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.     

Knockout of two BnaMAX1 homologs by CRISPR/Cas9‐targeted mutagenesis improves plant architecture and increases yield in rapeseed (Brassica napus L.)

Plant height and branch number are essential components of rapeseed plant architecture and are directly correlated with its yield. Presently, improvement of plant architecture is a major challenge in rapeseed breeding. In this study, we first verified that the two rapeseed BnaMAX1 genes had redundant functions resembling those of Arabidopsis MAX1, which regulates plant height and axillary bud outgrowth. Therefore, we designed two sgRNAs to edit these BnaMAX1 homologs using the CRISPR/Cas9 system. The T₀ plants were edited very efficiently (56.30%–67.38%) at the BnaMAX1 target sites resulting in homozygous, heterozygous, bi‐allelic and chimeric mutations. Transmission tests revealed that the mutations were passed on to the T₁ and T₂ progeny. We also obtained transgene‐free lines created by the CRISPR/Cas9 editing, and no mutations were detected in potential off‐target sites. Notably, simultaneous knockout of all four BnaMAX1 alleles resulted in semi‐dwarf and increased branching phenotypes with more siliques, contributing to increased yield per plant relative to wild type. Therefore, these semi‐dwarf and increased branching characteristics have the potential to help construct a rapeseed ideotype. Significantly, the editing resources obtained in our study provide desirable germplasm for further breeding of high yield in rapeseed.     

Homoeologous exchange is a major cause of gene presence/absence variation in the amphidiploid Brassica napus

Homoeologous exchanges (HEs) have been shown to generate novel gene combinations and phenotypes in a range of polyploid species. Gene presence/absence variation (PAV) is also a major contributor to genetic diversity. In this study, we show that there is an association between these two events, particularly in recent Brassica napus synthetic accessions, and that these represent a novel source of genetic diversity, which can be captured for the improvement of this important crop species. By assembling the pangenome of B. napus, we show that 38% of the genes display PAV behaviour, with some of these variable genes predicted to be involved in important agronomic traits including flowering time, disease resistance, acyl lipid metabolism and glucosinolate metabolism. This study is a first and provides a detailed characterization of the association between HEs and PAVs in B. napus at the pangenome level.     

Polymorphism in the neurofibromin gene,Nf1, is associated with antagonistic selection on wing size and development time in Drosophila melanogaster

In many invertebrates, body size shows genetically based clines, with size increasing in colder climates. Large body size is typically associated with prolonged development times. We consider variation in the CNS‐specific gene neurofibromin 1 (Nf1) and its association with body size and development time. We identified two major Nf1 haplotypes in natural populations, Nf1‐insertion‐A and Nf1‐deletion‐G. These haplotypes are characterized by a 45‐base insertion/deletion (INDEL) in Nf1 intron 2 and an A/G synonymous substitution (locus L17277). Linkage disequilibrium (LD) between the INDEL and adjacent sites is high but appears to be restricted within the Nf1 gene interval. In Australia, the frequency of the Nf1‐insertion‐A haplotype increases with latitude where wing size is larger, independent of the chromosomal inversion In(3R)Payne. Unexpectedly, the Nf1‐insertion‐A haplotype is negatively associated with wing size. We found that the Nf1‐insertion‐A haplotype is enriched in females with shorter development time. This suggests that the Nf1 haplotype cline may be driven by selection for development time rather than size; females from southern (higher latitude) D. melanogaster populations maintain a rapid development time despite being relatively larger, and the higher incidence of Nf1‐insertion‐A in Southern Australia may contribute to this pattern, whereas the effects of the Nf1 haplotypes on size may be countered by other loci with antagonistic effects on size and development time. Our results point to the potential complexity involved in identifying selection on genetic variants exhibiting pleiotropic effects when studies are based on spatial patterns or association studies.     

Identification and comprehensive analysis of the CLV3/ESR‐related (CLE) gene family in Brassica napus L.

• The CLE (CLAVATA3/ESR) gene family, encoding a group of small secretory peptides, plays important roles in cell‐to‐cell communication, thereby controlling a broad spectrum of development processes. The CLE family has been systematically characterized in some plants, but not in Brassica napus.
• In the present study, 116 BnCLE genes were identified in the B. napus genome, including seven unannotated, six incorrectly predicted and five multi‐CLE domain‐encoding genes. These BnCLE members were separated into seven distinct groups based on phylogenetic analysis, which might facilitate the functional characterization of the peptides.
• Further characterization of CLE pre‐propeptides revealed 31 unique CLE peptides from 45 BnCLE genes, which may give rise to distinct roles of BnCLE and expansion of the gene family. The biological activity of these unique CLE dodecamer peptides was tested further through in vitro peptide assays. Variations in several important residues were identified as key contributors to the functional differentiation of BnCLE and expansion of the gene family in B. napus. Expression profile analysis helped to characterize possible functional redundancy and sub‐functionalization among the BnCLE members.
• This study presents a comprehensive overview of the CLE gene family in B. napus and provides a foundation for future evolutionary and functional studies.

     

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.     

Down‐regulation of OsSPX1 caused semi‐male sterility,resulting in reduction of grain yield in rice

OsSPX1, a rice SPX domain gene, involved in the phosphate (Pi)‐sensing mechanism plays an essential role in the Pi‐signalling network through interaction with OsPHR2. In this study, we focused on the potential function of OsSPX1 during rice reproductive phase. Based on investigation of OsSPX1 antisense and sense transgenic rice lines in the paddy fields, we discovered that the down‐regulation of OsSPX1 caused reduction of seed‐setting rate and filled grain number. Through examination of anthers and pollens of the transgenic and wild‐type plants by microscopy, we found that the antisense of OsSPX1 gene led to semi‐male sterility, with lacking of mature pollen grains and phenotypes with a disordered surface of anthers and pollens. We further conducted rice whole‐genome GeneChip analysis to elucidate the possible molecular mechanism underlying why the down‐regulation of OsSPX1 caused deficiencies in anthers and pollens and lower seed‐setting rate in rice. The down‐regulation of OsSPX1 significantly affected expression of genes involved in carbohydrate metabolism and sugar transport, anther development, cell cycle, etc. These genes may be related to pollen fertility and male gametophyte development. Our study demonstrated that down‐regulation of OsSPX1 disrupted rice normal anther and pollen development by affecting carbohydrate metabolism and sugar transport, leading to semi‐male sterility, and ultimately resulted in low seed‐setting rate and grain yield.     

A sucrose non‐fermenting‐1‐related protein kinase‐1 gene,IbSnRK1, improves starch content,composition, granule size,degree of crystallinity and gelatinization in transgenic sweet potato

Sucrose non‐fermenting‐1‐related protein kinase‐1 (SnRK1) is an essential energy‐sensing regulator and plays a key role in the global control of carbohydrate metabolism. The SnRK1 gene has been found to increase starch accumulation in several plant species. However, its roles in improving starch quality have not been reported to date. In this study, we found that the IbSnRK1 gene was highly expressed in the storage roots of sweet potato and strongly induced by exogenous sucrose. Its expression followed the circandian rhythm. Its overexpression not only increased starch content, but also decreased proportion of amylose, enlarged granule size and improved degree of crystallinity and gelatinization in transgenic sweet potato, which revealed, for the first time, the important roles of SnRK1 in improving starch quality of plants. The genes involved in starch biosynthesis pathway were systematically up‐regulated, and the content of ADP‐glucose as an important precursor for starch biosynthesis and the activities of key enzymes were significantly increased in transgenic sweet potato. These findings indicate that IbSnRK1 improves starch content and quality through systematical up‐regulation of the genes and the increase in key enzyme activities involved in starch biosynthesis pathway in transgenic sweet potato. This gene has the potential to improve starch content and quality in sweet potato and other plants.     

Induction of telomere‐mediated chromosomal truncation and behavior of truncated chromosomes in Brassica napus

Engineered minichromosomes could be stably inherited and serve as a platform for simultaneously transferring and stably expressing multiple genes. Chromosomal truncation mediated by repeats of telomeric sequences is a promising approach for the generation of minichromosomes. In the present work, direct repetitive sequences of Arabidopsis telomere were used to study telomere‐mediated truncation of chromosomes in Brassica napus. Transgenes containing alien Arabidopsis telomere were successfully obtained, and Southern blotting and fluorescence in situ hybridization (FISH) results show that the transgenes resulted in successful chromosomal truncation in B. napus. In addition, truncated chromosomes were inherited at rates lower than that predicted by Mendelian rules. To determine the potential manipulations and applications of the engineered chromosomes, such as the stacking of multiple transgenes and the Cre/lox and FRT/FLP recombination systems, both amenable to genetic manipulations through site‐specific recombination in somatic cells, were tested for their ability to undergo recombination in B. napus. These results demonstrate that alien Arabidopsis telomere is able to mediate chromosomal truncation in B. napus. This technology would be feasible for chromosomal engineering and for studies on chromosome structure and function in B. napus.     

Increased expression of phospholipase Dα1 in guard cells decreases water loss with improved seed production under drought in Brassica napus

The activation of phospholipase Dα1 (PLDα1) produces lipid messenger phosphatidic acid and promotes stomatal closure in Arabidopsis. To explore the use of the PLDα1‐mediated signalling towards decreasing water loss in crop plants, we introduced Arabidopsis PLDα1 under the control of a guard cell–specific promoter AtKatI_pro into two canola (Brassica napus) cultivars. Multiple AtKatI_pro::PLDα1 lines in each cultivar displayed decreased water loss and improved biomass accumulation under hyperosmotic stress conditions, including drought and high salinity. Moreover, AtKatI_pro::PLDα1 plants produced more seeds than did WT plants in fields under drought. The results indicate that the guard cell–specific expression of PLDα1 has the potential to improve crop yield by enhancing drought tolerance.     

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.     

Thiol‐based redox proteins in abscisic acid and methyl jasmonate signaling in Brassica napus guard cells

Reversibly oxidized cysteine sulfhydryl groups serve as redox sensors or targets of redox sensing that are important in various physiological processes. However, little is known about redox‐sensitive proteins in guard cells and how they function in stomatal signaling. In this study, Brassica napus guard‐cell proteins altered by redox in response to abscisic acid (ABA) or methyl jasmonate (MeJA) were identified by complementary proteomics approaches, saturation differential in‐gel electrophoresis and isotope‐coded affinity tagging. In total, 65 and 118 potential redox‐responsive proteins were identified in ABA‐ and MeJA‐treated guard cells, respectively. All the proteins contain at least one cysteine, and over half of them are predicted to form intra‐molecular disulfide bonds. Most of the proteins fall into the functional groups of ‘energy’, ‘stress and defense’ and ‘metabolism’. Based on the peptide sequences identified by mass spectrometry, 30 proteins were common to ABA‐ and MeJA‐treated samples. A total of 44 cysteines were mapped in the identified proteins, and their levels of redox sensitivity were quantified. Two of the proteins, a sucrose non‐fermenting 1‐related protein kinase and an isopropylmalate dehydrogenase, were confirmed to be redox‐regulated and involved in stomatal movement. This study creates an inventory of potential redox switches, and highlights a protein redox regulatory mechanism in ABA and MeJA signal transduction in guard cells.     

Maternal control of seed weight in rapeseed (Brassica napus L.): the causal link between the size of pod (mother,source) and seed (offspring,sink)

Seed size/weight is one of the key traits related to plant domestication and crop improvement. In rapeseed (Brassica napus L.) germplasm, seed weight shows extensive variation, but its regulatory mechanism is poorly understood. To identify the key mechanism of seed weight regulation, a systematic comparative study was performed. Genetic, morphological and cytological evidence showed that seed weight was controlled by maternal genotype, through the regulation of seed size mainly via cell number. The physiological evidence indicated that differences in the pod length might result in differences in pod wall photosynthetic area, carbohydrates and the final seed weight. We also identified two pleiotropic major quantitative trait loci that acted indirectly on seed weight via their effects on pod length. RNA‐seq results showed that genes related to pod development and hormones were significantly differentially expressed in the pod wall; genes related to development, cell division, nutrient reservoir and ribosomal proteins were all up‐regulated in the seeds of the large‐seed pool. Finally, we proposed a potential seed weight regulatory mechanism that is specific to rapeseed and novel in plants. The results demonstrate a causal link between the size of the pod (mother, source) and the seed (offspring, sink) in rapeseed, which provides novel insight into the maternal control of seed weight and will open a new research field in plants.