Bioinformatics and functional analysis of EDS1 genes in Brassica napus in response to Plasmodiophora brassicae infection.

Enhanced Disease Susceptibility 1 (EDS1) is a key regulator of plant-pathogen-associated molecular pattern-triggered immunity (PTI) and effector-triggered immunity (ETI) responses. In the Brassica napus genome, we identified six novel EDS1 genes, among which four were responsive to clubroot infection, a major rapeseed disease resistant to chemical control. Developing resistant cultivars is a potent and economically viable strategy to control clubroot infection. Bioinformatics analysis revealed conserved domains and structural uniformity in Bna-EDS1 homologs. Bna-EDS1 promoters harbored elements associated with diverse phytohormones and stress responses, highlighting their crucial roles in plant defense. A functional analysis was performed with Bna-EDS1 overexpression and RNAi transgenic lines. Bna-EDS1 overexpression boosted resistance to clubroot and upregulated defense-associated genes (PR1, PR2, ICS1, and CBP60), while Bna-EDS1 RNAi increased plant susceptibility, indicating suppression of the defense signaling pathway downstream of NBS-LRRs. RNA-Seq analysis identified key transcripts associated with clubroot resistance, including phenylpropanoid biosynthesis. Activation of SA regulator NPR1, defense signaling markers PR1 and PR2, and upregulation of MYC-TFs suggested that EDS1-mediated clubroot resistance potentially involves the SA pathway. Our findings underscore the pivotal role of Bna-EDS1-dependent mechanisms in resistance of B. napus to clubroot disease, and provide valuable insights for fortifying resistance against Plasmodiophora brassicae infection in rapeseed.

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.

Genetic and Biochemical Investigation of Seed Fatty Acid Accumulation in Arabidopsis.

As a vegetable oil, consisting principally of triacylglycerols, is the major storage form of photosynthetically-fixed carbon in oilseeds which are of significant agricultural and industrial value. Photosynthesis in chlorophyll-containing green seeds, along with photosynthesis in leaves and other green organs, generates ATP and reductant (NADPH and NADH) needed for seed fatty acid production. However, contribution of seed photosynthesis to fatty acid accumulation in seeds have not been well-defined. Here, we report the contribution of seed-photosynthesis to fatty acid production by probing segregating green (photosynthetically-competent) and non-green or yellow (photosynthetically-non-competent) seeds in siliques of an Arabidopsis chlorophyll synthase mutant. Using this mutant, we found that yellow seeds lacking photosynthetic capacity reached 80% of amounts of oil in green seeds at maturity. Combining this with studies using shaded siliques, we determined that seed-photosynthesis accounts for 20% and silique and leaf/stem photosynthesis each account for ~40% of the ATP and reductant for seed oil production. Transmission electron microscopy (TEM) and pyridine nucleotides and ATP analyses revealed that seed photosynthesis provides ATP and reductant for oil production mostly during early development, as evidenced by delayed oil accumulation in non-green seeds. Transcriptomic analyses suggests that the oxidative pentose phosphate pathway could be the source of carbon, energy and reductants required for fatty acid synthesis beyond the early stages of seed development.

Identification and Characterization of Circular RNAs in Brassica rapa in Response to Plasmodiophora brassicae.

Plasmodiophora brassicae is a soil-borne pathogen that attacks the roots of cruciferous plants and causes clubroot disease. CircRNAs are noncoding RNAs, widely existing in plant and animal species. Although knowledge of circRNAs has been updated continuously and rapidly, information about circRNAs in the regulation of clubroot disease resistance is extremely limited in Brassica rapa. Here, Chinese cabbage (BJN 222) containing clubroot resistance genes (CRa) against P. brassicae Pb4 was susceptible to PbE. To investigate the mechanism of cicRNAs responsible for clubroot disease resistance in B. rapa, circRNA-seq was performed with roots of 'BJN 222' at 0, 8, and 23 days post-inoculated (dpi) with Pb4 and PbE. A total of 231 differentially expressed circRNAs were identified between the groups. Based on the differentially expressed circRNAs, the circRNA-miRNA-mRNA network was constructed using the target genes directly or indirectly related to plant resistance. Upregulated novel_circ_000495 suppressed the expression of miR5656-y, leading to the upregulation of Bra026508, which might cause plant resistance. Our results provide new insights into clubroot resistance mechanisms and lay a foundation for further studies exploring complex gene regulation networks in B. rapa.

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.

Somatic variants for seed and fruit set in grapevine.

BACKGROUND: Grapevine reproductive development has direct implications on yield. It also impacts on berry and wine quality by affecting traits like seedlessness, berry and bunch size, cluster compactness and berry skin to pulp ratio. Seasonal fluctuations in yield, fruit composition and wine attributes, which are largely driven by climatic factors, are major challenges for worldwide table grape and wine industry. Accordingly, a better understanding of reproductive processes such as gamete development, fertilization, seed and fruit set is of paramount relevance for managing yield and quality. With the aim of providing new insights into this field, we searched for clones with contrasting seed content in two germplasm collections. RESULTS: We identified eight variant pairs that seemingly differ only in seed-related characteristics while showing identical genotype when tested with the GrapeReSeq_Illumina_20K_SNP_chip and several microsatellites. We performed multi-year observations on seed and fruit set deriving from different pollination treatments, with special emphasis on the pair composed by Sangiovese and its seedless variant locally named Corinto Nero. The pollen of Corinto Nero failed to germinate in vitro and gave poor berry set when used to pollinate other varieties. Most berries from both open- and cross-pollinated Corinto Nero inflorescences did not contain seeds. The genetic analysis of seedlings derived from occasional Corinto Nero normal seeds revealed that the few Corinto Nero functional gametes are mostly unreduced. Moreover, three genotypes, including Sangiovese and Corinto Nero, were unexpectedly found to develop fruits without pollen contribution and occasionally showed normal-like seeds. Five missense single nucleotide polymorphisms were identified between Corinto Nero and Sangiovese from transcriptomic data. CONCLUSIONS: Our observations allowed us to attribute a seedlessness type to some variants for which it was not documented in the literature. Interestingly, the VvAGL11 mutation responsible for Sultanina stenospermocarpy was also discovered in a seedless mutant of Gouais Blanc. We suggest that Corinto Nero parthenocarpy is driven by pollen and/or embryo sac defects, and both events likely arise from meiotic anomalies. The single nucleotide polymorphisms identified between Sangiovese and Corinto Nero are suitable for testing as traceability markers for propagated material and as functional candidates for the seedless phenotype.

Promoter variations in a homeobox gene, BnA10.LMI1, determine lobed leaves in rapeseed (Brassica napus L.).

KEY MESSAGE: BnA10.LMI1 positively regulates the development of leaf lobes in Brassica napus, and cis-regulatory divergences cause the different allele effects. Leaf shape is an important agronomic trait, and large variations in this trait exist within the Brassica germplasm. The lobed leaf is a unique morphological characteristic for Brassica improvement. Nevertheless, the molecular basis of leaf lobing in Brassica is poorly understood. Here, we show that an incompletely dominant locus, BnLLA10, is responsible for the lobed-leaf shape in rapeseed. A LATE MERISTEM IDENTITY1 (LMI1)-like gene (BnA10.LMI1) encoding an HD-Zip I transcription factor is the causal gene underlying the BnLLA10 locus. Sequence analysis of parental alleles revealed no sequence variations in the coding sequences, whereas abundant variations were identified in the regulatory region. Consistent with this finding, the expression levels of BnLMI1 were substantially elevated in the lobed-leaf parent compared with its near-isogenic line. The knockout mutations of BnA10.LMI1 gene were induced using the CRISPR/Cas9 system in both HY (the lobed-leaf parent) and J9707 (serrated leaf) genetic backgrounds. BnA10.LMI1 null mutations in the HY background were sufficient to produce unlobed leaves, whereas null mutations in the J9707 background showed no obvious changes in leaf shape compared with the control. Collectively, our results indicate that BnA10.LMI1 positively regulates the development of leaf lobes in B. napus, with cis-regulatory divergences causing the different allelic effects, providing new insights into the molecular mechanism of leaf lobe formation in Brassica crops.

Transcriptome sequencing reveals key potential long non-coding RNAs related to duration of fertility trait in the uterovaginal junction of egg-laying hens.

Duration of fertility, (DF) is an important functional trait in poultry production and lncRNAs have emerged as important regulators of various process including fertility. In this study we applied a genome-guided strategy to reconstruct the uterovaginal junction (UVJ) transcriptome of 14 egg-laying birds with long- and short-DF (n = 7); and sought to uncover key lncRNAs related to duration of fertility traits by RNA-sequencing technology. Examination of RNA-seq data revealed a total of 9977 lncRNAs including 2576 novel lncRNAs. Differential expression (DE) analysis of lncRNA identified 223 lncRNAs differentially expressed between the two groups. DE-lncRNA target genes prediction uncovered over 200 lncRNA target genes and functional enrichment tests predict a potential function of DE-lncRNAs. Gene ontology classification and pathway analysis revealed 8 DE-lncRNAs, with the majority of their target genes enriched in biological functions such as reproductive structure development, developmental process involved in reproduction, response to cytokine, carbohydrate binding, chromatin organization, and immune pathways. Differential expression of lncRNAs and target genes were confirmed by qPCR. Together, these results significantly expand the utility of the UVJ transcriptome and our analysis identification of key lncRNAs and their target genes regulating DF will form the baseline for understanding the molecular functions of lncRNAs regulating DF.

Development of iFOX-hunting as a functional genomic tool and demonstration of its use to identify early senescence-related genes in the polyploid Brassica napus.

Functional genomic studies of many polyploid crops, including rapeseed (Brassica napus), are constrained by limited tool sets. Here we report development of a gain-of-function platform, termed 'iFOX (inducible Full-length cDNA OvereXpressor gene)-Hunting', for inducible expression of B. napus seed cDNAs in Arabidopsis. A Gateway-compatible plant gene expression vector containing a methoxyfenozide-inducible constitutive promoter for transgene expression was developed. This vector was used for cloning of random cDNAs from developing B. napus seeds and subsequent Agrobacterium-mediated transformation of Arabidopsis. The inducible promoter of this vector enabled identification of genes upon induction that are otherwise lethal when constitutively overexpressed and to control developmental timing of transgene expression. Evaluation of a subset of the resulting ~6000 Arabidopsis transformants revealed a high percentage of lines with full-length B. napus transgene insertions. Upon induction, numerous iFOX lines with visible phenotypes were identified, including one that displayed early leaf senescence. Phenotypic analysis of this line (rsl-1327) after methoxyfenozide induction indicated high degree of leaf chlorosis. The integrated B. napuscDNA was identified as a homolog of an Arabidopsis acyl-CoA binding protein (ACBP) gene designated BnACBP1-like. The early senescence phenotype conferred by BnACBP1-like was confirmed by constitutive expression of this gene in Arabidopsis and B. napus. Use of the inducible promoter in the iFOX line coupled with RNA-Seq analyses allowed mechanistic clues and a working model for the phenotype associated with BnACBP1-like expression. Our results demonstrate the utility of iFOX-Hunting as a tool for gene discovery and functional characterization of Brassica napus genome.

Cytological and morphological analysis of hybrids between Brassicoraphanus, and Brassica napus for introgression of clubroot resistant trait into Brassica napus L.

Interspecific hybridization is a powerful tool for improvement of crop species, it has the potential to broaden the genetic base and create new plant forms for breeding programs. Synthetic allopolyploid is a widely-used model for the study of genetic recombination and fixed heterosis in Brassica. In Brassica napus breeding, identification and introgression of new sources of clubroot resistance trait from wild or related species into it by hybridization is a long-term crop management strategy for clubroot disease. Radish (Raphanus sativus L.) is a close relative of the Brassica and most radish accessions are immune to the clubroot disease. A synthesized allotetraploid Brassicoraphanus (RRCC, 2n = 36) between R. sativus cv. HQ-04 (2n = 18, RR) and Brassica oleracea var. alboglabra (L.H Bailey) (2n = 18, CC) proved resistant of multiple clubroot disease pathogen P. brassicae. To predict the possibility to transfer the clubroot resistance trait from the RR subgenome of allotetraploid Brassicoraphanus (RRCC, 2n = 36) into Brassica napus (AACC, 2n = 38), we analyzed the frequency of chromosome pairings in the F1 hybrids produced from a cross between B. napus cv. HS5 and the allotetraploid, characterize the genomic composition of some backcrossed progeny (BC1) using GISH, BAC-FISH and AFLP techniques. The level of intergenomic pairing between A and R genomes in the F1 hybrid was high, allosyndetic bivalents formed in 73.53% PMCs indicative of significant level of homeologous recombination between two genomes and high probability of incorporating chromosomal segments/genes from R-genome into A/C-genomes. The BC1 plants inherited variant extra R chromosomes or fragments from allotetraploid as revealed by GISH and AFLP analysis. 13.51% BC2 individuals were resistant to clubroot disease, and several resistance lines had high pollen fertility, Overall, the genetic material presented in this work represents a potential new genetic resource for practical use in breeding B. napus clubroot resistant cultivars.

Transcriptome analysis during berry development provides insights into co-regulated and altered gene expression between a seeded wine grape variety and its seedless somatic variant.

BACKGROUND: Seedless grapes are greatly appreciated for fresh and dry fruit consumption. Parthenocarpy and stenospermocarpy have been described as the main phenomena responsible for seedlessness in Vitis vinifera. However, the key genes underpinning molecular and cellular processes that play a significant role in seed development are not well characterized. To identify important regulators and mechanisms that may be altered in the seedless phenotype, we performed a comprehensive transcriptional analysis to compare the transcriptomes of a popular seeded wine cultivar (wild-type) and its seedless somatic variant (mutant) at three key developmental stages. RESULTS: The transcriptomes revealed by Illumina mRNA-Seq technology had approximately 98% of grapevine annotated transcripts and about 80% of them were commonly expressed in the two lines. Differential gene expression analysis revealed a total of 1075 differentially expressed genes (DE) in the pairwise comparison of developmental stages, which included DE genes specific to the wild-type background, DE genes specific to the mutant background and DE genes commonly shared in both backgrounds. The analysis of differential expression patterns and functional category enrichment of wild-type and mutant DE genes highlighted significant coordination and enrichment of pollen and ovule developmental pathways. The expression of some selected DE genes was further confirmed by real-time RT-PCR analysis. CONCLUSIONS: This study represents the most comprehensive attempt to characterize the genetic bases of seed formation in grapevine. With a high throughput method, we have shown that a seeded wine grape and its seedless somatic variant are similar in several biological processes. Nevertheless, we could identify an inventory of genes with altered expression in the mutant compared to the wild-type, which may be responsible for the seedless phenotype. The genes located within known genomic regions regulating seed content may be used for the development of molecular tools to assist table grape breeding. Therefore the data reported here have provided a rich genomic resource for practical use and functional characterization of the genes that potentially underpin seedlessness in grapevine.