Winter warming post floral initiation delays flowering via bud dormancy activation and affects yield in a winter annual crop.

Winter annual life history is conferred by the requirement for vernalization to promote the floral transition and control the timing of flowering. Here we show using winter oilseed rape that flowering time is controlled by inflorescence bud dormancy in addition to vernalization. Winter warming treatments given to plants in the laboratory and field increase flower bud abscisic acid levels and delay flowering in spring. We show that the promotive effect of chilling reproductive tissues on flowering time is associated with the activity of two FLC genes specifically silenced in response to winter temperatures in developing inflorescences, coupled with activation of a BRANCHED1-dependent bud dormancy transcriptional module. We show that adequate winter chilling is required for normal inflorescence development and high yields in addition to the control of flowering time. Because warming during winter flower development is associated with yield losses at the landscape scale, our work suggests that bud dormancy activation may be important for effects of climate change on winter arable crop yields.

BABY BOOM regulates early embryo and endosperm development.

The BABY BOOM (BBM) AINTEGUMENTA-LIKE (AIL) AP2/ERF domain transcription factor is a major regulator of plant cell totipotency, as it induces asexual embryo formation when ectopically expressed. Surprisingly, only limited information is available on the role of BBM during zygotic embryogenesis. Here we reexamined BBM expression and function in the model plant Arabidopsis thaliana (Arabidopsis) using reporter analysis and newly developed CRISPR mutants. BBM was expressed in the embryo from the zygote stage and also in the maternal (nucellus) and filial (endosperm) seed tissues. Analysis of CRISPR mutant alleles for BBM (bbm-cr) and the redundantly acting AIL gene PLETHORA2 (PLT2) (plt2-cr) uncovered individual roles for these genes in the timing of embryo progression. We also identified redundant roles for BBM and PLT2 in endosperm proliferation and cellularization and the maintenance of zygotic embryo development. Finally, we show that ectopic BBM expression in the egg cell of Arabidopsis and the dicot crops Brassica napus and Solanum lycopersicon is sufficient to bypass the fertilization requirement for embryo development. Together these results highlight roles for BBM and PLT2 in seed development and demonstrate the utility of BBM genes for engineering asexual embryo development in dicot species.

Alleviating salinity stress in canola (Brassica napus L.) through exogenous application of salicylic acid.

Canola, a vital oilseed crop, is grown globally for food and biodiesel. With the enormous demand for growing various crops, the utilization of agriculturally marginal lands is emerging as an attractive alternative, including brackish-saline transitional lands. Salinity is a major abiotic stress limiting growth and productivity of most crops, and causing food insecurity. Salicylic acid (SA), a small-molecule phenolic compound, is an essential plant defense phytohormone that promotes immunity against pathogens. Recently, several studies have reported that SA was able to improve plant resilience to withstand high salinity. For this purpose, a pot experiment was carried out to ameliorate the negative effects of sodium chloride (NaCl) on canola plants through foliar application of SA. Two canola varieties Faisal (V1) and Super (V2) were assessed for their growth performance during exposure to high salinity i.e. 0 mM NaCl (control) and 200 mM NaCl. Three levels of SA (0, 10, and 20 mM) were applied through foliar spray. The experimental design used for this study was completely randomized design (CRD) with three replicates. The salt stress reduced the shoot and root fresh weights up to 50.3% and 47% respectively. In addition, foliar chlorophyll a and b contents decreased up to 61-65%. Meanwhile, SA treatment diminished the negative effects of salinity and enhanced the shoot fresh weight (49.5%), root dry weight (70%), chl. a (36%) and chl. b (67%). Plants treated with SA showed an increased levels of both enzymatic i.e. (superoxide dismutase (27%), peroxidase (16%) and catalase (34%)) and non-enzymatic antioxidants i.e. total soluble protein (20%), total soluble sugar (17%), total phenolic (22%) flavonoids (19%), anthocyanin (23%), and endogenous ascorbic acid (23%). Application of SA also increased the levels of osmolytes i.e. glycine betaine (31%) and total free proline (24%). Salinity increased the concentration of Na+ ions and concomitantly decreased the K+ and Ca2+ absorption in canola plants. Overall, the foliar treatments of SA were quite effective in reducing the negative effects of salinity. By comparing both varieties of canola, it was observed that variety V2 (Super) grew better than variety V1 (Faisal). Interestingly, 20 mM foliar application of SA proved to be effective in ameliorating the negative effects of high salinity in canola plants.

Effect of methyl jasmonate and GA3 on canola (Brassica napus L.) growth, antioxidants activity, and nutrient concentration cultivated in salt-affected soils.

Salinity stress is a significant challenge in agricultural production. When soil contains high salts, it can adversely affect plant growth and productivity due to the high concentration of soluble salts in the soil water. To overcome this issue, foliar applications of methyl jasmonate (MJ) and gibberellic acid (GA3) can be productive amendments. Both can potentially improve the plant's growth attributes and flowering, which are imperative in improving growth and yield. However, limited literature is available on their combined use in canola to mitigate salinity stress. That's why the current study investigates the impact of different levels of MJ (at concentrations of 0.8, 1.6, and 3.2 mM MJ) and GA3 (0GA3 and 5 mg/L GA3) on canola cultivated in salt-affected soils. Applying all the treatments in four replicates. Results indicate that the application of 0.8 mM MJ with 5 mg/L GA3 significantly enhances shoot length (23.29%), shoot dry weight (24.77%), number of leaves per plant (24.93%), number of flowering branches (26.11%), chlorophyll a (31.44%), chlorophyll b (20.28%) and total chlorophyll (27.66%) and shoot total soluble carbohydrates (22.53%) over control. Treatment with 0.8 mM MJ and 5 mg/L GA3 resulted in a decrease in shoot proline (48.17%), MDA (81.41%), SOD (50.59%), POD (14.81%) while increase in N (10.38%), P (15.22%), and K (8.05%) compared to control in canola under salinity stress. In conclusion, 0.8 mM MJ + 5 mg/L GA3 can improve canola growth under salinity stress. More investigations are recommended at the field level to declare 0.8 mM MJ + 5 mg/L GA3 as the best amendment for alleviating salinity stress in different crops.

Exploring silique number in Brassica napus L.: Genetic and molecular advances for improving yield.

Silique number is a crucial yield-related trait for the genetic enhancement of rapeseed (Brassica napus L.). The intricate molecular process governing the regulation of silique number involves various factors. Despite advancements in understanding the mechanisms regulating silique number in Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa), the molecular processes involved in controlling silique number in rapeseed remain largely unexplored. In this review, we identify candidate genes and review the roles of genes and environmental factors in regulating rapeseed silique number. We use genetic regulatory networks for silique number in Arabidopsis and grain number in rice to uncover possible regulatory pathways and molecular mechanisms involved in regulating genes associated with rapeseed silique number. A better understanding of the genetic network regulating silique number in rapeseed will provide a theoretical basis for the genetic improvement of this trait and genetic resources for the molecular breeding of high-yielding rapeseed.

Genetic dissection of Brassica napus seed vigor after aging.

KEY MESSAGE: Stable and novel QTLs that affect seed vigor under different storage durations were discovered, and BnaOLE4, located in the interval of cqSW-C2-3, increased seed vigor after aging. Seed vigor is an important trait in crop breeding; however, the underlying molecular regulatory mechanisms governing this trait in rapeseed remain largely unknown. In the present study, vigor-related traits were analyzed in seeds from a doubled haploid (DH) rapeseed (Brassica napus) population grown in 2 different environments using seeds stored for 7, 5, and 3 years under natural storage conditions. A total of 229 quantitative trait loci (QTLs) were identified and were found to explain 3.78%-17.22% of the phenotypic variance for seed vigor-related traits after aging. We further demonstrated that seed vigor-related traits were positively correlated with oil content (OC) but negatively correlated with unsaturated fatty acids (FAs). Some pleiotropic QTLs that collectively regulate OC, FAs, and seed vigor, such as uq.A8, uq.A3-2, uq.A9-2, and uq.C3-1, were identified. The transcriptomic results from extreme pools of DH lines with distinct seed vigor phenotypes during accelerated aging revealed that various biological pathways and metabolic processes (such as glutathione metabolism and reactive oxygen species) were involved in seed vigor. Through integration of QTL analysis and RNA-Seq, a regulatory network for the control of seed vigor was constructed. Importantly, a candidate (BnaOLE4) from cqSW-C2-3 was selected for functional analysis, and transgenic lines overexpressing BnaOLE4 showed increased seed vigor after artificial aging. Collectively, these results provide novel information on QTL and potential candidate genes for molecular breeding for improved seed storability.

Envirotyping within a multi-environment trial allowed identifying genetic determinants of winter oilseed rape yield stability.

KEY MESSAGE: A comprehensive environmental characterization allowed identifying stable and interactive QTL for seed yield: QA09 and QC09a were detected across environments; whereas QA07a was specifically detected on the most stressed environments. A main challenge for rapeseed consists in maintaining seed yield while adapting to climate changes and contributing to environmental-friendly cropping systems. Breeding for cultivar adaptation is one of the keys to meet this challenge. Therefore, we propose to identify the genetic determinant of seed yield stability for winter oilseed rape using GWAS coupled with a multi-environmental trial and to interpret them in the light of environmental characteristics. Due to a comprehensive characterization of a multi-environmental trial using 79 indicators, four contrasting envirotypes were defined and used to identify interactive and stable seed yield QTL. A total of four QTLs were detected, among which, QA09 and QC09a, were stable (detected at the multi-environmental trial scale or for different envirotypes and environments); and one, QA07a, was specifically detected into the most stressed envirotype. The analysis of the molecular diversity at QA07a showed a lack of genetic diversity within modern lines compared to older cultivars bred before the selection for low glucosinolate content. The results were discussed in comparison with other studies and methods as well as in the context of breeding programs.

Genomic and phenomic predictions help capture low-effect alleles promoting seed germination in oilseed rape in addition to QTL analyses.

KEY MESSAGE: Phenomic prediction implemented on a large diversity set can efficiently predict seed germination, capture low-effect favorable alleles that are not revealed by GWAS and identify promising genetic resources. Oilseed rape faces many challenges, especially at the beginning of its developmental cycle. Achieving rapid and uniform seed germination could help to ensure a successful establishment and therefore enabling the crop to compete with weeds and tolerate stresses during the earliest developmental stages. The polygenic nature of seed germination was highlighted in several studies, and more knowledge is needed about low- to moderate-effect underlying loci in order to enhance seed germination effectively by improving the genetic background and incorporating favorable alleles. A total of 17 QTL were detected for seed germination-related traits, for which the favorable alleles often corresponded to the most frequent alleles in the panel. Genomic and phenomic predictions methods provided moderate-to-high predictive abilities, demonstrating the ability to capture small additive and non-additive effects for seed germination. This study also showed that phenomic prediction estimated phenotypic values closer to phenotypic values than GEBV. Finally, as the predictive ability of phenomic prediction was less influenced by the genetic structure of the panel, it is worth using this prediction method to characterize genetic resources, particularly with a view to design prebreeding populations.

Genetic control of root morphology in response to nitrogen across rapeseed diversity.

Rapeseed (Brassica napus L.) is an oil-containing crop of great economic value but with considerable nitrogen requirement. Breeding root systems that efficiently absorb nitrogen from the soil could be a driver to ensure genetic gains for more sustainable rapeseed production. The aim of this study is to identify genomic regions that regulate root morphology in response to nitrate availability. The natural variability offered by 300 inbred lines was screened at two experimental locations. Seedlings grew hydroponically with low or elevated nitrate levels. Fifteen traits related to biomass production and root morphology were measured. On average across the panel, a low nitrate level increased the root-to-shoot biomass ratio and the lateral root length. A large phenotypic variation was observed, along with important heritability values and genotypic effects, but low genotype-by-nitrogen interactions. Genome-wide association study and bulk segregant analysis were used to identify loci regulating phenotypic traits. The first approach nominated 319 SNPs that were combined into 80 QTLs. Three QTLs identified on the A07 and C07 chromosomes were stable across nitrate levels and/or experimental locations. The second approach involved genotyping two groups of individuals from an experimental F2 population created by crossing two accessions with contrasting lateral root lengths. These individuals were found in the tails of the phenotypic distribution. Co-localized QTLs found in both mapping approaches covered a chromosomal region on the A06 chromosome. The QTL regions contained some genes putatively involved in root organogenesis and represent selection targets for redesigning the root morphology of rapeseed.

Lipid and sugar metabolism play an essential role in pollen development and male sterility: a case analysis in Brassica napus.

AIMS: The genic male sterility (GMS) system is an important strategy for generating heterosis in plants. To better understand the essential role of lipid and sugar metabolism and to identify additional candidates for pollen development and male sterility, transcriptome and metabolome analysis of a GMS line of 1205AB in B. napus was used as a case study. DATA RESOURCES GENERATED: To characterize the GMS system, the transcriptome and metabolome profiles were generated for 24 samples and 48 samples of 1205AB in B. napus, respectively. Transcriptome analysis yielded a total of 156.52 Gb of clean data and revealed the expression levels of 109,541 genes and 8,501 novel genes. In addition, a total of 1,353 metabolites were detected in the metabolomic analysis, including 784 in positive ion mode and 569 in negative ion mode. KEY RESULTS: A total of 15,635 differentially expressed genes (DEGs) and 83 differential metabolites (DMs) were identified from different comparison groups, most of which were involved in lipid and sugar metabolism. The combination of transcriptome and metabolome analysis revealed 49 orthologous GMS genes related to lipid metabolism and 46 orthologous GMS genes related to sugar metabolism, as well as 45 novel genes. UTILITY OF THE RESOURCE: The transcriptome and metabolome profiles and their analysis provide useful reference data for the future discovery of additional GMS genes and the development of more robust male sterility breeding systems for use in the production of plant hybrids.

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;1Q 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.

An adaptive spacing of root-zone hole fertilization to improve production and fertilizer utilization of rapeseed.

BACKGROUND: Root-zone hole fertilization has a positive impact on enhancing crop production and fertilization efficiency. However, a suitable spacing for hole fertilization in rapeseed cultivation is unclear. To explore an adaptive hole spacing for improving rapeseed yield and fertilization efficiency, field experiments were conducted. Four spacings of hole fertilization were designed: 10 (FD10), 20 (FD20), 30 (FD30) and 40 cm (FD40), using no fertilization (F0) and deep-banded placement of fertilizer (DBP) as controls. The burial depth was 10 cm for FD and DBP treatments. RESULTS: Compared to DBP, hole fertilization impacted soil microenvironment, crop growth and yield components, resulting in a significant increase of 28.4% in seed yield and 25.6% in oil yield. Seed yield in FD20 (4345.43 kg ha-1) increased by 4.3%, 9.4% and 15.1% compared to FD10, FD30 and FD40, respectively. Fertilizer partial factor productivity under FD20 was 4.2%, 8.6% and 13.9% greater than FD10, FD30 and FD40, respectively; whereas the increase for agronomic efficiency was 6.0%, 12.7% and 21.0%, and the increase for N recovery efficiency was 39.5%, 52.5% and 62.9%, respectively. CONCLUSION: Fertilization with a hole spacing of 17 cm is a promising practice to maintain high production and fertilization efficiency when cultivating rapeseed. These results provide a theoretical foundation and scientific basis for improving rapeseed productivity and fertilizer utilization. © 2024 Society of Chemical Industry.

Gene expression profiling reveals transcription factor networks and subgenome bias during Brassica napus seed development.

We profiled the global gene expression landscape across the reproductive lifecycle of Brassica napus. Comparative analysis of this nascent amphidiploid revealed the contribution of each subgenome to plant reproduction. Whole-genome transcription factor networks identified BZIP11 as a transcriptional regulator of early B. napus seed development. Knockdown of BZIP11 using RNA interference resulted in a similar reduction in gene activity of predicted gene targets, and a reproductive-lethal phenotype. Global mRNA profiling revealed lower accumulation of Cn subgenome transcripts relative to the An subgenome. Subgenome-specific transcription factor networks identified distinct transcription factor families enriched in each of the An and Cn subgenomes early in seed development. Analysis of laser-microdissected seed subregions further reveal subgenome expression dynamics in the embryo, endosperm and seed coat of early stage seeds. Transcription factors predicted to be regulators encoded by the An subgenome are expressed primarily in the seed coat, whereas regulators encoded by the Cn subgenome were expressed primarily in the embryo. Data suggest subgenome bias are characteristic features of the B. napus seed throughout development, and that such bias might not be universal across the embryo, endosperm and seed coat of the developing seed. Transcriptional networks spanning both the An and Cn genomes of the whole B. napus seed can identify valuable targets for seed development research and that -omics level approaches to studying gene regulation in B. napus can benefit from both broad and high-resolution analyses.

Non-specific phospholipase C4 hydrolyzes phosphosphingolipids and phosphoglycerolipids and promotes rapeseed growth and yield.

Phosphorus is a major nutrient vital for plant growth and development, with a substantial amount of cellular phosphorus being used for the biosynthesis of membrane phospholipids. Here, we report that NON-SPECIFIC PHOSPHOLIPASE C4 (NPC4) in rapeseed (Brassica napus) releases phosphate from phospholipids to promote growth and seed yield, as plants with altered NPC4 levels showed significant changes in seed production under different phosphate conditions. Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated nuclease 9 (Cas9)-mediated knockout of BnaNPC4 led to elevated accumulation of phospholipids and decreased growth, whereas overexpression (OE) of BnaNPC4 resulted in lower phospholipid contents and increased plant growth and seed production. We demonstrate that BnaNPC4 hydrolyzes phosphosphingolipids and phosphoglycerolipids in vitro, and plants with altered BnaNPC4 function displayed changes in their sphingolipid and glycerolipid contents in roots, with a greater change in glycerolipids than sphingolipids in leaves, particularly under phosphate deficiency conditions. In addition, BnaNPC4-OE plants led to the upregulation of genes involved in lipid metabolism, phosphate release, and phosphate transport and an increase in free inorganic phosphate in leaves. These results indicate that BnaNPC4 hydrolyzes phosphosphingolipids and phosphoglycerolipids in rapeseed to enhance phosphate release from membrane phospholipids and promote growth and seed production.

Fatty acid export protein BnFAX6 functions in lipid synthesis and axillary bud growth in Brassica napus.

Sugar is considered as the primary regulator of plant apical dominance, whereby the outgrowth of axillary buds is inhibited by the shoot tip. However, there are some deficiencies in this theory. Here, we reveal that Fatty Acid Export 6 (BnFAX6) functions in FA transport, and linoleic acid or its derivatives acts as a signaling molecule in regulating apical dominance of Brassica napus. BnFAX6 is responsible for mediating FA export from plastids. Overexpression of BnFAX6 in B. napus heightened the expression of genes involved in glycolysis and lipid biosynthesis, promoting the flow of photosynthetic products to the biosynthesis of FAs (including linoleic acid and its derivatives). Enhancing expression of BnFAX6 increased oil content in seeds and leaves and resulted in semi-dwarf and increased branching phenotypes with more siliques, contributing to increased yield per plant relative to wild-type. Furthermore, decapitation led to the rapid flow of the carbon from photosynthetic products to FA biosynthesis in axillary buds, consistent with the overexpression of BnFAX6 in B. napus. In addition, free FAs, especially linoleic acid, were rapidly transported from leaves to axillary buds. Increasing linoleic acid in axillary buds repressed expression of a key transcriptional regulator responsible for maintaining bud dormancy, resulting in bud outgrowth. Taken together, we uncovered that BnFAX6 mediating FA export from plastids functions in lipid biosynthesis and in axillary bud dormancy release, possibly through enhancing linoleic acid level in axillary buds of B. napus.

Integrated dominance mechanisms regulate reproductive architecture in Arabidopsis thaliana and Brassica napus.

The production of seed in flowering plants is complicated by the need to first invest in reproductive shoots, inflorescences, flowers, and fruit. Furthermore, in many species, it will be months between plants generating flowers and setting seed. How can plants therefore produce an optimal seed-set relative to environmental resources when the "reproductive architecture" that supports seed-set needs to be elaborated so far in advance? Here, we address this question by investigating the spatio-temporal control of reproductive architecture in Arabidopsis (Arabidopsis thaliana) and Brassica napus. We show that resource and resource-related signals such as substrate volume play a key role in determining the scale of reproductive effort, and that this is reflected in the earliest events in reproductive development, which broadly predict the subsequent reproductive effort. We show that a series of negative feedbacks both within and between developmental stages prevent plants from over-committing to early stages of development. These feedbacks create a highly plastic, homeostatic system in which additional organs can be produced in the case of reproductive failure elsewhere in the system. We propose that these feedbacks represent an "integrated dominance" mechanism that allows resource use to be correctly sequenced between developmental stages to optimize seed set.

Targeted mutagenesis of EOD3 gene in Brassica napus L. regulates seed production.

Seed size and number are central to the evolutionary fitness of plants and are also crucial for seed production of crops. However, the molecular mechanisms of seed production control are poorly understood in Brassica crops. Here, we report the gene cloning, expression analysis, and functional characterization of the EOD3/CYP78A6 gene in rapeseed. BnaEOD3 has four copies located in two subgenomes, which exhibited a steady higher expression during seed development with differential expression among copies. The targeted mutations of BnaEOD3 gene were efficiently generated by stable transformation of the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat) vector. These mutations were stably transmitted to T1 and T2 generations and a large collection of homozygous mutants with combined loss-of-function alleles across four BnaEOD3 copies were created for phenotyping. All mutant T1 lines had shorter siliques, smaller seeds, and an increased number of seeds per silique, in which the quadrable mutants showed the most significant changes in these traits. Consequently, the seed weight per plant in the quadrable mutants increased by 13.9% on average compared with that of wild type, indicating that these BnaEOD3 copies have redundant functions in seed development in rapeseed. The phenotypes of the different allelic combinations of BnaEOD3 copies also revealed gene functional differentiation among the two subgenomes. Cytological observations indicated that the BnaEOD3 could act maternally to promote cotyledon cell expansion and proliferation to regulate seed growth in rapeseed. Collectively, our findings reveal the quantitative involvement of the different BnaEOD3 copies function in seed development, but also provided valuable resources for rapeseed breeding programs.

Suppression of Metacaspase- and Autophagy-Dependent Cell Death Improves Stress-Induced Microspore Embryogenesis in Brassica napus.

Microspore embryogenesis is a biotechnological process that allows us to rapidly obtain doubled-haploid plants for breeding programs. The process is initiated by the application of stress treatment, which reprograms microspores to embark on embryonic development. Typically, a part of the microspores undergoes cell death that reduces the efficiency of the process. Metacaspases (MCAs), a phylogenetically broad group of cysteine proteases, and autophagy, the major catabolic process in eukaryotes, are critical regulators of the balance between cell death and survival in various organisms. In this study, we analyzed the role of MCAs and autophagy in cell death during stress-induced microspore embryogenesis in Brassica napus. We demonstrate that this cell death is accompanied by the transcriptional upregulation of three BnMCA genes (BnMCA-Ia, BnMCA-IIa and BnMCA-IIi), an increase in MCA proteolytic activity and the activation of autophagy. Accordingly, inhibition of autophagy and MCA activity, either individually or in combination, suppressed cell death and increased the number of proembryos, indicating that both components play a pro-cell death role and account for decreased efficiency of early embryonic development. Therefore, MCAs and/or autophagy can be used as new biotechnological targets to improve in vitro embryogenesis in Brassica species and doubled-haploid plant production in crop breeding and propagation programs.

Characterization and fine mapping of a new dwarf mutant in Brassica napus.

BACKGROUND: Plant height is an important plant characteristic closely related to yield performance of many crops. Reasonable reduction of plant height of crops is beneficial for improving yield and enhancing lodging resistance. RESULTS: In the present study, we described the Brassica napus dwarf mutant bnd2 that was isolated using ethyl methanesulfonate (EMS) mutagenesis. Compared to wild type (WT), bnd2 exhibited reduced height and shorter hypocotyl and petiole leaves. By crossing the bnd2 mutant with the WT strain, we found that the ratio of the mutant to the WT in the F2 population was close to 1:3, indicating that bnd2 is a recessive mutation of a single locus. Following bulked segregant analysis (BSA) by resequencing, BND2 was found to be located in the 13.77-18.08 Mb interval of chromosome A08, with a length of 4.31 Mb. After fine mapping with single nucleotide polymorphism (SNP) and insertion/deletion (InDel) markers, the gene was narrowed to a 140-Kb interval ranging from 15.62 Mb to 15.76 Mb. According to reference genome annotation, there were 27 genes in the interval, of which BnaA08g20960D had an SNP type variation in the intron between the mutant and its parent, which may be the candidate gene corresponding to BND2. The hybrid line derived from a cross between the mutant bnd2 and the commercial cultivar L329 had similar plant height but higher grain yield compared to the commercial cultivar, suggesting that the allele bnd2 is beneficial for hybrid breeding of lodging resistant and high yield rapeseed. CONCLUSION: In this study, we identified a novel dwarf mutant of rapeseed with a new locus, which may be useful for functional analyses of genetic mechanisms of plant architecture and grain yield in rapeseed.

Identification of miRNAs and their target genes in genic male sterility lines in Brassica napus by small RNA sequencing.

BACKGROUND: Brassica napus is the third leading source of edible oil in the world. Genic male sterility (GMS) lines provide crucial material for harnessing heterosis for rapeseed. GMS lines have been used successfully for rapeseed hybrid production in China. MicroRNAs (miRNAs) play crucial regulatory roles in various plant growth, development, and stress response processes. However, reports on miRNAs that regulate the pollen development of GMS lines in B. napus are few. RESULTS: In this study, 12 small RNA and transcriptome libraries were constructed and sequenced for the flower buds from the fertile and sterile lines of two recessive GMS (RGMS) lines, namely, "6251AB" and "6284AB". At the same time, 12 small RNA and transcriptome libraries were also constructed and sequenced for the flower buds from the fertile and sterile lines of two dominant GMS (DGMS) lines, namely, "4001AB" and "4006AB". Based on the results, 46 known miRNAs, 27 novel miRNAs on the other arm of known pre-miRNAs, and 44 new conserved miRNAs were identified. Thirty-five pairs of novel miRNA-3p/miRNA-5p were found. Among all the identified miRNAs, fifteen differentially expressed miRNAs with over 1.5-fold change between flower buds of sterile and fertile lines were identified, including six differentially expressed miRNAs between "4001A" and "4001B", two differentially expressed miRNAs between "4006A" and "4006B", four differentially expressed miRNAs between "6251A" and "6251B", and ten differentially expressed miRNAs between "6284A" and "6284B". The correlation analysis of small RNA and transcriptome sequencing was conducted. And 257 candidate target genes were predicted for the 15 differentially expressed miRNAs. The results of 5' modified RACE indicated that BnaA09g48720D, BnaA09g11120D, and BnaCnng51960D were cleaved by bna-miR398a-3p, bna-miR158-3p and bna-miR159a, respectively. Among the differentially expressed miRNAs, miR159 was chosen to analyze its function. Overexpression of bna-miR159 in Arabidopsis resulted in decreased seed setting rate, and shortened siliques, illustrating that miR159 may regulate the fertility and silique development in rapeseed. CONCLUSIONS: Our findings provide an overview of miRNAs that are potentially involved in GMS and pollen development. New information on miRNAs and their related target genes are provided to exploit the GMS mechanism and reveal the miRNA networks in B. napus.