Transcriptional Interactions of Single B-Subgenome Chromosome with C-Subgenome in B. oleracea-nigra Additional Lines.

Serial monosomic alien addition lines (MAALs) provide an ideal system to elucidate the transcriptomic interactions between the alien chromosomes and recipient genome under aneuploidy. Herein, five available Brassica oleracea-nigra MAALs (CCB1, CCB4, CCB5, CCB6, CCB8), their derived B. oleracea plants (non-MAALs), and two parents were analyzed for their gene expressions by using high-throughput technology. Compared to parental B. oleracea, all MAALs showed various numbers of DEGs, but CCB8 gave much higher DEGs; the number of downregulated DEGs was slightly higher than the number of upregulated ones, except for in relation to CCB8. All derived B. oleracea plants also gave certain numbers of DEGs, despite these being much lower than in the respective MAALs. Compared to B. nigra, in all five MAALs more DEGs were downregulated than upregulated. Trans-effects were likely more prevailing than cis-effects, and these DEGs were predominantly associated with material transport by dysregulating the cellular component. Meanwhile, the orthologous genes on alien chromosomes could only play a feeble compensatory role for those gene pairs in C-subgenome, and different levels of the expressed genes had a greater tendency towards downregulation. These results revealed transcriptional aneuploidy response patterns between two genomes and suggested that cis- and trans-mechanisms synergistically regulated alien gene transcriptions after distant hybridization.

Fine mapping of genes controlling pigment accumulation in oilseed rape (Brassica napus L.).

Purple/red appearance is one of the common phenotypic variations in leaves, stems, and siliques of oilseed rape (Brassica napus L.) but very rare in flowers. In this study, the causal genes for the purple/red traits in stems and flowers in two accessions of oilseed rape (DH_PR and DH_GC001, respectively) derived from the wide hybridization were fine mapped, and candidate genes were determined by methods combined with bulked segregant analysis (BSA) and RNA-seq analysis. Both traits of purple stem and red flowers were mapped to the locus as AtPAP2 homologous genes (BnaPAP2.C6a and BnaPAP2.A7b, respectively) belonging to the R2R3-MYB family. Sequence comparisons of full-length allelic genes revealed several InDels and SNPs in intron 1 as well as exons, and completely different promoter region of BnaPAP2.C6a and a 211 bp insertion was identified in the promoter region of BnaPAP2.A7b of DH_GC001. Our results not only contribute to a better understanding of anthocyanin inheritance in B. napus, but also provide a useful toolbox for future breeding of cultivars with purple/red traits through the combination of different functional alleles and homologs. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-023-01365-5.

Unraveling Large and Polyploidy Genome of the Crucifer Orychophragmus violaceus in China, a Potential Oil Crop.

The genus Orychophragmus in the Brassicaceae family includes the types with 2n = 20, 22, 24, and 48. The species O. violaceus (L.) O. E. Schulz has 2n = 24 and is widely cultivated as an ornamental plant in China. This review summarizes the research progress of its genome structure and evolution in the context of cytogenetics and genome sequencing. This species has a large genome size of ~1 Gb and longer chromosomes than those of Brassica species, which is attributable to the burst of TE insertions. Even more, one tetraploidization event from about 600-800 million years ago is elucidated to occur during its genome evolution, which is consistent with the polyploidy nature of its genome revealed by the meiotic pairing patterns. Its chromosomes are still characterized by a larger size and deeper staining than those from Brassica species in their intergeneric hybrids, which is likely related to their inherent differences between genome structures and cytology. Its genome is dissected by the development of additional alien lines, and some traits are located on individual chromosomes. Due to the abundant dihydroxy fatty acids in its seed oil with superior lubricant properties and wide environmental adaptations, this plant promises to be utilized as one new oil crop in the future.

Genome assembly of the Brassicaceae diploid Orychophragmus violaceus reveals complex whole-genome duplication and evolution of dihydroxy fatty acid metabolism.

Orychophragmus violaceus is a Brassicaceae species widely cultivated in China, particularly as a winter cover crop in northern China because of its low-temperature tolerance and low water demand. Recently, O. violaceus has also been cultivated as a potential industrial oilseed crop because of its abundant 24-carbon dihydroxy fatty acids (diOH-FAs), which contribute to superior high-temperature lubricant properties. In this study, we performed de novo assembly of the O. violaceus genome. Whole-genome synteny analysis of the genomes of its relatives demonstrated that O. violaceus is a diploid that has undergone an extra whole-genome duplication (WGD) after the Brassicaceae-specific α-WGD event, with a basic chromosome number of x = 12. Formation of diOH-FAs is hypothesized to have occurred after the WGD event. Based on the genome and the transcriptome data from multiple stages of seed development, we predicted that OvDGAT1-1 and OvDGAT1-2 are candidate genes for the regulation of diOH-FA storage in O. violaceus seeds. These results may greatly facilitate the development of heat-tolerant and eco-friendly plant-based lubricants using O. violaceus seed oil and improve our understanding of the genomic evolution of Brassicaceae.

Sinapis genomes provide insights into whole-genome triplication and divergence patterns within tribe Brassiceae.

Sinapis alba and Sinapis arvensis are mustard crops within the Brassiceae tribe of the Brassicaceae family, and represent an important genetic resource for crop improvement. We performed the de novo assembly of Brassica nigra, S. alba, and S. arvensis, and conducted comparative genomics to investigate the pattern of genomic evolution since an ancient whole-genome triplication event. Both Sinapis species retained evidence of the Brassiceae whole-genome triplication approximately 20.5 million years ago (Mya), with subgenome dominance observed in gene density, gene expression, and selective constraint. While S. alba diverged from the ancestor of Brassica and Raphanus at approximately 12.5 Mya, the divergence time of S. arvensis and B. nigra was approximately 6.5 Mya. S. arvensis and B. nigra had greater collinearity compared with their relationship to either Brassica rapa or Brassica oleracea. Two chromosomes of S. alba (Sal03 and Sal08) were completely collinear with two ancestral chromosomes proposed in the Ancestral Crucifer Karyotype (ACK) genomic block model, the first time this has been observed in the Brassiceae. These results are consistent with S. alba representing a relatively ancient lineage of the species evolved from the common ancestor of tribe Brassiceae, and suggest that the phylogeny of the Brassica and Sinapis genera requires some revision. Our study provides new insights into the genome evolution and phylogenetic relationships of Brassiceae and provides genomic information for genetic improvement of these plants.

Transcriptomic and iTRAQ-Based Quantitative Proteomic Analyses of inap CMS in Brassica napus L.

Brassica napus inap cytoplasmic male sterility (CMS) is a novel sterile line with potential application in rapeseed hybrid breeding. Sterile cytoplasm was obtained from Isatis indigotica through somatic fusion and then recurrent backcrossing with B. napus. Previous studies have shown that inap CMS abortion occurred before the stamen primordia (stage 4-5), but the genetic mechanism of sterility needs to be studied. RNA-seq analyses were performed on the floral buds at two stages (0-5 and 6-8), before and after the formation of stamen primordium. As a result, a total of 1769 and 594 differentially expressed genes (DEGs) were detected in the CMS line compared to its maintainer line at the two stages, respectively. In accordance with the CMS phenotype, the up- and downstream regulators of the stamen identity genes AP3 and PI were up- and downregulated in the CMS line, respectively. Furthermore, isobaric tags for relative and absolute quantitation (iTRAQ) analysis showed that a total of 760 differentially abundant proteins (DAPs) were identified in flower buds at stages 0-8, and most of the proteins related to the anther development, oxidative phosphorylation, and programmed cell death (PCD) were downregulated in inap CMS. In combined transcriptomic and proteomic analysis, a total of 32 DEGs/DAPs were identified, of which 7 common DEGs/DAPs had the same expression trend at stage 0-8 of flower development. The downregulation of genes related to the energy deficiency, hormone signal transduction, and the maintenance of mitochondrial metabolic homeostasis at stage 0-5 might disturb the normal differentiation of stamen primordium, resulting in carpelloid stamen of inap CMS. The study will help provide insights into the molecular mechanism of this new male sterility.

Production and cytology of Brassica autoallohexaploids with two and four copies of two subgenomes.

KEY MESSAGE: Different digenomic Brassica autoallohexaploids were produced from the crosses of three allotetraploids and ancestral diploids and characterized for the cytological behavior of two subgenomes with two and four copies. Interspecific hybridization and allopolyploidization present an important pathway for plant evolution and breeding. In this study, different types of digenomic autoallohexaploids with two or four copies of two subgenomes (AAAACC, AACCCC, AAAABB, BBBBCC, BBCCCC) were synthesized by the crosses between three Brassica allotetraploids and their diploid progenitors and the chromosome doubling, and their meiotic behaviors were analyzed by fluorescence in situ hybridization (FISH). These autoallohexaploids showed some variations in pollen fertility and seed-sets and produced both euploid and aneuploid progenies with some chromosomes lost. Two subgenomes in these autoallohexaploids showed some aberrant pairings and segregations, and the degrees of meiotic regularity were negatively associated with the genome affinities. The chromosomes of the subgenome with four copies formed few quadrivalents with the average number < 2, and mainly paired as bivalents, and majority of the chromosomes from the subgenome with two copies gave the expected bivalents. The different extents of the equal and unequal segregations corresponded to the chromosome pairings. The development and cytological investigation of these autoallohexaploids provide not only the new germplasm for genetic research and breeding but also the new clues for the genome behavior and interplay between these subgenomes with different copies.

R gene triplication confers European fodder turnip with improved clubroot resistance.

Clubroot is one of the most important diseases for many important cruciferous vegetables and oilseed crops worldwide. Different clubroot resistance (CR) loci have been identified from only limited species in Brassica, making it difficult to compare and utilize these loci. European fodder turnip ECD04 is considered one of the most valuable resources for CR breeding. To explore the genetic and evolutionary basis of CR in ECD04, we sequenced the genome of ECD04 using de novo assembly and identified 978 candidate R genes. Subsequently, the 28 published CR loci were physically mapped to 15 loci in the ECD04 genome, including 62 candidate CR genes. Among them, two CR genes, CRA3.7.1 and CRA8.2.4, were functionally validated. Phylogenetic analysis revealed that CRA3.7.1 and CRA8.2.4 originated from a common ancestor before the whole-genome triplication (WGT) event. In clubroot susceptible Brassica species, CR-gene homologues were affected by transposable element (TE) insertion, resulting in the loss of CR function. It can be concluded that the current functional CR genes in Brassica rapa and non-functional CR genes in other Brassica species were derived from a common ancestral gene before WGT. Finally, a hypothesis for CR gene evolution is proposed for further discussion.

QTL Identification for Stem Fiber, Strength and Rot Resistance in a DH Population from an Alien Introgression of Brassica napus.

Stem fiber, stem strength and stem-rot resistance are important agronomic traits in Brassica napus. To understand the molecular mechanism that controls the stem-related traits, we investigated the stem lignin (ADL), cellulose (Cel), hemicellulose (Hem) content, S/G monolignol ratio (SG), stem breaking force (BF), breaking strength (F) and Sclerotinia sclerotiorum resistance (SSR). Each trait was significantly positively or negatively correlated with more than three of the other six traits. QTL mapping for ADL, Cel, Hem, SG, BF, F and SSR were performed using a doubled haploid population derived from an intertribal B. napus introgression line 'Y689' crossed with B. napus cv. 'Westar'. A total of 67 additive QTL were identified and integrated into 55 consensus QTL by meta-analysis. Among the 55 consensus QTL, 23 (41.8%) QTL were co-located and were integrated into 11 unique QTL. The QTL by environment (Q × E) interactions were analyzed and 22 combined QTL were identified. In addition, candidate genes within the QTL intervals were proposed based on the known function of Arabidopsis orthologs. These results provided valuable information for improving lodging resistance, S. sclerotiorum resistance and mechanized harvesting of B. napus.

Integrating GWAS, linkage mapping and gene expression analyses reveal the genetic control of first branch height in Brassica napus L.

Rapeseed (Brassica napus L.) is a crucial oil crop cultivated worldwide. First branch height, an essential component of rapeseed plant architecture, has an important effect on yield and mechanized harvesting; however, the underlying genetic mechanism remains unclear. In this study, based on the 60K single nucleotide polymorphism array and a recombinant inbred lines population derived from M083 and 888-5, a total of 19 QTLs were detected in five environments, distributed on linkage groups A02, A09, A10, C06, and C07, which explained phenotypic variation ranging from 4.87 to 29.87%. Furthermore, 26 significant SNPs were discovered on Chr.A02 by genome-wide association study in a diversity panel of 324 re-sequencing accessions. The major QTL of the first branch height trait was co-located on Chr.A02 by integrating linkage mapping and association mapping. Eleven candidate genes were screened via allelic variation analysis, inter-subgenomic synteny analysis, and differential expression of genes in parental shoot apical meristem tissues. Among these genes, BnaA02g13010D, which encodes a TCP transcription factor, was confirmed as the target gene according to gene function annotation, haplotype analysis, and full-length gene sequencing, which revealed that TATA insertion/deletion in the promoter region was closely linked to significantly phenotypic differences BnaA02.TCP1 M083 overexpression resulted in decreased branch height and increased branch number in Arabidopsis. These results provide a genetic basis for first branch height and the ideal architecture of B. napus.