant recognition of insects is largely limited to a few resistance (R) genes against sap-sucking insects. Hypersensitive response (HR) characterizes monogenic plant traits relying on R genes in several pathosystems. HR-like cell death can be triggered by eggs of cabbage white butterflies (Pieris spp.), pests of cabbage crops (Brassica spp.), reducing egg survival and representing an effective plant resistance trait before feeding damage occurs. Here, we performed genetic mapping of HR-like cell death induced by Pieris brassicae eggs in the black mustard Brassica nigra (B. nigra). We show that HR-like cell death segregates as a Mendelian trait and identified a single dominant locus on chromosome B3, named PEK (Pieris  egg- killing). Eleven genes are located in an approximately 50 kb region, including a cluster of genes encoding intracellular TIR-NBS-LRR (TNL) receptor proteins. The PEK locus is highly polymorphic between the parental accessions of our mapping populations and among B. nigra reference genomes. Our study is the first one to identify a single locus potentially involved in HR-like cell death induced by insect eggs in B. nigra. Further fine-mapping, comparative genomics and validation of the PEK locus will shed light on the role of these TNL receptors in egg-killing HR.

Interploidy Introgression Shaped Adaptation during the Origin and Domestication History of Brassica napus.

Polyploidy is recurrent across the tree of life and known as an evolutionary driving force in plant diversification and crop domestication. How polyploid plants adapt to various habitats has been a fundamental question that remained largely unanswered. Brassica napus is a major crop cultivated worldwide, resulting from allopolyploidy between unknown accessions of diploid B. rapa and B. oleracea. Here, we used whole-genome resequencing data of accessions representing the majority of morphotypes and ecotypes from the species B. rapa, B. oleracea, and B. napus to investigate the role of polyploidy during domestication. To do so, we first reconstructed the phylogenetic history of B. napus, which supported the hypothesis that the emergence of B. napus derived from the hybridization of European turnip of B. rapa and wild B. oleracea. These analyses also showed that morphotypes of swede and Siberian kale (used as vegetable and fodder) were domesticated before rapeseed (oil crop). We next observed that frequent interploidy introgressions from sympatric diploids were prominent throughout the domestication history of B. napus. Introgressed genomic regions were shown to increase the overall genetic diversity and tend to be localized in regions of high recombination. We detected numerous candidate adaptive introgressed regions and found evidence that some of the genes in these regions contributed to phenotypic diversification and adaptation of different morphotypes. Overall, our results shed light on the origin and domestication of B. napus and demonstrate interploidy introgression as an important mechanism that fuels rapid diversification in polyploid species.

Fine mapping of meiotic crossovers in Brassica oleracea reveals patterns and variations depending on direction and combination of crosses.

Meiotic recombination is crucial for assuring proper segregation of parental chromosomes and generation of novel allelic combinations. As this process is tightly regulated, identifying factors influencing rate, and distribution of meiotic crossovers (COs) is of major importance, notably for plant breeding programs. However, high-resolution recombination maps are sparse in most crops including the Brassica genus and knowledge about intraspecific variation and sex differences is lacking. Here, we report fine-scale resolution recombination landscapes for 10 female and 10 male crosses in Brassica oleracea, by analyzing progenies of five large four-way-cross populations from two reciprocally crossed F1s per population. Parents are highly diverse inbred lines representing major crops, including broccoli, cauliflower, cabbage, kohlrabi, and kale. We produced approximately 4.56T Illumina data from 1248 progenies and identified 15 353 CO across the 10 reciprocal crosses, 51.13% of which being mapped to <10 kb. We revealed fairly similar Mb-scale recombination landscapes among all cross combinations and between the sexes, and provided evidence that these landscapes are largely independent of sequence divergence. We evidenced strong influence of gene density and large structural variations on CO formation in B. oleracea. Moreover, we found extensive variations in CO number depending on the direction and combination of the initial parents crossed with, for the first time, a striking interdependency between these factors. These data improve our current knowledge on meiotic recombination and are important for Brassica breeders.

Genetic dissection of morphological variation in rosette leaves and leafy heads in cabbage (Brassica oleracea var. capitata).

KEY MESSAGE: Correlations between morphological traits of cabbage rosette leaves and heads were found. Genome-wide association studies of these traits identified 50 robust quantitative trait loci in multiple years. Half of these loci affect both organs. Cabbage (Brassica oleracea var. capitata) is an economically important vegetable crop cultivated worldwide. Cabbage plants go through four vegetative stages: seedling, rosette, folding and heading. Rosette leaves are the largest leaves of cabbage plants and provide most of the energy needed to produce the leafy head. To understand the relationship and the genetic basis of leaf development and leafy head formation, 308 cabbage accessions were scored for rosette leaf and head traits in three-year field trials. Significant correlations were found between morphological traits of rosette leaves and heads, namely leaf area with the head area, height and width, and leaf width with the head area and head height, when heads were harvested at a fixed number of days after sowing. Fifty robust quantitative trait loci (QTLs) for rosette leaf and head traits distributed over all nine chromosomes were identified with genome-wide association studies. All these 50 loci were identified in multiple years and generally affect multiple traits. Twenty-five of the QTL were associated with both rosette leaf and leafy head traits. We discuss thirteen candidate genes identified in these QTL that are expressed in heading leaves, with an annotation related to auxin and other phytohormones, leaf development, and leaf polarity that likely play a role in leafy head development or rosette leaf expansion.

Genetic analysis reveals three novel QTLs underpinning a butterfly egg-induced hypersensitive response-like cell death in Brassica rapa.

BACKGROUND: Cabbage white butterflies (Pieris spp.) can be severe pests of Brassica crops such as Chinese cabbage, Pak choi (Brassica rapa) or cabbages (B. oleracea). Eggs of Pieris spp. can induce a hypersensitive response-like (HR-like) cell death which reduces egg survival in the wild black mustard (B. nigra). Unravelling the genetic basis of this egg-killing trait in Brassica crops could improve crop resistance to herbivory, reducing major crop losses and pesticides use. Here we investigated the genetic architecture of a HR-like cell death induced by P. brassicae eggs in B. rapa. RESULTS: A germplasm screening of 56 B. rapa accessions, representing the genetic and geographical diversity of a B. rapa core collection, showed phenotypic variation for cell death. An image-based phenotyping protocol was developed to accurately measure size of HR-like cell death and was then used to identify two accessions that consistently showed weak (R-o-18) or strong cell death response (L58). Screening of 160 RILs derived from these two accessions resulted in three novel QTLs for Pieris brassicae-induced cell death on chromosomes A02 (Pbc1), A03 (Pbc2), and A06 (Pbc3). The three QTLs Pbc1-3 contain cell surface receptors, intracellular receptors and other genes involved in plant immunity processes, such as ROS accumulation and cell death formation. Synteny analysis with A. thaliana suggested that Pbc1 and Pbc2 are novel QTLs associated with this trait, while Pbc3 also contains an ortholog of LecRK-I.1, a gene of A. thaliana previously associated with cell death induced by a P. brassicae egg extract. CONCLUSIONS: This study provides the first genomic regions associated with the Pieris egg-induced HR-like cell death in a Brassica crop species. It is a step closer towards unravelling the genetic basis of an egg-killing crop resistance trait, paving the way for breeders to further fine-map and validate candidate genes.

Evidence for two domestication lineages supporting a middle-eastern origin for Brassica oleracea crops from diversified kale populations.

Brassica oleracea displays enormous phenotypic variation, including vegetables like cabbage, broccoli, cauliflower, kohlrabi, kales etc. Its domestication has not been clarified, despite several genetic studies and investigations of ancient literature. We used 14 152 high-quality SNP markers for population genetic studies and species-tree estimation (treating morphotypes as "species") using SVD-quartets coalescent-modelling of a collection of 912 globally distributed accessions representing ten morphotypes of B. oleracea, wild B. oleracea accessions and nine related C9 Brassica species. Our genealogical tree provided evidence for two domestication lineages, the "leafy head" lineage (LHL) and the "arrested inflorescence" lineage (AIL). It also showed that kales are polyphyletic with regards to B. oleracea morphotypes, which fits ancient literature describing highly diverse kale types at around 400 BC. The SVD-quartets species tree topology showed that different kale clades are sister to either the LHL or the AIL. Cabbages from the middle-east formed the first-branching cabbage-clade, supporting the hypothesis that cabbage domestication started in the middle-east, which is confirmed by archeological evidence and historic writings. We hypothesize that cabbages and cauliflowers stem from kales introduced from Western Europe to the middle-east, possibly transported with the tin-trade routes in the Bronze age, to be re-introduced later into Europe. Cauliflower is the least diverse morphotype showing strong genetic differentiation with other morphotypes except broccoli, suggesting a strong genetic bottleneck. Genetic diversity reduced from landraces to modern hybrids for almost all morphotypes. This comprehensive Brassica C-group germplasm collection provides valuable genetic resources and a sound basis for B. oleracea breeding.

Breeding Beyond Monoculture: Putting the "Intercrop" Into Crops.

Intercropping is both a well-established and yet novel agricultural practice, depending on one's perspective. Such perspectives are principally governed by geographic location and whether monocultural practices predominate. Given the negative environmental effects of monoculture agriculture (loss of biodiversity, reliance on non-renewable inputs, soil degradation, etc.), there has been a renewed interest in cropping systems that can reduce the impact of modern agriculture while maintaining (or even increasing) yields. Intercropping is one of the most promising practices in this regard, yet faces a multitude of challenges if it is to compete with and ultimately replace the prevailing monocultural norm. These challenges include the necessity for more complex agricultural designs in space and time, bespoke machinery, and adapted crop cultivars. Plant breeding for monocultures has focused on maximizing yield in single-species stands, leading to highly productive yet specialized genotypes. However, indications suggest that these genotypes are not the best adapted to intercropping systems. Re-designing breeding programs to accommodate inter-specific interactions and compatibilities, with potentially multiple different intercropping partners, is certainly challenging, but recent technological advances offer novel solutions. We identify a number of such technology-driven directions, either ideotype-driven (i.e., "trait-based" breeding) or quantitative genetics-driven (i.e., "product-based" breeding). For ideotype breeding, plant growth modeling can help predict plant traits that affect both inter- and intraspecific interactions and their influence on crop performance. Quantitative breeding approaches, on the other hand, estimate breeding values of component crops without necessarily understanding the underlying mechanisms. We argue that a combined approach, for example, integrating plant growth modeling with genomic-assisted selection and indirect genetic effects, may offer the best chance to bridge the gap between current monoculture breeding programs and the more integrated and diverse breeding programs of the future.

Genome sequencing sheds light on the contribution of structural variants to Brassica oleracea diversification.

BACKGROUND: Brassica oleracea includes several morphologically diverse, economically important vegetable crops, such as the cauliflower and cabbage. However, genetic variants, especially large structural variants (SVs), that underlie the extreme morphological diversity of B. oleracea remain largely unexplored. RESULTS: Here we present high-quality chromosome-scale genome assemblies for two B. oleracea morphotypes, cauliflower and cabbage. Direct comparison of these two assemblies identifies ~ 120 K high-confidence SVs. Population analysis of 271 B. oleracea accessions using these SVs clearly separates different morphotypes, suggesting the association of SVs with B. oleracea intraspecific divergence. Genes affected by SVs selected between cauliflower and cabbage are enriched with functions related to response to stress and stimulus and meristem and flower development. Furthermore, genes affected by selected SVs and involved in the switch from vegetative to generative growth that defines curd initiation, inflorescence meristem proliferation for curd formation, maintenance and enlargement, are identified, providing insights into the regulatory network of curd development. CONCLUSIONS: This study reveals the important roles of SVs in diversification of different morphotypes of B. oleracea, and the newly assembled genomes and the SVs provide rich resources for future research and breeding.

Genetic analysis of the "head top shape" quality trait of Chinese cabbage and its association with rosette leaf variation.

The agricultural and consumer quality of Chinese cabbage is determined by its shape. The shape is defined by the folding of the heading leaves, which defines the head top shape (HTS). The overlapping HTS, in which the heading leaves curve inward and overlap at the top, is the shape preferred by consumers. To understand the genetic regulation of HTS, we generated a large segregating F2 population from a cross between pak choi and Chinese cabbage, with phenotypes ranging from nonheading to heading with either outward curving or inward curving overlapping heading leaves. HTS was correlated with plant height, outer/rosette leaf length, and petiole length. A high-density genetic map was constructed. Quantitative trait locus (QTL) analysis resulted in the identification of 22 QTLs for leafy head-related traits, which included five HTS QTLs. Bulked segregant analysis (BSA) was used to confirm HTS QTLs and identify candidate genes based on informative single-nucleotide polymorphisms. Interestingly, the HTS QTLs colocalized with QTLs for plant height, outer/rosette leaf, and petiole length, consistent with the observed phenotypic correlations. Combined QTL analysis and BSA laid a foundation for molecular marker-assisted breeding of Chinese cabbage HTS and directions for further research on the genetic regulation of this trait.

Genome-wide transcriptome analysis reveals molecular pathways involved in leafy head formation of Chinese cabbage (Brassica rapa).

Chinese cabbage plants go through seedling and rosette stages before forming their leafy head. Chinese cabbage plants resemble pak-choi plants at their seedling stage, but in their rosette stage the leaves of Chinese cabbage differentiate, as they increase in size with shorter petioles. In order to understand the molecular pathways that play a role in leafy head formation, transcript abundance of young emerging leaves was profiled during development of two Chinese cabbage genotypes and a single pak-choi genotype. The two Chinese cabbages differed in many aspects, among others earliness, leaf size and shape, leaf numbers, and leafy head shape. Genome-wide transcriptome analysis clearly separated the seedling stages of all three genotypes together with the later stages from pak-choi, from the later developmental stages of both Chinese cabbages (rosette, folding, and heading). Weighted correlation network analysis and hierarchical clustering using Euclidean distances resulted in gene clusters with transcript abundance patterns distinguishing the two Chinese cabbages from pak-choi. Three clusters included genes with transcript abundance affected by both genotype and developmental stage, whereas two clusters showed only genotype effects. This included a genotype by developmental stage cluster highly enriched with the MapMan category photosynthesis, with high expression during rosette and folding in Chinese cabbages and low expression in the heading inner leaves that are not exposed to light. The other clusters contained many genes in the MapMan categories Cell, showing again differences between pak-choi and both Chinese cabbages. We discuss how this relates to the differences in leaf blade growth between Chinese cabbage and pak-choi, especially at the rosette stage. Overall, comparison of the transcriptome between leaves of two very different Chinese cabbages with pak-choi during plant development allowed the identification of specific gene categories associated with leafy head formation.

Glucosinolate variability between turnip organs during development.

Turnip (Brassica rapa spp. rapa) is an important vegetable species, with a unique physiology. Several plant parts, including both the turnip tubers and leaves, are important for human consumption. During the development of turnip plants, the leaves function as metabolic source tissues, while the tuber first functions as a sink, while later the tuber turns into a source for development of flowers and seeds. In the present study, chemical changes were determined for two genotypes with different genetic background, and included seedling, young leaves, mature leaves, tuber surface, tuber core, stalk, flower and seed tissues, at seven different time points during plant development. As a basis for understanding changes in glucosinolates during plant development, the profile of glucosinolates was analysed using liquid chromatography (LC) coupled to mass spectrometry (MS). This analysis was complemented by a gene expression analysis, focussed on GLS biosynthesis, which could explain part of the observed variation, pointing to important roles of specific gene orthologues for defining the chemical differences. Substantial differences in glucosinolate profiles were observed between above-ground tissues and turnip tuber, reflecting the differences in physiological role. In addition, differences between the two genotypes and between tissues that were harvested early or late during the plant lifecycle. The importance of the observed differences in glucosinolate profile for the ecophysiology of the turnip and for breeding turnips with optimal chemical profiles is discussed.

Genetic dissection of flowering time in Brassica rapa responses to temperature and photoperiod.

The Brassica rapa (B. rapa) species displays enormous phenotypic diversity, with leafy vegetables, storage root vegetables and oil crops. These different crops all have different flowering time, which determine their growing season and cultivation area. Little is known about the effects of diverse temperature and day-lengths on flowering time QTL associated with FLC paralogues. We phenotyped the flowering time of a doubled haploid population, established from a cross between Yellow sarson and Pak choi under diverse environmental conditions. We identified flowering-time QTL (fQTL) in different photoperiod and temperature regimes in the greenhouse, and studied their colocation with known flowering time genes. As several fQTL colocalized with FLC paralogues, we studied the expression patterns of four FLC paralogues during the course of vernalization in parental lines. Under all environmental conditions tested the major fQTL that mapped to the BrFLC2_A02 locus was detected, however its effect decreased when plants were grown at low temperatures. Another fQTL that mapped to the FLC paralogue, BrFLC5_A03 was also identified under all tested environments, while no fQTL colocated with BrFLC1_A10 or BrFLC3_A03. Furthermore, the vernalization treatment decreased expression of all BrFLC paralogues in the parental lines, and showed the lowest transcript level after 28 days of vernalization. Transcript abundance stayed low after returning the plants for seven days to normal growth temperature. Interestingly, transcript abundance of BrFLC3_A03 and BrFLC5_A03 was repressed much stronger and already reached lowest levels after 14d in the early-flowering type YS-143. This study improves understanding of the effects of daylength and vernalization on flowering time in B. rapa and the role of the different BrFLC paralogues therein.

What makes turnips: anatomy, physiology and transcriptome during early stages of its hypocotyl-tuber development.

Brassica species are characterized by their tremendous intraspecific diversity, exemplified by leafy vegetables, oilseeds, and crops with enlarged inflorescences or above ground storage organs. In contrast to potato tubers that are edible storage organs storing energy as starch and are the vegetative propagation modules, the storage organs of turnips, grown from true seed, are swollen hypocotyls with varying degrees of root and stem that mainly store glucose and fructose. To highlight their anatomical origin, we use the term "hypocotyl-tuber" for these turnip vegetative storage organs. We combined cytological, physiological, genetic and transcriptomic approaches, aiming to identify the initial stages, molecular pathways and regulatory genes for hypocotyl-tuber induction in turnips (B. rapa subsp. rapa). We first studied the development of the hypocotyl zone of turnip and Pak choi and found that 16 days after sowing (DAS) morphological changes occurred in the xylem which indicated the early tuberization stage. Tissue culture experiments showed a clear effect of auxin on hypocotyl-tuber growth. Differentially expressed genes between 1 and 6 weeks after sowing in turnip hypocotyls, located in genomic regions involved in tuber initiation and/or tuber growth defined by QTL and selective sweeps for tuber formation, were identified as candidate genes that were studied in more detail for their role in hypocotyl-tuber formation. This included a Bra-FLOR1 paralogue with increased expression 16 DAS, when the hypocotyl starts swelling, suggesting dual roles for duplicated flowering time genes in flowering and hypocotyl-tuber induction. Bra-CYP735A2 was identified for its possible role in tuber growth via trans-zeatin. Weigthed Co-expression Network Analysis (WGCNA) identified 59 modules of co-expressed genes. Bra-FLOR1 and Bra-CYP735A2 were grouped in a module that included several genes involved in carbohydrate transport and metabolism, cell-wall growth, auxin regulation and secondary metabolism that serve as starting points to illuminate the transcriptional regulation of hypocotyl-tuber formation and development.

Genetic Analysis of Chinese Cabbage Reveals Correlation Between Rosette Leaf and Leafy Head Variation.

To understand the genetic regulation of the domestication trait leafy-head formation of Chinese cabbages, we exploit the diversity within Brassica rapa. To improve our understanding of the relationship between variation in rosette-leaves and leafy heads, we phenotyped a diversity set of 152 Chinese cabbages. This showed correlation between rosette-leaf traits and both head traits and heading capacity. Interestingly, the leaf number of the mature head is not correlated to heading degree nor head shape. We then chose a non-heading pak choi genotype to cross to a Chinese cabbage to generate populations segregating for the leafy head traits. Both a large F2 (485 plants) and a smaller Doubled Haploid (88 lines) mapping population were generated. A high density DH-88 genetic map using the Brassica SNP array and an F2 map with a subset of these SNPs and InDel markers was used for quantitative trait locus (QTL) analysis. Thirty-one quantitative trait loci (QTLs) were identified for phenotypes of rosette-leaves in time and both heading degree and several heading traits. On chromosome A06 in both DH-88 and F2-485 QTLs for rosette leaf length and petiole length at different developmental days and an F2 QTL for head height co-located. Variation in head height, width and weight all correlate with variation in heading degree with co-locating QTLs, respectively, on chromosome A03, A05, and A08 in F2-485. The correlation between rosette-leaf and heading traits provides not only insight in the leaf requirements to form a head, but also can be used for selection by Chinese cabbage breeders.

Splicing-related genes are alternatively spliced upon changes in ambient temperatures in plants.

Plants adjust their development and architecture to small variations in ambient temperature. In a time in which temperatures are rising world-wide, the mechanism by which plants are able to sense temperature fluctuations and adapt to it, is becoming of special interest. By performing RNA-sequencing on two Arabidopsis accession and one Brassica species exposed to temperature alterations, we showed that alternative splicing is an important mechanism in ambient temperature sensing and adaptation. We found that amongst the differentially alternatively spliced genes, splicing related genes are enriched, suggesting that the splicing machinery itself is targeted for alternative splicing when temperature changes. Moreover, we showed that many different components of the splicing machinery are targeted for ambient temperature regulated alternative splicing. Mutant analysis of a splicing related gene that was differentially spliced in two of the genotypes showed an altered flowering time response to different temperatures. We propose a two-step mechanism where temperature directly influences alternative splicing of the splicing machinery genes, followed by a second step where the altered splicing machinery affects splicing of downstream genes involved in the adaptation to altered temperatures.

A Phylogenetic Analysis of Chloroplast Genomes Elucidates the Relationships of the Six Economically Important Brassica Species Comprising the Triangle of U.

The Brassica genus comprises many economically important worldwide cultivated crops. The well-established model of the Brassica genus, U's triangle, consists of three basic diploid plant species (Brassica rapa, Brassica oleracea, and Brassica nigra) and three amphidiploid species (Brassica napus, Brassica juncea, and Brassica carinata) that arose through interspecific hybridizations. Despite being extensively studied because of its commercial relevance, several aspects of the origin of the Brassica species and the relationships within and among these six species still remain open questions. Here, we successfully de novo assembled 60 complete chloroplast genomes of Brassica genotypes of all six species. A complete map of the single nucleotide variants and insertions and deletions in the chloroplast genomes of different Brassica species was produced. The chloroplast genome consists of a Large and a Small Single Copy (LSC and SSC) region between two inverted repeats, and while these regions of chloroplast genomes have very different molecular evolutionary rates, phylogenetic analyses of different regions yielded no contradicting topologies and separated the Brassica genus into four clades. B. carinata and B. juncea share their chloroplast genome with one of their hybridization donors B. nigra and B. rapa, respectively, which fits the U model. B. rapa, surprisingly, shows evidence of two types of chloroplast genomes, with one type specific to some Italian broccoletto accessions. B. napus clearly has evidence for two independent hybridization events, as it contains either B. rapa chloroplast genomes. The divergence estimation suggests that B. nigra and B. carinata diverged from the main Brassica clade 13.7 million years ago (Mya), while B. rapa and B. oleracea diverged at 2.18 Mya. The use of the complete chloroplast DNA sequence not only provides insights into comparative genome analysis but also paves the way for a better understanding of the phylogenetic relationships within the Brassica genus.