A CRISPR/Cas9-induced male-sterile line facilitating easy hybrid production in polyploid rapeseed (Brassica napus).

Rapeseed is a globally significant oilseed crop cultivated to meet the increasing demand for vegetable oil. In order to enhance yield and sustainability, breeders have adopted the development of rapeseed hybrids as a common strategy. However, current hybrid production systems in rapeseed have various limitations, necessitating the development of a simpler and more efficient approach. In this study, we propose a novel method involving the targeted disruption of Defective in Anther Dehiscence1 of Brassica napus (BnDAD1), an essential gene in the jasmonic acid biosynthesis pathway, using CRISPR/Cas9 technology, to create male-sterile lines. BnDAD1 was found to be dominantly expressed in the stamen of rapeseed flower buds. Disrupting BnDAD1 led to decreased levels of α-linolenic acid and jasmonate in the double mutants, resulting in defects in anther dehiscence and pollen maturation. By crossing the double mutant male-sterile lines with male-fertile lines, a two-line system was demonstrated, enabling the production of F 1 seeds. The male-sterile trait of the bndad1 double mutant lines was maintainable by applying exogenous methyl jasmonate and subsequently self-pollinating the flowers. This breakthrough holds promising potential for harnessing heterosis in rapeseed and offers a simpler and more efficient method for producing hybrid seeds.

Genomic and transcriptome analyses reveal potential contributors to erucic acid biosynthesis in seeds of rapeseed (Brassica napus).

KEY MESSAGE: Through comprehensive genomic and transcriptomic analyses, we identified a set of 23 genes that act up- or downstream of erucic acid content (EAC) production in rapeseed seeds. We selected example genes to showcase the distribution of single nucleotide polymorphisms, haplotypes associated with EAC phenotypes, and the creation of molecular markers differentiating low EAC and high EAC genotypes. Erucic acid content (EAC) is a crucial trait in rapeseed, with low LEAC oil recognized for its health benefits and high EA oil holding industrial value. Despite its significance, the genomic consequences of intensive LEAC-cultivar selection and the genetic basis underlying EA regulation remain largely unexplored. To address this knowledge gap, we conducted selective signal analyses, genome-wide association studies (GWAS), and transcriptome analyses. Our investigation unveiled the genetic footprints resulting from LEAC selection in germplasm populations, drawing attention to specific loci that contribute to enriching diversity. By integrating GWAS and transcriptome analyses, we identified a set of 23 genes that play a significant role in determining EAC in seeds or are downstream consequences of EA-level alterations. These genes have emerged as promising candidates for elucidating the potential mechanisms governing EAC in rapeseed. To exemplify the findings, we selected specific genes to demonstrate the distribution of single nucleotide polymorphisms and haplotypes associated with different EAC phenotypes. Additionally, we showcased to develop molecular markers distinguishing between LEAC and high EAC genotypes.

Genome-wide association study reveals the genetic basis for petal-size formation in rapeseed (Brassica napus) and CRISPR-Cas9-mediated mutagenesis of BnFHY3 for petal-size reduction.

Petals in rapeseed (Brassica napus) serve multiple functions, including protection of reproductive organs, nutrient acquisition, and attraction of pollinators. However, they also cluster densely at the top, forming a thick layer that absorbs and reflects a considerable amount of photosynthetically active radiation. Breeding genotypes with large, small, or even petal-less varieties, requires knowledge of primary genes for allelic selection and manipulation. However, our current understanding of petal-size regulation is limited, and the lack of markers and pre-breeding materials hinders targeted petal-size breeding. Here, we conducted a genome-wide association study on petal size using 295 diverse accessions. We identified 20 significant single nucleotide polymorphisms and 236 genes associated with petal-size variation. Through a cross-analysis of genomic and transcriptomic data, we focused on 14 specific genes, from which molecular markers for diverging petal-size features can be developed. Leveraging CRISPR-Cas9 technology, we successfully generated a quadruple mutant of Far-Red Elongated Hypocotyl 3 (q-bnfhy3), which exhibited smaller petals compared to the wild type. Our study provides insights into the genetic basis of petal-size regulation in rapeseed and offers abundant potential molecular markers for breeding. The q-bnfhy3 mutant unveiled a novel role of FHY3 orthologues in regulating petal size in addition to previously reported functions.

Temporal control of the Aux/IAA genes BnIAA32 and BnIAA34 mediates Brassica napus dual shade responses.

Precise responses to changes in light quality are crucial for plant growth and development. For example, hypocotyls of shade-avoiding plants typically elongate under shade conditions. Although this typical shade-avoidance response (TSR) has been studied in Arabidopsis (Arabidopsis thaliana), the molecular mechanisms underlying shade tolerance are poorly understood. Here we report that B. napus (Brassica napus) seedlings exhibit dual shade responses. In addition to the TSR, B. napus seedlings also display an atypical shade response (ASR), with shorter hypocotyls upon perception of early-shade cues. Genome-wide selective sweep analysis indicated that ASR is associated with light and auxin signaling. Moreover, genetic studies demonstrated that phytochrome A (BnphyA) promotes ASR, whereas BnphyB inhibits it. During ASR, YUCCA8 expression is activated by early-shade cues, leading to increased auxin biosynthesis. This inhibits hypocotyl elongation, as young B. napus seedlings are highly sensitive to auxin. Notably, two non-canonical AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA) repressor genes, BnIAA32 and BnIAA34, are expressed during this early stage. BnIAA32 and BnIAA34 inhibit hypocotyl elongation under shade conditions, and mutations in BnIAA32 and BnIAA34 suppress ASR. Collectively, our study demonstrates that the temporal expression of BnIAA32 and BnIAA34 determines the behavior of B. napus seedlings following shade-induced auxin biosynthesis.

Transcriptomic Analysis of the Reduction in Seed Oil Content through Increased Nitrogen Application Rate in Rapeseed (Brassica napus L.).

Nitrogen is essential for improving the seed oil yield of rapeseed (Brassica napus L.). However, the molecular mechanism by which increased nitrogen rates impact seed oil content is largely unknown. Therefore, a field experiment was conducted to determine how three nitrogen application rates (120, 240, and 360 kg ha-1) regulated seed oil content via transcriptomic analysis. The results showed that the seed yield and the protein and total N contents increased from N1 to N3, with average increases of 57.2%, 16.9%, and 79.5%, respectively. However, the seed oil content significantly decreased from N1 to N3, with an average decrease of 8.6%. These results were repeated over a number of years. The quantity of oil protein bodies observed under a transmission electron microscope was in accordance with the ultimate seed oil and protein contents. As the nitrogen application rate increased, a substantial number of genes involved in the photosynthesis, glycolysis, and phenylpropanoid biosynthesis pathways were up-regulated, as were TF families, such as AP2/ERF, MYB, and NAC. The newly identified genes were mainly involved in carbohydrate, lipid, and amino acid metabolism. Metabolic flux analysis showed that most of the genes involved in glycolysis and fatty acid biosynthesis had higher transcript levels in the early development stages. Our results provide new insights into the molecular regulation of rapeseed seed oil content through increased nitrogen application rates.

Genotypic variation of tocopherol content in a representative genetic population and genome-wide association study on tocopherol in rapeseed (Brassica napus).

Tocopherols (Tocs) are a kind of lipid-soluble substance required for the normal physiological function of mammals, particularly their antioxidant capacity. Rapeseed (Brassica napus) oil is an important source of exogenous Tocs. However, the genotypic differences in the total Toc contents, the Toc composition in the seeds, and the molecular markers associated with the seed Toc remain largely unknown. Here, we selected 290 rapeseed accessions based on the resequencing of 991 genomes in a worldwide collection of rapeseed germplasm. The contents of the four Toc isoforms, namely, α-, ß-, γ-, and δ-Tocs, were also measured. Results show that the total Toc content and the γ-/α-Toc ratio varied greatly across the accessions from 85.34 to 387.00 mg/mg and 0.65 to 5.03, respectively. Furthermore, we conducted genome-wide association studies on the Tocs, which identified 28 and 73 single nucleotide polymorphisms significantly associated with the variation of total Toc content and γ-/α-Toc ratio, respectively. Bna.C02.VTE4, a putative orthologue of Arabidopsis VITAMIN E DEFICIENT 4, was tightly associated with the γ-/α-Toc ratio. This study recommends specific genetic materials with particularly high total Toc and/or low γ-/α-Toc ratio and the molecular markers and haplotypes associated with these quality traits for rapeseed breeding. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-023-01394-0.

Syntaxin of plants71 plays essential roles in plant development and stress response via regulating pH homeostasis.

SYP71, a plant-specific Qc-SNARE with multiple subcellular localization, is essential for symbiotic nitrogen fixation in nodules in Lotus, and is implicated in plant resistance to pathogenesis in rice, wheat and soybean. Arabidopsis SYP71 is proposed to participate in multiple membrane fusion steps during secretion. To date, the molecular mechanism underlying SYP71 regulation on plant development remains elusive. In this study, we clarified that AtSYP71 is essential for plant development and stress response, using techniques of cell biology, molecular biology, biochemistry, genetics, and transcriptomics. AtSYP71-knockout mutant atsyp71-1 was lethal at early development stage due to the failure of root elongation and albinism of the leaves. AtSYP71-knockdown mutants, atsyp71-2 and atsyp71-3, had short roots, delayed early development, and altered stress response. The cell wall structure and components changed significantly in atsyp71-2 due to disrupted cell wall biosynthesis and dynamics. Reactive oxygen species homeostasis and pH homeostasis were also collapsed in atsyp71-2. All these defects were likely resulted from blocked secretion pathway in the mutants. Strikingly, change of pH value significantly affected ROS homeostasis in atsyp71-2, suggesting interconnection between ROS and pH homeostasis. Furthermore, we identified AtSYP71 partners and propose that AtSYP71 forms distinct SNARE complexes to mediate multiple membrane fusion steps in secretory pathway. Our findings suggest that AtSYP71 plays an essential role in plant development and stress response via regulating pH homeostasis through secretory pathway.

Moderate Salinity Stress Increases the Seedling Biomass in Oilseed Rape (Brassica napus L.).

Oilseed rape (Brassica napus L.), an important oil crop of the world, suffers various abiotic stresses including salinity stress during the growth stage. While most of the previous studies paid attention to the adverse effects of high salinity stress on plant growth and development, as well as their underlying physiological and molecular mechanisms, less attention was paid to the effects of moderate or low salinity stress. In this study, we first tested the effects of different concentrations of NaCl solution on the seedling growth performance of two oilseed rape varieties (CH336, a semi-winter type, and Bruttor, a spring type) in pot cultures. We found that moderate salt concentrations (25 and 50 mmol L-1 NaCl) can stimulate seedling growth by a significant increase (10~20%, compared to controls) in both above- and underground biomasses, as estimated at the early flowering stage. We then performed RNA-seq analyses of shoot apical meristems (SAMs) from six-leaf-aged seedlings under control (CK), low (LS, 25 mmol L-1), and high (HS, 180 mmol L-1) salinity treatments in the two varieties. The GO and KEGG enrichment analyses of differentially expressed genes (DEGs) demonstrated that such a stimulating effect on seedling growth by low salinity stress may be caused by a more efficient capacity for photosynthesis as compensation, accompanied by a reduced energy loss for the biosynthesis of secondary metabolites and redirecting of energy to biomass formation. Our study provides a new perspective on the cultivation of oilseed rape in saline regions and new insights into the molecular mechanisms of salt tolerance in Brassica crops. The candidate genes identified in this study can serve as targets for molecular breeding selection and genetic engineering toward enhancing salt tolerance in B. napus.

Phenotyping of Silique Morphology in Oilseed Rape Using Skeletonization with Hierarchical Segmentation.

Silique morphology is an important trait that determines the yield output of oilseed rape (Brassica napus L.). Segmenting siliques and quantifying traits are challenging because of the complicated structure of an oilseed rape plant at the reproductive stage. This study aims to develop an accurate method in which a skeletonization algorithm was combined with the hierarchical segmentation (SHS) algorithm to separate siliques from the whole plant using 3-dimensional (3D) point clouds. We combined the L1-median skeleton with the random sample consensus for iteratively extracting skeleton points and optimized the skeleton based on information such as distance, angle, and direction from neighborhood points. Density-based spatial clustering of applications with noise and weighted unidirectional graph were used to achieve hierarchical segmentation of siliques. Using the SHS, we quantified the silique number (SN), silique length (SL), and silique volume (SV) automatically based on the geometric rules. The proposed method was tested with the oilseed rape plants at the mature stage grown in a greenhouse and field. We found that our method showed good performance in silique segmentation and phenotypic extraction with R 2 values of 0.922 and 0.934 for SN and total SL, respectively. Additionally, SN, total SL, and total SV had the statistical significance of correlations with the yield of a plant, with R values of 0.935, 0.916, and 0.897, respectively. Overall, the SHS algorithm is accurate, efficient, and robust for the segmentation of siliques and extraction of silique morphological parameters, which is promising for high-throughput silique phenotyping in oilseed rape breeding.

Structural variations and environmental specificities of flowering time-related genes in Brassica napus.

KEY MESSAGE: We found that the flowering time order of accessions in a genetic population considerably varied across environments, and homolog copies of essential flowering time genes played different roles in different locations. Flowering time plays a critical role in determining the life cycle length, yield, and quality of a crop. However, the allelic polymorphism of flowering time-related genes (FTRGs) in Brassica napus, an important oil crop, remains unclear. Here, we provide high-resolution graphics of FTRGs in B. napus on a pangenome-wide scale based on single nucleotide polymorphism (SNP) and structural variation (SV) analyses. A total of 1337 FTRGs in B. napus were identified by aligning their coding sequences with Arabidopsis orthologs. Overall, 46.07% of FTRGs were core genes and 53.93% were variable genes. Moreover, 1.94%, 0.74%, and 4.49% FTRGs had significant presence-frequency differences (PFDs) between the spring and semi-winter, spring and winter, and winter and semi-winter ecotypes, respectively. SNPs and SVs across 1626 accessions of 39 FTRGs underlying numerous published qualitative trait loci were analyzed. Additionally, to identify FTRGs specific to an eco-condition, genome-wide association studies (GWASs) based on SNP, presence/absence variation (PAV), and SV were performed after growing and observing the flowering time order (FTO) of plants in a collection of 292 accessions at three locations in two successive years. It was discovered that the FTO of plants in a genetic population changed a lot across various environments, and homolog copies of some key FTRGs played different roles in different locations. This study revealed the molecular basis of the genotype-by-environment (G × E) effect on flowering and recommended a pool of candidate genes specific to locations for breeding selection.

Genome-wide-association study and transcriptome analysis reveal the genetic basis controlling the formation of leaf wax in Brassica napus.

Cuticular wax protects plants from various biotic and abiotic stresses. However, the genetic network of wax biosynthesis and the environmental factors influencing leaf wax production in rapeseed (Brassica napus) remains unclear. Here, we demonstrated the role of leaf wax in the resistance to Sclerotinia infection in rapeseed. We found that leaves grown under high light intensity had higher expression of genes involved in wax biosynthesis, and produced more wax on the leaf surface, compared with those grown under low light conditions. Genome-wide association study (GWAS) identified 89 single nucleotide polymorphisms significantly associated with leaf wax coverage. A cross-analysis between GWAS and differentially expressed genes (DEGs) in the leaf epidermis of the accessions with contrasting differences in wax content revealed 17 candidate genes that control this variation in rapeseed. Selective sweep analysis combined with DEG analysis unveiled 510 candidate genes with significant selective signatures. From the candidate genes, we selected BnaA02.LOX4, a putative lipoxygenase, and BnaCnn.CER1, BnaA02.CER3, BnaC02.CER3, and BnaA01.CER4 (ECERIFERUM1-4) that were putatively responsible for wax biosynthesis, to analyse the allelic forms and haplotypes corresponding to high or low leaf wax coverage. These data enrich our knowledge about wax formation, and provide a gene pool for breeding an ideal leaf wax content in rapeseed.

CRISPR/Cas9-mediated editing of double loci of BnFAD2 increased the seed oleic acid content of rapeseed (Brassica napus L.).

Seed oleic acid is an important quality trait sought in rapeseed breeding programs. Many methods exist to increase seed oleic acid content, such as the CRISPR/Cas9-mediated genome editing system, yet there is no report on seed oleic acid content improvement via this system's precise editing of the double loci of BnFAD2. Here, a precise CRISPR/Cas9-mediated genome editing of the encoded double loci (A5 and C5) of BnFAD2 was established. The results demonstrated high efficiency of regeneration and transformation, with the rapeseed genotype screened in ratios of 20.18% and 85.46%, respectively. The total editing efficiency was 64.35%, whereas the single locus- and double locus-edited ratios were 21.58% and 78.42%, respectively. The relative proportion of oleic acid with other fatty acids in seed oil of mutants was significantly higher for those that underwent the editing on A5 copy than that on C5 copy, but it was still less than 80%. For double locus-edited mutants, their relative proportion of oleic acid was more than 85% in the T1 and T4 generations. A comparison of the sequences between the double locus-edited mutants and reference showed that no transgenic border sequences were detected from the transformed vector. Analysis of the BnFAD2 sequence on A5 and C5 at the mutated locus of double loci mutants uncovered evidence for base deletion and insertion, and combination. Further, no editing issue of FAD2 on the copy of A1 was detected on the three targeted editing regions. Seed yield, yield component, oil content, and relative proportion of oleic acid between one selected double loci-edited mutant and wild type were also compared. These results showed that although the number of siliques per plant of the wild type was significantly higher than those of the mutant, the differences in seed yield and oil content were not significant between them, albeit with the mutant having a markedly higher relative proportion of oleic acid. Altogether, our results confirmed that the established CRISPR/Cas9-mediated genome editing of double loci (A5 and C5) of the BnFAD2 can precisely edit the targeted genes, thereby enhancing the seed oleic acid content to a far greater extent than can a single locus-editing system.

BnaA03.ANS Identified by Metabolomics and RNA-seq Partly Played Irreplaceable Role in Pigmentation of Red Rapeseed (Brassica napus) Petal.

Colorful flowers of rapeseed (Brassica napus L.) have been a hotspot for researchers, but the underlying mechanisms of pigment formation still need to be clarified. In this study, two stages of unopened rapeseed petals with red, white, and yellow colors were selected to identify the metabolites and genes involved in red pigment formation. Metabolomic analysis showed that flavonoids enriched the most co-differentially accumulated metabolites among all categories, and showed higher accumulation in red petal rapeseed than in white and yellow petal ones. RNA-seq analysis showed that among co-differentially expressed genes involved in red pigment formation, genes involved in anthocyanin (belonging to flavonoids) biosynthesis pathway were largely regulated by ANS, DFR, and UF3GT. The expression of those genes was higher in red petals of rapeseed than in white and yellow petals ones as well. Results of RNA interference of BnaA03.ANS in red rapeseed altered petal colors from raspberry red to beige red and zinc yellow under different interference levels, with the contents of pelargonidin, cyanidin, lutein, neoxanthin, ß-carotene, and lycopene significantly decreased. However, overexpression of BnaA03.ANS in yellow rapeseed petals did not change the color of yellow petals. This study confirmed the important function of flavonoids, especially anthocyanins on red pigment formation, and for the first time, identified the irreplaceable role of BnaA03.ANS on red-flowered rapeseed.

Stomatal morphological variation contributes to global ecological adaptation and diversification of Brassica napus.

MAIN CONCLUSION: Stomatal density and guard cell length of 274 global core germplasms of rapeseed reveal that the stomatal morphological variation contributes to global ecological adaptation and diversification of Brassica napus. Stomata are microscopic structures of plants for the regulation of CO2 assimilation and transpiration. Stomatal morphology has changed substantially in the adaptation to the external environment during land plant evolution. Brassica napus is a major crop to produce oil, livestock feed and biofuel in the world. However, there are few studies on the regulatory genes controlling stomatal development and their interaction with environmental factors as well as the genetic mechanism of adaptive variation in B. napus. Here, we characterized stomatal density (SD) and guard cell length (GL) of 274 global core germplasms at seedling stage. It was found that among the significant phenotypic variation, European germplasms are mostly winter rapeseed with high stomatal density and small guard cell length. However, the germplasms from Asia (especially China) are semi-winter rapeseed, which is characterized by low stomatal density and large guard cell length. Through selective sweep analysis and homology comparison, we identified several candidate genes related to stomatal density and guard cell length, including Epidermal Patterning Factor2 (EPF2; BnaA09g23140D), Epidermal Patterning Factor Like4 (EPFL4; BnaC01g22890D) and Suppressor of LLP1 (SOL1 BnaC01g22810D). Haplotype and phylogenetic analysis showed that natural variation in EPF2, EPFL4 and SOL1 is closely associated with the winter, spring, and semi-winter rapeseed ecotypes. In summary, this study demonstrated for the first time the relation between stomatal phenotypic variation and ecological adaptation in rapeseed, which is useful for future molecular breeding of rapeseed in the context of evolution and domestication of key stomatal traits and global climate change.

The genome and gene editing system of sea barleygrass provide a novel platform for cereal domestication and stress tolerance studies.

The tribe Triticeae provides important staple cereal crops and contains elite wild species with wide genetic diversity and high tolerance to abiotic stresses. Sea barleygrass (Hordeum marinum Huds.), a wild Triticeae species, thrives in saline marshlands and is well known for its high tolerance to salinity and waterlogging. Here, a 3.82-Gb high-quality reference genome of sea barleygrass is assembled de novo, with 3.69 Gb (96.8%) of its sequences anchored onto seven chromosomes. In total, 41 045 high-confidence (HC) genes are annotated by homology, de novo prediction, and transcriptome analysis. Phylogenetics, non-synonymous/synonymous mutation ratios (Ka/Ks), and transcriptomic and functional analyses provide genetic evidence for the divergence in morphology and salt tolerance among sea barleygrass, barley, and wheat. The large variation in post-domestication genes (e.g. IPA1 and MOC1) may cause interspecies differences in plant morphology. The extremely high salt tolerance of sea barleygrass is mainly attributed to low Na+ uptake and root-to-shoot translocation, which are mainly controlled by SOS1, HKT, and NHX transporters. Agrobacterium-mediated transformation and CRISPR/Cas9-mediated gene editing systems were developed for sea barleygrass to promote its utilization for exploration and functional studies of hub genes and for the genetic improvement of cereal crops.

MAG2 and MAL Regulate Vesicle Trafficking and Auxin Homeostasis With Functional Redundancy.

Auxin is a central phytohormone and controls almost all aspects of plant development and stress response. Auxin homeostasis is coordinately regulated by biosynthesis, catabolism, transport, conjugation, and deposition. Endoplasmic reticulum (ER)-localized MAIGO2 (MAG2) complex mediates tethering of arriving vesicles to the ER membrane, and it is crucial for ER export trafficking. Despite important regulatory roles of MAG2 in vesicle trafficking, the mag2 mutant had mild developmental abnormalities. MAG2 has one homolog protein, MAG2-Like (MAL), and the mal-1 mutant also had slight developmental phenotypes. In order to investigate MAG2 and MAL regulatory function in plant development, we generated the mag2-1 mal-1 double mutant. As expected, the double mutant exhibited serious developmental defects and more alteration in stress response compared with single mutants and wild type. Proteomic analysis revealed that signaling, metabolism, and stress response in mag2-1 mal-1 were affected, especially membrane trafficking and auxin biosynthesis, signaling, and transport. Biochemical and cell biological analysis indicated that the mag2-1 mal-1 double mutant had more serious defects in vesicle transport than the mag2-1 and mal-1 single mutants. The auxin distribution and abundance of auxin transporters were altered significantly in the mag2-1 and mal-1 single mutants and mag2-1 mal-1 double mutant. Our findings suggest that MAG2 and MAL regulate plant development and auxin homeostasis by controlling membrane trafficking, with functional redundancy.

Evolutionary Analysis of the YABBY Gene Family in Brassicaceae.

The YABBY gene family is one of the plant transcription factors present in all seed plants. The family members were extensively studied in various plants and shown to play important roles in plant growth and development, such as the polarity establishment in lateral organs, the formation and development of leaves and flowers, and the response to internal plant hormone and external environmental stress signals. In this study, a total of 364 YABBY genes were identified from 37 Brassicaceae genomes, of which 15 were incomplete due to sequence gaps, and nine were imperfect (missing C2C2 zinc-finger or YABBY domain) due to sequence mutations. Phylogenetic analyses resolved these YABBY genes into six compact clades except for a YAB3-like gene identified in Aethionema arabicum. Seventeen Brassicaceae species each contained a complete set of six basic YABBY genes (i.e., 1 FIL, 1 YAB2, 1 YAB3, 1 YAB5, 1 INO and 1 CRC), while 20 others each contained a variable number of YABBY genes (5-25) caused mainly by whole-genome duplication/triplication followed by gene losses, and occasionally by tandem duplications. The fate of duplicate YABBY genes changed considerably according to plant species, as well as to YABBY gene type. These YABBY genes were shown to be syntenically conserved across most of the Brassicaceae species, but their functions might be considerably diverged between species, as well as between paralogous copies, as demonstrated by the promoter and expression analysis of YABBY genes in two Brassica species (B. rapa and B. oleracea). Our study provides valuable insights for understanding the evolutionary story of YABBY genes in Brassicaceae and for further functional characterization of each YABBY gene across the Brassicaceae species.

Prediction of heterosis in the recent rapeseed (Brassica napus) polyploid by pairing parental nucleotide sequences.

The utilization of heterosis is a successful strategy in increasing yield for many crops. However, it consumes tremendous manpower to test the combining ability of the parents in fields. Here, we applied the genomic-selection (GS) strategy and developed models that significantly increase the predictability of heterosis by introducing the concept of a regional parental genetic-similarity index (PGSI) and reducing dimension in the calculation matrix in a machine-learning approach. Overall, PGSI negatively affected grain yield and several other traits but positively influenced the thousand-seed weight of the hybrids. It was found that the C subgenome of rapeseed had a greater impact on heterosis than the A subgenome. We drew maps with overviews of quantitative-trait loci that were responsible for the heterosis (h-QTLs) of various agronomic traits. Identifications and annotations of genes underlying high impacting h-QTLs were provided. Using models that we elaborated, combining abilities between an Ogu-CMS-pool member and a potential restorer can be simulated in silico, sidestepping laborious work, such as testing crosses in fields. The achievements here provide a case of heterosis prediction in polyploid genomes with relatively large genome sizes.

Modelling of gene loss propensity in the pangenomes of three Brassica species suggests different mechanisms between polyploids and diploids.

Plant genomes demonstrate significant presence/absence variation (PAV) within a species; however, the factors that lead to this variation have not been studied systematically in Brassica across diploids and polyploids. Here, we developed pangenomes of polyploid Brassica napus and its two diploid progenitor genomes B. rapa and B. oleracea to infer how PAV may differ between diploids and polyploids. Modelling of gene loss suggests that loss propensity is primarily associated with transposable elements in the diploids while in B. napus, gene loss propensity is associated with homoeologous recombination. We use these results to gain insights into the different causes of gene loss, both in diploids and following polyploidization, and pave the way for the application of machine learning methods to understanding the underlying biological and physical causes of gene presence/absence.

Genome-wide association study reveals a patatin-like lipase relating to the reduction of seed oil content in Brassica napus.

BACKGROUND: Rapeseed (Brassica napus L.) is an important oil crop world-widely cultivated, and seed oil content (SOC) is one of the most important traits for rapeseed. To increase SOC, many efforts for promoting the function of genes on lipid biosynthesis pathway have been previously made. However, seed oil formation is a dynamic balance between lipid synthesis and breakdown. It is, therefore, also reasonable to weaken or eliminate the function of genes involved in lipid degradation for a higher final SOC. RESULTS: We applied a genome-wide association study (GWAS) on SOC in a collection of 290 core germplasm accessions. A total of 2,705,480 high-quality SNPs were used in the GWAS, and we identified BnaC07g30920D, a patatin-like lipase (PTL) gene, that was associated with SOC. In particular, six single-nucleotide-polymorphisms (SNPs) in the promoter region of BnaC07g30920D were associated with the significant reduction of SOC, leading to a 4.7-6.2% reduction of SOCs. We performed in silico analysis to show a total of 40 PTLs, which were divided into four clades, evenly distributed on the A and C subgenomes of Brassica napus. RNA-seq analysis unveiled that BnPTLs were preferentially expressed in reproductive tissues especially maturing seeds. CONCLUSIONS: We identified BnaC07g30920D, a BnPTL gene, that was associated with SOC using GWAS and performed in silico analysis of 40 PTLs in Brassica napus. The results enrich our knowledge about the SOC formation in rapeseed and facilitate the future study in functional characterization of BnPTL genes.