Natural variation in BnaA9.NF-YA7 contributes to drought tolerance in Brassica napus L.

Rapeseed (Brassica napus) is one of the important oil crops worldwide. Its production is often threatened by drought stress. Here, we identify a transcription factor (BnaA9.NF-YA7) that negatively regulates drought tolerance through genome-wide association study in B. napus. The presence of two SNPs within a CCAAT cis element leads to downregulation of BnaA9.NF-YA7 expression. In addition, the M63I (G-to-C) substitution in the transactivation domain can activate low level expression of BnaA4.DOR, which is an inhibitory factor of ABA-induced stomatal closure. Furthermore, we determine that Bna.ABF3/4s directly regulate the expression of BnaA9.NF-YA7, and BnaA9.NF-YA7 indirectly suppresses the expression of Bna.ABF3/4s by regulation of Bna.ASHH4s. Our findings uncover that BnaA9.NF-YA7 serves as a supplementary role for ABA signal balance under drought stress conditions, and provide a potential molecular target to breed drought-tolerant B. napus cultivars.

Screening of microRNAs and target genes involved in Sclerotinia sclerotiorum (Lib.) infection in Brassica napus L.

BACKGROUND: Rapeseed (Brassica napus L.) is the third largest source of vegetable oil in the world, and Sclerotinia sclerotiorum (Lib.) is a major soil-borne fungal plant pathogen that infects more than 400 plant species, including B. napus. Sclerotinia stem rot caused an annual loss of 10 - 20% in rapeseed yield. Exploring the molecular mechanisms in response to S. sclerotiorum infection in B. napus is beneficial for breeding and cultivation of resistant varieties. To gain a better understanding of the mechanisms regarding B. napus tolerance to Sclerotinia stem rot, we employed a miRNAome sequencing approach and comprehensively investigated global miRNA expression profile among five relatively resistant lines and five susceptible lines of oilseed at 0, 24, and 48 h post-inoculation. RESULTS: In this study, a total of 40 known and 1105 novel miRNAs were differentially expressed after S. sclerotiorum infection, including miR156, miR6028, miR394, miR390, miR395, miR166, miR171, miR167, miR164, and miR172. Furthermore, 8,523 genes were predicted as targets for these differentially expressed miRNAs. These target genes were mainly associated with disease resistance (R) genes, signal transduction, transcription factors, and hormones. Constitutively expressing miR156b (OX156b) plants strengthened Arabidopsis resistance against S. sclerotiorum accompanied by smaller necrotic lesions, whereas blocking miR156 expression in Arabidopsis (MIM156) led to greater susceptibility to S. sclerotiorum disease, associated with extensive cell death of necrotic lesions. CONCLUSIONS: This study reveals the distinct difference in miRNA profiling between the relatively resistant lines and susceptible lines of B. napus in response to S. sclerotiorum. The identified differentially expressed miRNAs related to sclerotinia stem rot resistance are involved in regulating resistance to S. sclerotiorum in rapeseed by targeting genes related to R genes, signal transduction, transcription factors, and hormones. miR156 positively modulates the resistance to S. sclerotiorum infection by restricting colonization of S. sclerotiorum mycelia. This study provides a broad view of miRNA expression changes after S. sclerotiorum infection in oilseed and is the first to elucidate the function and mechanism underlying the miR156 response to S. sclerotiorum infection in oilseed rape.

Comparative genomic analyses reveal the genetic basis of the yellow-seed trait in Brassica napus.

Yellow-seed trait is a desirable breeding characteristic of rapeseed (Brassica napus) that could greatly improve seed oil yield and quality. However, the underlying mechanisms controlling this phenotype in B. napus plants are difficult to discern because of their complexity. Here, we assemble high-quality genomes of yellow-seeded (GH06) and black-seeded (ZY821). Combining in-depth fine mapping of a quantitative trait locus (QTL) for seed color with other omics data reveal BnA09MYB47a, encoding an R2R3-MYB-type transcription factor, as the causal gene of a major QTL controlling the yellow-seed trait. Functional studies show that sequence variation of BnA09MYB47a underlies the functional divergence between the yellow- and black-seeded B. napus. The black-seed allele BnA09MYB47aZY821, but not the yellow-seed allele BnA09MYB47aGH06, promotes flavonoid biosynthesis by directly activating the expression of BnTT18. Our discovery suggests a possible approach to breeding B. napus for improved commercial value and facilitates flavonoid biosynthesis studies in Brassica crops.

Meta-analysis of seed weight QTLome using a consensus and highly dense genetic map in Brassica napus L.

KEY MESSAGE: We report here the discovery of high-confidence MQTL regions and of putative candidate genes associated with seed weight in B. napus using a highly dense consensus genetic map and by comparing various large-scale multiomics datasets. Seed weight (SW) is a direct determinant of seed yield in Brassica napus and is controlled by many loci. To unravel the main genomic regions associated with this complex trait, we used 13 available genetic maps to construct a consensus and highly dense map, comprising 40,401 polymorphic markers and 9191 genetic bins, harboring a cumulative length of 3047.8 cM. Then, we performed a meta-analysis using 639 projected SW quantitative trait loci (QTLs) obtained from studies conducted since 1999, enabling the identification of 57 meta-QTLS (MQTLs). The confidence intervals of our MQTLs were 9.8 and 4.3 times lower than the average CIs of the original QTLs for the A and C subgenomes, respectively, resulting in the detection of some key genes and several putative novel candidate genes associated with SW. By comparing the genes identified in MQTL intervals with multiomics datasets and coexpression analyses of common genes, we defined a more reliable and shorter list of putative candidate genes potentially involved in the regulation of seed maturation and SW. As an example, we provide a list of promising genes with high expression levels in seeds and embryos (e.g., BnaA03g04230D, BnaC03g08840D, BnaA10g29580D and BnaA03g27410D) that can be more finely studied through functional genetics experiments or that may be useful for MQTL-assisted breeding for SW. The high-density genetic consensus map and the single nucleotide polymorphism (SNP) physical map generated from the latest B. napus cv. Darmor-bzh v10 assembly will be a valuable resource for further mapping and map-based cloning of other important traits.

Comparative transcriptome and co-expression network analysis revealed the genes associated with senescence and polygalacturonase activity involved in pod shattering of rapeseed.

BACKGROUND: The pod shattering (PS) trait negatively affects the crop yield in rapeseed especially under dry conditions. To better understand the trait and cultivate higher resistance varieties, it's necessary to identify key genes and unravel the PS mechanism thoroughly. RESULTS: In this study, we conducted a comparative transcriptome analysis between two materials significantly different in silique shatter resistance lignin deposition and polygalacturonase (PG) activity. Here, we identified 10,973 differentially expressed genes at six pod developmental stages. We found that the late pod development stages might be crucial in preparing the pods for upcoming shattering events. GO enrichment results from K-means clustering and weighed gene correlation network analysis (WGCNA) both revealed senescence-associated genes play an important role in PS. Two hub genes Bna.A05ABI5 and Bna.C03ERF/AP2-3 were selected from the MEyellow module, which possibly regulate the PS through senescence-related mechanisms. Further investigation found that senescence-associated transcription factor Bna.A05ABI5 upregulated the expression of SAG2 and ERF/AP2 to control the shattering process. In addition, the upregulation of Bna.C03ERF/AP2-3 is possibly involved in the transcription of downstream SHP1/2 and LEA proteins to trigger the shattering mechanism. We also analyzed the PS marker genes and found Bna.C07SHP1/2 and Bna.PG1/2 were significantly upregulated in susceptible accession. Furthermore, the role of auxin transport by Bna.WAG2 was also observed, which could reduce the PG activity to enhance the PS resistance through the cell wall loosening process. CONCLUSION: Based on comparative transcriptome evaluation, this study delivers insights into the regulatory mechanism primarily underlying the variation of PS in rapeseed. Taken together, these results provide a better understanding to increase the yield of rapeseed by reducing the PS through better engineered crops.

Integrated genetic mapping and transcriptome analysis reveal the BnaA03.IAA7 protein regulates plant architecture and gibberellin signaling in Brassica napus L.

KEY MESSAGE: A novel mutation in the BnaA03.IAA7 protein reduces plant height and enhances gibberellin signaling in Brassica napus L. Rapeseed (Brassica napus) is an excellent and important source for vegetable oil production, but its production is severely affected by lodging. Lodging hinders mechanization and decreases yield, and an ideal solution is semidwarf breeding. Limited by germplasm resources, semidwarf breeding developed slowly in rapeseed. In the current study, a mutant called sdA03 was isolated from EMS-mutagenized lines of Zhongshuang 11 (ZS11). The inheritance analysis showed that phenotypes of sdA03 were controlled by a single semidominant gene. Genetic mapping, RNA-seq and candidate gene analysis identified BnaA03.IAA7 as a candidate gene, and a function test confirmed that the mutated BnaA03.iaa7 regulates plant architecture in a dose-dependent manner. Yeast two-hybrid and transient expression experiments illustrated the P87L substitution in the GWPPV/I degron motif of BnaA03.iaa7 impaired the interaction between BnaA03.IAA7 and TIR1 proteins, and BnaA03.iaa7 prevented ARF from activating the auxin signaling pathway.The gibberellin (GA) content was higher in sdA03 hypocotyls than in those of ZS11. Further expression analysis showed more active gibberellin signaling in hypocotyl and richer expression of GA synthetic genes in root and cotyledon of sdA03 seedlings. Finally, a marker was developed based on the SNP found in BnaA03.iaa7 and used in molecular breeding. The study enriched our understanding of the architectural regulation of rapeseed and provided germplasm resources for breeding.

Genetic mapping and physiological analysis of chlorophyll-deficient mutant in Brassica napus L.

BACKGROUND: Leaf color mutants have reduced photosynthetic efficiency, which has severely negative impacts on crop growth and economic product yield. There are different chlorophyll mutants in Arabidopsis and crops that can be used for genetic control and molecular mechanism studies of chlorophyll biosynthesis, chloroplast development and photoefficiency. Chlorophyll mutants in Brassica napus are mostly used for mapping and location research but are rarely used for physiological research. The chlorophyll-deficient mutant in this experiment were both genetically mapped and physiologically analyzed. RESULTS: In this study, yellow leaf mutant of Brassica napus L. mutated by ethyl methyl sulfone (EMS) had significantly lower chlorophyll a, b and carotenoid contents than the wild type, and the net photosynthetic efficiency, stomatal conductance and transpiration rate were all significantly reduced. The mutant had sparse chloroplast distribution and weak autofluorescence. The granule stacks were reduced, and the shape was extremely irregular, with more broken stromal lamella. Transcriptome data analysis enriched the differentially expressed genes mainly in phenylpropane and sugar metabolism. The mutant was mapped to a 2.72 Mb region on A01 by using BSA-Seq, and the region was validated by SSR markers. CONCLUSIONS: The mutant chlorophyll content and photosynthetic efficiency were significantly reduced compared with those of the wild type. Abnormal chloroplasts and thylakoids less connected to the stroma lamella appeared in the mutant. This work on the mutant will facilitate the process of cloning the BnaA01.cd gene and provide more genetic and physiological information concerning chloroplast development in Brassica napus.

Knockout of the lignin pathway gene BnF5H decreases the S/G lignin compositional ratio and improves Sclerotinia sclerotiorum resistance in Brassica napus.

Ferulate-5-hydroxylase is a key enzyme involved in the conversion of the guaiacyl monolignol to the syringyl monolignol in angiosperms. The monolignol ratio has been proposed to affect biomass recalcitrance and the resistance to plant disease. Stem rot caused by the fungus Sclerotinia sclerotiorum in Brassica napus causes severe losses in its production. To date, there is no information about the effect of the lignin monomer ratio on the resistance to S. sclerotiorum in B. napus. Four dominantly expressed ferulate-5-hydroxylase genes were concertedly knocked out by CRISPR/Cas9 in B. napus, and three mutant lines were generated. The S/G lignin compositional ratio was decreased compared to that of the wild type based on the results of Mӓule staining and 2D-NMR profiling in KO-7. The resistance to S. sclerotiorum in stems and leaves increased for the three f5h mutant lines compared with WT. Furthermore, we found that the stem strength of f5h mutant lines was significantly increased. Overall, we demonstrate for the first time that decreasing the S/G ratio by knocking out of the F5H gene improves S. sclerotiorum resistance in B. napus and increases stem strength.

Genome-wide association study and transcriptome analysis dissect the genetic control of silique length in Brassica napus L.

BACKGROUND: Rapeseed is the third-largest oilseed crop after soybeans and palm that produces vegetable oil for human consumption and biofuel for industrial production. Silique length (SL) is an important trait that is strongly related to seed yield in rapeseed. Although many studies related to SL have been reported in rapeseed, only a few candidate genes have been found and cloned, and the genetic mechanisms regulating SL in rapeseed remain unclear. Here, we dissected the genetic basis of SL by genome-wide association studies (GWAS) combined with transcriptome analysis. RESULTS: We identified quantitative trait locus (QTL) for SL using a recombinant inbred line (RIL) population and two independent GWAS populations. Major QTLs on chromosomes A07, A09, and C08 were stably detected in all environments from all populations. Several candidate genes related to starch and sucrose metabolism, plant hormone signal transmission and phenylpropanoid biosynthesis were detected in the main QTL intervals, such as BnaA9.CP12-2, BnaA9.NST2, BnaA7.MYB63, and BnaA7.ARF17. In addition, the results of RNA-seq and weighted gene co-expression network analysis (WGCNA) showed that starch and sucrose metabolism, photosynthesis, and secondary cell wall biosynthesis play an important role in the development of siliques. CONCLUSIONS: We propose that photosynthesis, sucrose and starch metabolism, plant hormones, and lignin content play important roles in the development of rapeseed siliques.

Genetic mapping high protein content QTL from soybean 'Nanxiadou 25' and candidate gene analysis.

BACKGROUND: Soybean is a globally important legume crop that provides a primary source of high-quality vegetable protein and oil. Seed protein content (SPC) is a valuable quality trait controlled by multiple genes in soybean. RESULTS: In this study, we performed quantitative trait loci (QTL) mapping, QTL-seq, and RNA sequencing (RNA-seq) to reveal the genes controlling protein content in the soybean by using the high protein content variety Nanxiadou 25. A total of 50 QTL for SPC distributed on 14 chromosomes except chromosomes 4, 12, 14, 17, 18, and 19 were identified by QTL mapping using 178 recombinant inbred lines (RILs). Among these QTL, the major QTL qSPC_20-1 and qSPC_20-2 on chromosome 20 were repeatedly detected across six tested environments, corresponding to the location of the major QTL detected using whole-genome sequencing-based QTL-seq. 329 candidate DEGs were obtained within the QTL region of qSPC_20-1 and qSPC_20-2 via gene expression profile analysis. Nine of which were associated with SPC, potentially representing candidate genes. Clone sequencing results showed that different single nucleotide polymorphisms (SNPs) and indels between high and low protein genotypes in Glyma.20G088000 and Glyma.16G066600 may be the cause of changes in this trait. CONCLUSIONS: These results provide the basis for research on candidate genes and marker-assisted selection (MAS) in soybean breeding for seed protein content.

Metabolite Profiling and Transcriptome Analysis Provide Insight into Seed Coat Color in Brassica juncea.

The allotetraploid species Brassica juncea (mustard) is grown worldwide as oilseed and vegetable crops; the yellow seed-color trait is particularly important for oilseed crops. Here, to examine the factors affecting seed coat color, we performed a metabolic and transcriptomic analysis of yellow- and dark-seeded B. juncea seeds. In this study, we identified 236 compounds, including 31 phenolic acids, 47 flavonoids, 17 glucosinolates, 38 lipids, 69 other hydroxycinnamic acid compounds, and 34 novel unknown compounds. Of these, 36 compounds (especially epicatechin and its derivatives) accumulated significantly different levels during the development of yellow- and dark-seeded B. juncea. In addition, the transcript levels of BjuDFR, BjuANS,BjuBAN, BjuTT8, and BjuTT19 were closely associated with changes to epicatechin and its derivatives during seed development, implicating this pathway in the seed coat color determinant in B. juncea. Furthermore, we found numerous variations of sequences in the TT8A genes that may be associated with the stability of seed coat color in B. rapa, B. napus, and B. juncea, which might have undergone functional differentiation during polyploidization in the Brassica species. The results provide valuable information for understanding the accumulation of metabolites in the seed coat color of B. juncea and lay a foundation for exploring the underlying mechanism.

Comparative transcriptomic analysis of seed coats with high and low lignin contents reveals lignin and flavonoid biosynthesis in Brassica napus.

BACKGROUND: Brassica napus L. (2n = 38, AACC) is one of the most important oil crops and sources of protein for animal feed worldwide. Lignin is a large molecule aromatic polymer and a major cell wall component. However, lignin in the seed coat reduces the availability and restricts the development of rapeseed cake. Therefore, it is critical to reduce the lignin content of the seed coat. Here, high-lignin (H-lignin) and low-lignin (L-lignin) content recombinant inbred lines (RILs) were selected from an RIL population for analysis. RESULTS: The cross-section results indicated that the seed coat of the H-lignin lines was thicker than that of the L-lignin lines, especially the palisade layer. The seed coats and embryos at 35, 40 and 46 days after flowering (DAF) were subjected to RNA sequencing (RNA-Seq), and the expression of the BnPAL and BnC4H gene families in the lignin pathway was significantly higher in the H-lignin seed coat than in the L-lignin seed coat. The Bn4CL gene family also showed this trend. In addition, among the genes related to plant hormone synthesis, BnaC02g01710D was upregulated and BnaA07g11700D and BnaC09g00190D were downregulated in H-lignin lines. Some transcription factors were upregulated, such as BnNAC080, BnNAC083, BnMYB9, BnMYB9-1, BnMYB60 and BnMYB60-1, while BnMYB91 was downregulated in H-lignin lines. Moreover, most genes of the flavonoid pathway, such as BnCHS and BnDFR, were strongly expressed in H-lignin seed coat. CONCLUSIONS: In Our study, some key genes such as hormone synthesis genes, transcription factors and miRNAs related to lignin and flavonoid biosynthesis were identified. A regulatory model of B. napus seed coat lignin was proposed. These results provide new insight into lignin and flavonoid biosynthesis in B. napus.

Comprehensive analysis of polygalacturonase genes offers new insights into their origin and functional evolution in land plants.

Polygalacturonase (PG) is a hydrolase that participates in pectin degradation, pod shattering and fruit softening. Here, we identified 2786 PG genes across 54 plants, which could be divided into three groups. Evolutionary analysis suggested that PG family originated from the charophyte green algae, and Subgroups A2-A4 evolved from the Subgroup A1 after the tracheophyte-angiosperm split. Whole-genome duplication was the major force leading to PG gene expansion. Interestingly, the PG genes continuously expanded in eudicots, whereas it contracted in monocots after the eudicot-monocot split. PG genes in Group A are expressed at high levels in floral organs, whereas genes in Groups B and C are expressed at high levels in various tissues. Moreover, three BnaPG15 members were found for their potential possibility in pod shattering in Brassica napus. Our results provide new insight into the evolutionary history of PG family, and their potentially functional role in plants.

Integrating GWAS, linkage mapping and gene expression analyses reveals the genetic control of growth period traits in rapeseed (Brassica napus L.).

BACKGROUND: Brassica napus is one of the most important oilseed crops, and also an important biofuel plant due to its low air pollution and renewability. Growth period are important traits that affect yield and are crucial for its adaptation to different environments in B. napus. RESULTS: To elucidate the genetic basis of growth period traits, genome-wide association analysis (GWAS) and linkage mapping were employed to detect the quantitative trait loci (QTL) for days to initial flowering (DIF), days to final flowering (DFF), flowering period (FP), maturity time (MT), and whole growth period (GP). A total of 146 SNPs were identified by association mapping, and 83 QTLs were identified by linkage mapping using the RIL population. Among these QTLs, 19 were pleiotropic SNPs related to multiple traits, and six (q18DFF.A03-2, q18MT.A03-2, q17DFF.A05-1, q18FP.C04, q17DIF.C05 and q17GP.C09) were consistently detected using both mapping methods. Additionally, we performed RNA sequencing to analyze the differential expression of gene (DEG) transcripts between early- and late-flowering lines selected from the RIL population, and the DEGs were integrated with association mapping and linkage analysis to confirm their roles in the growth period. Consequently, 12 candidate genes associated with growth period traits were identified in B. napus. Among these genes, seven have polymorphic sites in the coding sequence and the upstream 2-kb sequence based on the resequencing data. The haplotype BnaSOC1.A05-Haplb and BnaLNK2.C06-Hapla showed more favorable phenotypic traits. CONCLUSIONS: The candidate genes identified in this study will contribute to our genetic understanding of growth period traits and can be used as targets for target mutations or marker-assisted breeding for rapeseed adapted to different environments.

Characterization of cold stress responses in different rapeseed ecotypes based on metabolomics and transcriptomics analyses.

The winter oilseed ecotype is more tolerant to low temperature than the spring ecotype. Transcriptome and metabolome analyses of leaf samples of five spring Brassica napus L. (B. napus) ecotype lines and five winter B. napus ecotype lines treated at 4 °C and 28 °C were performed. A total of 25,460 differentially expressed genes (DEGs) of the spring oilseed ecotype and 28,512 DEGs of the winter oilseed ecotype were identified after cold stress; there were 41 differentially expressed metabolites (DEMs) in the spring and 47 in the winter oilseed ecotypes. Moreover, more than 46.2% DEGs were commonly detected in both ecotypes, and the extent of the changes were much more pronounced in the winter than spring ecotype. By contrast, only six DEMs were detected in both the spring and winter oilseed ecotypes. Eighty-one DEMs mainly belonged to primary metabolites, including amino acids, organic acids and sugars. The large number of specific genes and metabolites emphasizes the complex regulatory mechanisms involved in the cold stress response in oilseed rape. Furthermore, these data suggest that lipid, ABA, secondary metabolism, signal transduction and transcription factors may play distinct roles in the spring and winter ecotypes in response to cold stress. Differences in gene expression and metabolite levels after cold stress treatment may have contributed to the cold tolerance of the different oilseed ecotypes.

Genetic Mapping Combined with a Transcriptome Analysis to Screen for Candidate Genes Responsive to Abscisic Acid Treatment in Brassica napus Embryos During Seed Germination.

Brassica napus embryos contain precursor tissues for the leaves, stem, and root, as well as the cotyledons, and these precursor tissues play key roles in seed germination, seedling survival, and subsequent seedling growth. Abscisic acid (ABA) plays a prominent role in the inhibition of seed germination. The underlying molecular mechanisms of the embryo responses to ABA stress followed by inhibited seed germination have not been reported in B. napus to date. In this study, we conducted quantitative trait locus (QTL) analysis of B. napus seed in response to ABA stress using 170 recombinant inbred lines. Furthermore, we performed transcriptome sequencing (RNA-seq) analyses by using B. napus ZS11 embryos under sterile deionized water (control) and 10 mg/L (10A), 20 mg/L (20A), and 30 mg/L (30A) ABA treatment conditions. In total, 10 QTLs were screened for explaining 2.70-6.73% of the phenotypic variation under ABA stress. In addition, 1495, 3332, and 3868 differentially expressed genes (DEGs) were identified in the "control vs 10A," "control vs 20A," and "control vs 30A" comparisons, respectively. Gene Ontology (GO) enrichment analysis indicated that DEG functions are mainly related to response to stimuli, response to oxygen-containing compounds, response to lipids, and the transport and seed dormancy processes. These DEGs mainly participated in the response to plant hormone signal transduction, starch and sucrose metabolism, cutin, suberine, and wax biosynthesis, and phenylpropanoid biosynthesis processes pathways. Our results provide a foundation for further explorations of the molecular regulatory mechanisms of B. napus embryos in response to abiotic stress during the seed germination stage.

Comparative Analysis of the Metabolic Profiles of Yellow- versus Black-Seeded Rapeseed Using UPLC-HESI-MS/MS and Transcriptome Analysis.

The high levels of secondary metabolites in rapeseed play important roles in determining the oil quality and feeding value. Here, we characterized the metabolic profiles in seeds of various yellow- and black-seeded rapeseed accessions. Two hundred and forty-eight features were characterized, including 31 phenolic acids, 54 flavonoids, 24 glucosinolates, 65 lipid compounds, and 74 other polar compounds. The most abundant phenolic acids and various flavonoids (epicatechin, isorhamnetin, kaempferol, quercetin, and their derivatives) were widely detected and showed significant differences in distribution between the yellow- and black-seeded rapeseed. Furthermore, the related genes (e.g., BnTT3, BnTT18, BnTT10, BnTT12, and BnBAN) involved in the proanthocyanidin pathway had lower expression levels in yellow-seeded rapeseed, strongly suggesting that the seed coat color could be mainly determined by the levels of epicatechin and their derivatives. These results improve our understanding of the primary constituents of rapeseed and lay the foundation for breeding novel varieties with a high nutritional value.

Genome-wide exploration and characterization of miR172/euAP2 genes in Brassica napus L. for likely role in flower organ development.

BACKGROUND: APETALA2-like genes encode plant-specific transcription factors, some of which possess one microRNA172 (miR172) binding site. The miR172 and its target euAP2 genes are involved in the process of phase transformation and flower organ development in many plants. However, the roles of miR172 and its target AP2 genes remain largely unknown in Brassica napus (B. napus). RESULTS: In this study, 19 euAP2 and four miR172 genes were identified in the B. napus genome. A sequence analysis suggested that 17 euAP2 genes were targeted by Bna-miR172 in the 3' coding region. EuAP2s were classified into five major groups in B.napus. This classification was consistent with the exon-intron structure and motif organization. An analysis of the nonsynonymous and synonymous substitution rates revealed that the euAP2 genes had gone through purifying selection. Whole genome duplication (WGD) or segmental duplication events played a major role in the expansion of the euAP2 gene family. A cis-regulatory element (CRE) analysis suggested that the euAP2s were involved in the response to light, hormones, stress, and developmental processes including circadian control, endosperm and meristem expression. Expression analysis of the miR172-targeted euAP2s in nine different tissues showed diverse spatiotemporal expression patterns. Most euAP2 genes were highly expressed in the floral organs, suggesting their specific functions in flower development. BnaAP2-1, BnaAP2-5 and BnaTOE1-2 had higher expression levels in late-flowering material than early-flowering material based on RNA-seq and qRT-PCR, indicating that they may act as floral suppressors. CONCLUSIONS: Overall, analyses of the evolution, structure, tissue specificity and expression of the euAP2 genes were peformed in B.napus. Based on the RNA-seq and experimental data, euAP2 may be involved in flower development. Three euAP2 genes (BnaAP2-1, BnaAP2-5 and BnaTOE1-2) might be regarded as floral suppressors. The results of this study provide insights for further functional characterization of the miR172 /euAP2 module in B.napus.

Genome-Wide Identification of the LAC Gene Family and Its Expression Analysis Under Stress in Brassica napus.

Lignin is an important biological polymer in plants that is necessary for plant secondary cell wall ontogenesis. The laccase (LAC) gene family catalyzes lignification and has been suggested to play a vital role in the plant kingdom. In this study, we identified 45 LAC genes from the Brassica napus genome (BnLACs), 25 LAC genes from the Brassica rapa genome (BrLACs) and 8 LAC genes from the Brassica oleracea genome (BoLACs). These LAC genes could be divided into five groups in a cladogram and members in same group had similar structures and conserved motifs. All BnLACs contained hormone- and stress- related elements determined by cis-element analysis. The expression of BnLACs was relatively higher in the root, seed coat and stem than in other tissues. Furthermore, BnLAC4 and its predicted downstream genes showed earlier expression in the silique pericarps of short silique lines than long silique lines. Three miRNAs (miR397a, miR397b and miR6034) target 11 BnLACs were also predicted. The expression changes of BnLACs under series of stresses were further investigated by RNA sequencing (RNA-seq) and quantitative real-time polymerase chain reaction (qRT-PCR). The study will give a deeper understanding of the LAC gene family evolution and functions in B. napus.