Genetic variation of BnaA3.NIP5;1 expressing in the lateral root cap contributes to boron deficiency tolerance in Brassica napus

Boron (B) is essential for vascular plants. Rapeseed (Brassica napus) is the second leading crop source for vegetable oil worldwide, but its production is critically dependent on B supplies. BnaA3.NIP5;1 was identified as a B-efficient candidate gene in B. napus in our previous QTL fine mapping. However, the molecular mechanism through which this gene improves low-B tolerance remains elusive. Here, we report genetic variation in BnaA3.NIP5;1 gene, which encodes a boric acid channel, is a key determinant of low-B tolerance in B. napus. Transgenic lines with increased BnaA3.NIP5;1 expression exhibited improved low-B tolerance in both the seedling and maturity stages. BnaA3.NIP5;1 is preferentially polar-localized in the distal plasma membrane of lateral root cap (LRC) cells and transports B into the root tips to promote root growth under B-deficiency conditions. Further analysis revealed that a CTTTC tandem repeat in the 5’UTR of BnaA3.NIP5;1 altered the expression level of the gene, which is tightly associated with plant growth and seed yield. Field tests with natural populations and near-isogenic lines (NILs) confirmed that the varieties carried BnaA3.NIP5;1^Q allele significantly improved seed yield. Taken together, our results provide novel insights into the low-B tolerance of B. napus, and the elite allele of BnaA3.NIP5;1 could serve as a direct target for breeding low-B-tolerant cultivars.     

A CACTA‐like transposable element in the upstream region of BnaA9.CYP78A9 acts as an enhancer to increase silique length and seed weight in rapeseed

Rapeseed (Brassica napus L.) is a model plant for polyploid crop research and the second‐leading source of vegetable oil worldwide. Silique length (SL) and seed weight are two important yield‐influencing traits in rapeseed. Using map‐based cloning, we isolated qSLWA9, which encodes a P450 monooxygenase (BnaA9.CYP78A9) and functions as a positive regulator of SL. The expression level of BnaA9.CYP78A9 in silique valves of the long‐silique variety is much higher than that in the regular‐silique variety, which results in elongated cells and a prolonged phase of silique elongation. Plants of the long‐silique variety and transgenic plants with high expression of BnaA9.CYP78A9 had a higher concentration of auxin in the developing silique; this induced a number of auxin‐related genes but no genes in well‐known auxin biosynthesis pathways, suggesting that BnaA9.CYP78A9 may influence auxin concentration by affecting auxin metabolism or an unknown auxin biosynthesis pathway. A 3.7‐kb CACTA‐like transposable element (TE) inserted in the 3.9‐kb upstream regulatory sequence of BnaA9.CYP78A9 elevates the expression level, suggesting that the CACTA‐like TE acts as an enhancer to stimulate high gene expression and silique elongation. Marker and sequence analysis revealed that the TE in B. napus had recently been introgressed from Brassica rapa by interspecific hybridization. The insertion of the TE is consistently associated with long siliques and large seeds in both B. napus and B. rapa collections. However, the frequency of the CACTA‐like TE in rapeseed varieties is still very low, suggesting that this allele has not been widely used in rapeseed breeding programs and would be invaluable for yield improvement in rapeseed breeding.     

Dissection of genetic architecture for glucosinolate accumulations in leaves and seeds of Brassica napus by genome‐wide association study

Glucosinolates (GSLs), whose degradation products have been shown to be increasingly important for human health and plant defence, compose important secondary metabolites found in the order Brassicales. It is highly desired to enhance pest and disease resistance by increasing the leaf GSL content while keeping the content low in seeds of Brassica napus, one of the most important oil crops worldwide. Little is known about the regulation of GSL accumulation in the leaves. We quantified the levels of 9 different GSLs and 15 related traits in the leaves of 366 accessions and found that the seed and leaf GSL content were highly correlated (r = 0.79). A total of 78 loci were associated with GSL traits, and five common and eleven tissue‐specific associated loci were related to total leaf and seed GSL content. Thirty‐six candidate genes were inferred to be involved in GSL biosynthesis. The candidate gene BnaA03g40190D (BnaA3.MYB28) was validated by DNA polymorphisms and gene expression analysis. This gene was responsible for high leaf/low seed GSL content and could explain 30.62% of the total leaf GSL variation in the low seed GSL panel and was not fixed during double‐low rapeseed breeding. Our results provide new insights into the genetic basis of GSL variation in leaves and seeds and may facilitate the metabolic engineering of GSLs and the breeding of high leaf/low seed GSL content in B. napus.     

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.     

Genetic changes in a novel breeding population of Brassica napus synthesized from hundreds of crosses between B. rapa and B. carinata

Introgression of genomic variation between and within related crop species is a significant evolutionary approach for population differentiation, genome reorganization and trait improvement. Using the Illumina Infinium Brassica 60K SNP array, we investigated genomic changes in a panel of advanced generation new‐type Brassica napus breeding lines developed from hundreds of interspecific crosses between 122 Brassica rapa and 74 Brassica carinata accessions, and compared them with representative accessions of their three parental species. The new‐type B. napus population presented rich genetic diversity and abundant novel genomic alterations, consisting of introgressions from B. rapa and B. carinata, novel allelic combinations, reconstructed linkage disequilibrium patterns and haplotype blocks, and frequent deletions and duplications (nonrandomly distributed), particularly in the C subgenome. After a much shorter, but very intensive, selection history compared to traditional B. napus, a total of 15 genomic regions with strong selective sweeps and 112 genomic regions with putative signals of selective sweeps were identified. Some of these regions were associated with important agronomic traits that were selected for during the breeding process, while others were potentially associated with restoration of genome stability and fertility after interspecific hybridization. Our results demonstrate how a novel method for population‐based crop genetic improvement can lead to rapid adaptation, restoration of genome stability and positive responses to artificial selection.     

The Brassica napus receptor‐like protein RLM2 is encoded by a second allele of the LepR3/Rlm2 blackleg resistance locus

Leucine‐rich repeat receptor‐like proteins (LRR‐RLPs) are highly adaptable parts of the signalling apparatus for extracellular detection of plant pathogens. Resistance to blackleg disease of Brassica spp. caused by Leptosphaeria maculans is largely governed by host race‐specific R‐genes, including the LRR‐RLP gene LepR3. The blackleg resistance gene Rlm2 was previously mapped to the same genetic interval as LepR3. In this study, the LepR3 locus of the Rlm2 Brassica napus line ‘Glacier DH24287’ was cloned, and B. napus transformants were analysed for recovery of the Rlm2 phenotype. Multiple B. napus, B. rapa and B. juncea lines were assessed for sequence variation at the locus. Rlm2 was found to be an allelic variant of the LepR3 LRR‐RLP locus, conveying race‐specific resistance to L. maculans isolates harbouring AvrLm2. Several defence‐related LRR‐RLPs have previously been shown to associate with the RLK SOBIR1 to facilitate defence signalling. Bimolecular fluorescence complementation (BiFC) and co‐immunoprecipitation of RLM2‐SOBIR1 studies revealed that RLM2 interacts with SOBIR1 of Arabidopsis thaliana when co‐expressed in Nicotiana benthamiana. The interaction of RLM2 with AtSOBIR1 is suggestive of a conserved defence signalling pathway between B. napus and its close relative A. thaliana.     

High‐throughput multiplex cpDNA resequencing clarifies the genetic diversity and genetic relationships among Brassica napus,Brassica rapa and Brassica oleracea

Brassica napus (rapeseed) is a recent allotetraploid plant and the second most important oilseed crop worldwide. The origin of B. napus and the genetic relationships with its diploid ancestor species remain largely unresolved. Here, chloroplast DNA (cpDNA) from 488 B. napus accessions of global origin, 139 B. rapa accessions and 49 B. oleracea accessions were populationally resequenced using Illumina Solexa sequencing technologies. The intraspecific cpDNA variants and their allelic frequencies were called genomewide and further validated via EcoTILLING analyses of the rpo region. The cpDNA of the current global B. napus population comprises more than 400 variants (SNPs and short InDels) and maintains one predominant haplotype (Bncp1). Whole‐genome resequencing of the cpDNA of Bncp1 haplotype eliminated its direct inheritance from any accession of the B. rapa or B. oleracea species. The distribution of the polymorphism information content (PIC) values for each variant demonstrated that B. napus has much lower cpDNA diversity than B. rapa; however, a vast majority of the wild and cultivated B. oleracea specimens appeared to share one same distinct cpDNA haplotype, in contrast to its wild C‐genome relatives. This finding suggests that the cpDNA of the three Brassica species is well differentiated. The predominant B. napus cpDNA haplotype may have originated from uninvestigated relatives or from interactions between cpDNA mutations and natural/artificial selection during speciation and evolution. These exhaustive data on variation in cpDNA would provide fundamental data for research on cpDNA and chloroplasts.     

Genome-wide association study dissects the genetic control of plant height and branch number in response to low-phosphorus stress in Brassica napus

Background and AimsOilseed rape (Brassica napus) is one of the most important oil crops worldwide. Phosphorus (P) deficiency severely decreases the plant height and branch number of B. napus. However, the genetic bases controlling plant height and branch number in B. napus under P deficiency remain largely unknown. This study aims to mine candidate genes for plant height and branch number by genome-wide association study (GWAS) and determine low-P-tolerance haplotypes.MethodsAn association panel of B. napus was grown in the field with a low P supply (P, 0 kg ha⁻¹) and a sufficient P supply (P, 40 kg ha⁻¹) across 2 years and plant height and branch number were investigated. More than five million single-nucleotide polymorphisms (SNPs) were used to conduct GWAS of plant height and branch number at two contrasting P supplies.Key ResultsA total of 2127 SNPs were strongly associated (P < 6·25 × 10⁻⁰⁷) with plant height and branch number at two P supplies. There was significant correlation between phenotypic variation and the number of favourable alleles of associated loci on chromosomes A10 (chrA10_821671) and C08 (chrC08_27999846), which will contribute to breeding improvement by aggregating these SNPs. BnaA10g09290D and BnaC08g26640D were identified to be associated with chrA10_821671 and chrC08_27999846, respectively. Candidate gene association analysis and haplotype analysis showed that the inbred lines carrying ATT at BnaA10g09290Hap1 and AAT at BnaC08g26640Hap1 had greater plant height than lines carrying other haplotype alleles at low P supply.ConclusionOur results demonstrate the power of GWAS in identifying genes of interest in B. napus and provided insights into the genetic basis of plant height and branch number at low P supply in B. napus. Candidate genes and favourable haplotypes may facilitate marker-based breeding efforts aimed at improving P use efficiency in B. napus.     

Comparative mitochondrial genome analysis reveals the evolutionary rearrangement mechanism in Brassica

The genus Brassica has many species that are important for oil, vegetable and other food products. Three mitochondrial genome types (mitotype) originated from its common ancestor. In this paper, a B. nigra mitochondrial main circle genome with 232,407 bp was generated through de novo assembly. Synteny analysis showed that the mitochondrial genomes of B. rapa and B. oleracea had a better syntenic relationship than B. nigra. Principal components analysis and development of a phylogenetic tree indicated maternal ancestors of three allotetraploid species in Us triangle of Brassica. Diversified mitotypes were found in allotetraploid B. napus, in which napus‐type B. napus was derived from B. oleracea, while polima‐type B. napus was inherited from B. rapa. In addition, the mitochondrial genome of napus‐type B. napus was closer to botrytis‐type than capitata‐type B. oleracea. The sub‐stoichiometric shifting of several mitochondrial genes suggested that mitochondrial genome rearrangement underwent evolutionary selection during domestication and/or plant breeding. Our findings clarify the role of diploid species in the maternal origin of allotetraploid species in Brassica and suggest the possibility of breeding selection of the mitochondrial genome.