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Rapeseed (Brassica napus L.) is one of the most important oil crops in terms of economics, ecology, and nutrition. For the purpose of selecting the most suitable canola genotypes for quantitative and qualitative traits, an experiment was conducted in Damavand region with the presence of nine genotypes and the examination of nine traits with three replications. The results of analysis of variance showed that the effect of genotype in terms of all traits had significant differences at the level of 0.01 and 0.05. Additionally, the results of the average comparison indicated that Zargol and Hyola 401 genotypes were more favorable than other cultivars in terms of all traits. According to the three analyses related to the examination of the traits, it was concluded that the grain yield trait was positively correlated with the harvest index trait, the biological yield trait was positively correlated with the oil percentage trait, and the leaf width trait was positively correlated with the number of days to 50% flowering. Using principal component analysis (PCA), the first three components explained more than 81 percent of the variance in the data, and the first and second components had positive coefficients for Zargol and Hyola 401 genotypes. On the basis of the graphical analysis, the Zargol and Sunday genotypes were selected as the best genotypes. In comparison with the cluster analysis and heat map drawn on the data, the genotypes were grouped into two main groups based on traits. Accordingly, Zargol genotypes are considered stable genotypes in terms of their traits and are suitable for cultivation and agricultural research.

Among several mechanisms involved in the plant stress response, synthesis of guanosine tetra and pentaphosphates (alarmones), homologous to the bacterial stringent response, is of crucial importance. Plant alarmones affect, among others, photosynthetic activity, metabolite accumulation, and nutrient remobilization, and thus regulate plant growth and development. The plant RSH ( RelA / SpoT homolog) genes, that encode synthetases and/or hydrolases of alarmones, have been characterized in a limited number of plant species, e.g., Arabidopsis thaliana , Oryza sativa, and Ipomoea nil . Here, we used dry-to-wet laboratory research approaches to characterize RSH family genes in the polyploid plant Brassica napus . There are 12 RSH genes in the genome of rapeseed that belong to four types of RSH genes: 6 RSH1 , 2 RSH2 , 3 RSH3 , and 1 CRSH . BnRSH genes contain 13-24 introns in RSH1 , 2-6 introns in RSH2 , 1-6 introns in RSH3 , and 2-3 introns in the CRSH genes. In the promoter regions of the RSH genes, we showed the presence of regulatory elements of the response to light, plant hormones, plant development, and abiotic and biotic stresses. The wet-lab analysis showed that expression of BnRSH genes is generally not significantly affected by salt stress, but that the presence of PGPR bacteria, mostly of Serratia sp., increased the expression of BnRSH significantly. The obtained results show that BnRSH genes are differently affected by biotic and abiotic factors, which indicates their different functions in plants.

Background: Understanding the relationship between physiological traits with yield and yield components is an essential step towards developing high-yielding and high-quality canola (Brassica napus L.) cultivars. This study aimed to explore further the relationship between some physiological features, including radiation use efficiency (RUE), and seed yield in canola., Results: Significant differences were found among cultivars regarding maximum leaf area index (LAI max ) and required days to achieve maximum LAI (DLAI max ). All cultivars obtained the minimum LAI required to intercept 90% of the incident radiation, but at different times. Some cultivars like SW102 and Shirali had the same fraction of intercepted photosynthetically active radiation (IPAR) when LAI was maximal, but SW102 had higher IPAR. This indicated that SW102 was more efficient in irradiation capacity and may have a higher photosynthesis rate when exposed to the high irradiation conditions. The average canola RUE in the current study was 3.80 and 3.63 g MJ -1 m -2 in 2014 and 2015, respectively. In general, the crop growth rate was higher in the first year than in the second year due to the fewer cloudy days and more incident radiation., Conclusion: Results indicated that duration of growth, crop growth rate, and harvest index were crucial for enhancing biomass and seed yield. Also, a relatively high correlation was found between the RUE and DLAI max . The cultivars that reached their maximum LAI later demonstrated higher RUE, and consequently had higher biological and seed yield. The results obtained could be used to develop an improved canola crop growth model and breeding programs. © 2021 Society of Chemical Industry., (© 2021 Society of Chemical Industry.)

Catalase (CAT) is an antioxidant enzyme expressed by the CAT gene family and exists in almost all aerobic organisms. Environmental stresses induce the generation of reactive oxygen species (ROS) that eventually hinder plant growth and development. The CAT enzyme translates the hydrogen peroxide (H 2 O 2 ) to water (H 2 O) and reduce the ROS levels to shelter the cells' death. So far, the CAT gene family has not been reported in rapeseed ( Brassica napus L.). Therefore, a genome-wide comprehensive analysis was conducted to classify the CAT genes in the rapeseed genome. The current study identified 14 BnCAT genes in the rapeseed genome. Based on phylogenetic and synteny analysis, the BnCATs belong to four groups (Groups I-IV). A gene structure and conserved motif analysis showed that Group I, Group II, and Group IV possess almost the same intron/exon pattern, and an equal number of motifs, while Group III contains diverse structures and contain 15 motifs. By analyzing the cis -elements in the promoters, we identified five hormone-correlated responsive elements and four stress-related responsive elements. Further, six putative bna-miRNAs were also identified, targeting three genes ( BnCAT4, BnCAT6, and BnCAT8 ). Gene ontology (GO) enrichment analysis showed that the BnCAT genes were largely related to cellular organelles, ROS response, stimulus response, stress response, and antioxidant enzymes. Almost 10 BnCAT genes showed higher expression levels in different tissues, i.e., root, leaf, stem, and silique. The expression analysis showed that BnCAT1-BnCAT3 and BnCAT11-BnCAT13 were significantly upregulated by cold, salinity, abscisic acid (ABA), and gibberellic acid (GA) treatment, but not by drought and methyl jasmonate (MeJA). Notably, most of the genes were upregulated by waterlogging stress, except BnCAT6, BnCAT9 , and BnCAT10 . Our results opened new windows for future investigations and provided insights into the CAT family genes in rapeseed.

Regulatory SNPs (rSNPs) are a special class of SNPs which have a high potential to affect the phenotype due to their impact on DNA-binding of transcription factors (TFs). Thus, the knowledge about such rSNPs and TFs could provide essential information regarding different genetic programs, such as tissue development or environmental stress responses. In this study, we use a multi-omics approach by combining genomics, transcriptomics, and proteomics data of two different Brassica napus L. cultivars, namely Zhongshuang11 (ZS11) and Zhongyou821 (ZY821), with high and low oil content, respectively, to monitor the regulatory interplay between rSNPs, TFs and their corresponding genes in the tissues flower, leaf, stem, and root. By predicting the effect of rSNPs on TF-binding and by measuring their association with the cultivars, we identified a total of 41,117 rSNPs, of which 1141 are significantly associated with oil content. We revealed several enriched members of the TF families DOF, MYB, NAC, or TCP, which are important for directing transcriptional programs regulating differential expression of genes within the tissues. In this work, we provide the first genome-wide collection of rSNPs for B. napus and their impact on the regulation of gene expression in vegetative and floral tissues, which will be highly valuable for future studies on rSNPs and gene regulation.

Background: Transcription factors GATAs are involved in plant developmental processes and respond to environmental stresses through binding DNA regulatory regions to regulate their downstream genes. However, little information on the GATA genes in Brassica napus is available. The release of the reference genome of B. napus provides a good opportunity to perform a genome-wide characterization of GATA family genes in rapeseed., Results: In this study, 96 GATA genes randomly distributing on 19 chromosomes were identified in B. napus, which were classified into four subfamilies based on phylogenetic analysis and their domain structures. The amino acids of BnGATAs were obvious divergence among four subfamilies in terms of their GATA domains, structures and motif compositions. Gene duplication and synteny between the genomes of B. napus and A. thaliana were also analyzed to provide insights into evolutionary characteristics. Moreover, BnGATAs showed different expression patterns in various tissues and under diverse abiotic stresses. Single nucleotide polymorphisms (SNPs) distributions of BnGATAs in a core collection germplasm are probably associated with functional disparity under environmental stress condition in different genotypes of B. napus., Conclusion: The present study was investigated genomic structures, evolution features, expression patterns and SNP distributions of 96 BnGATAs. The results enrich our understanding of the GATA genes in rapeseed.

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., (© 2020 German Society for Plant Sciences and The Royal Botanical Society of the Netherlands.)

Soil salinity is a main abiotic stress in agriculture worldwide. The Na + /H + antiporters (NHXs) play pivotal roles in intracellular Na + excretion and vacuolar Na + compartmentalization, which are important for plant salt stress resistance (SSR). However, few systematic analyses of NHXs has been reported in allotetraploid rapeseed so far. Here, a total of 18 full-length NHX homologs, representing seven subgroups (NHX1-NHX8 without NHX5), were identified in the rapeseed genome (A n A n C n C n ). Number variations of BnaNHXs might indicate their significantly differential roles in the regulation of rapeseed SSR. BnaNHXs were phylogenetically divided into three evolutionary clades, and the members in the same subgroups had similar physiochemical characteristics, gene/protein structures, and conserved Na + transport motifs. Darwin´s evolutionary pressure analysis suggested that BnaNHXs suffered from strong purifying selection. The cis-element analysis revealed the differential transcriptional regulation of NHXs between the model Arabidopsis and B. napus. Differential expression of BnaNHXs under salt stress, different nitrogen forms (ammonium and nitrate), and low phosphate indicated their potential involvement in the regulation of rapeseed SSR. Global landscapes of BnaNHXs will give an integrated understanding of their family evolution and molecular features, which will provide elite gene resources for the genetic improvement of plant SSR through regulating the NHX-mediated Na + transport., Competing Interests: The authors declare no conflict of interest.

Background: Strong artificial and natural selection causes the formation of highly conserved haplotypes that harbor agronomically important genes. GWAS combination with haplotype analysis has evolved as an effective method to dissect the genetic architecture of complex traits in crop species., Results: We used the 60 K Brassica Infinium SNP array to perform a genome-wide analysis of haplotype blocks associated with oleic acid (C18:1) in rapeseed. Six haplotype regions were identified as significantly associated with oleic acid (C18:1) that mapped to chromosomes A02, A07, A08, C01, C02, and C03. Additionally, whole-genome sequencing of 50 rapeseed accessions revealed three genes (BnmtACP2-A02, BnABCI13-A02 and BnECI1-A02) in the A02 chromosome haplotype region and two genes (BnFAD8-C02 and BnSDP1-C02) in the C02 chromosome haplotype region that were closely linked to oleic acid content phenotypic variation. Moreover, the co-expression network analysis uncovered candidate genes from these two different haplotype regions with potential regulatory interrelationships with oleic acid content accumulation., Conclusions: Our results suggest that several candidate genes are closely linked, which provides us with an opportunity to develop functional haplotype markers for the improvement of the oleic acid content in rapeseed.

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.

The plant specific LATERAL ORGAN BOUNDARIES (LOB)-domain (LBD) proteins belong to a family of transcription factors that play important roles in plant growth and development, as well as in responses to various stresses. However, a comprehensive study of LBDs in Brassica napus has not yet been reported. In the present study, 126 BnLBD genes were identified in B. napus genome using bioinformatics analyses. The 126 BnLBDs were phylogenetically classified into two groups and nine subgroups. Evolutionary analysis indicated that whole genome duplication (WGD) and segmental duplication played important roles in the expansion of the BnLBD gene family. On the basis of the RNA-seq analyses, we identified BnLBD genes with tissue or developmental specific expression patterns. Through cis -acting element analysis and hormone treatment, we identified 19 BnLBD genes with putative functions in plant response to abscisic acid (ABA) treatment. This study provides a comprehensive understanding on the origin and evolutionary history of LBDs in B. napus , and will be helpful in further functional characterisation of BnLBDs .

PbBa8.1 and CRb are two clubroot-resistant genes that are important for canola breeding in China. Previously, we combined these resistant genes and developed a pyramid-based, homozygous recurrent inbred line (618R), the results of which showed strong resistance to Plasmodiophora brassicae field isolates; however, the genetic mechanisms of resistance were unclear. In the present work, we conducted comparative RNA sequencing (RNA-Seq) analysis between 618R and its parental lines (305R and 409R) in order to uncover the transcriptomic response of the superior defense mechanisms of 618R and to determine how these two different resistant genes coordinate with each other. Here, we elucidated that the number and expression of differentially expressed genes (DEGs) in 618R are significantly higher than in the parental lines, and PbBa8.1 shares more DEGs and plays a dominant role in the pyramided line. The common DEGs among the lines largely exhibit non-additive expression patterns and enrichment in resistance pathways. Among the enriched pathways, plant-pathogen interaction, plant hormone signaling transduction, and secondary metabolites are the key observation. However, the expressions of the salicylic acid (SA) signaling pathway and reactive oxygen species (ROS) appear to be crucial regulatory components in defense response. Our findings provide comprehensive transcriptomic insight into understanding the interactions of resistance gene pyramids in single lines and can facilitate the breeding of improved resistance in Brassica napus .

Rapeseed (Brassica napus L.) is one of the most economically important oilseed crops worldwide. In Japan, it has been cultivated for more than a century and has formed many feral populations. The aim of this study was to elucidate the genetic diversity of feral rapeseeds by genotyping 537 individuals (among which 130 were determined to be genetically modified) sampled from various regions in Japan. Analysis of 30 microsatellite markers amplified 334 alleles and indicated moderate genetic diversity and high inbreeding (expected heterozygosity, 0.50; observed heterozygosity, 0.16; inbreeding coefficient within individuals, 0.68) within the feral populations. The Mantel test showed only an insignificant weak positive correlation between geographic distance and genetic distance. Analysis of molecular variance showed a greater genetic diversity among individuals than between populations. These results are in accordance with population structure assessed by using principal coordinate analysis and the program STRUCTURE, which showed that the 537 individuals could be assigned to 8 genetic clusters with very large genetic differences among individuals within the same geographic population, and that among feral individuals, many are closely related to rapeseed accessions in the NARO Genebank but some have unknown origins. These unique feral rapeseeds are likely to be affected by strong selection pressure. The results for genetically modified individuals also suggest that they have two different sources and have a considerable degree of diversity, which might be explained by hybridization with nearby individuals and separation of hybrid cultivars. The information obtained in this study could help improve the management of feral rapeseed plants in Japan., Competing Interests: The authors have declared that no competing interests exist.

Insect pollination of flowers should change the within-season allocation of resources in plants. But the nature of this life-history response, particularly regarding allocation to roots, photosynthetic structures, and flowers, is empirically unresolved. This study uses a greenhouse experiment to investigate the effect of insect pollination on the reproductive output of 23 varieties of a globally important crop-canola (Brassica napus). Overall, insect pollination modified the functional characteristics (flower timing & effort, plant size & shape, seed packaging, root biomass) of canola, increasing seed production and quality, and pollinator dependence. Reproductive output and pollinator dependence were defined by strong trait trade-offs, which ranged from more pollinator-dependent plants favouring early reproductive effort, to less pollinator-dependent plants favouring a prolonged phenology with smaller plant size and lower seed quality. Seed production decreased with pollinator dependence in the absence of pollinators. The agricultural preference for hybrid varieties will increase seed production compared to open-pollinated varieties, but, even so, pollinators typically enhance seed production of both types. Our study elucidates how insect pollination alters the character and function of a globally important crop, supporting optimization of yield via intensification of insect pollination, and highlights the beneficial effects of insect pollination early in the season.

The sophisticated uptake and translocation regulation of the essential element boron (B) in plants is ensured by two transmembrane transporter families: the Nodulin26-like Intrinsic Protein (NIP) and BOR transporter family. Though the agriculturally important crop Brassica napus is highly sensitive to B deficiency, and NIPs and BORs have been suggested to be responsible for B efficiency in this species, functional information of these transporter subfamilies is extremely rare. Here, we molecularly characterized the NIP and BOR1 transporter family in the European winter-type cv. Darmor-PBY018. Our transport assays in the heterologous oocyte and yeast expression systems as well as in growth complementation assays in planta demonstrated B transport activity of NIP5, NIP6, NIP7 and BOR1 isoforms. Moreover, we provided functional and quantitative evidence that also members of the NIP2, NIP3 and NIP4 groups facilitate the transport of B. A detailed B- and tissue-dependent B-transporter expression map was generated by quantitative polymerase chain reaction. We showed that NIP5 isoforms are highly upregulated under B-deficient conditions in roots, but also in shoot tissues. Moreover, we detected transcripts of several B-permeable NIPs from various groups in floral tissues that contribute to the B distribution within the highly B deficiency-sensitive flowers., (© 2019 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

Clubroot disease, caused by Plasmodiophora brassicae Woronin, is a major threat to the production of Brassica' crops. Resistance to different P. brassicae pathotypes has been reported in the A genome, chromosome A08; however, the molecular mechanism of this resistance, especially the involvement of long noncoding RNAs (lncRNAs), is not understood. We have used a strand-specific lncRNA-Seq approach to catalog lncRNAs from the roots of clubroot-susceptible and -resistant Brassica napus lines. In total, 530 differentially expressed (DE) lncRNAs were identified, including 88% of long intergenic RNAs and 11% natural antisense transcripts. Sixteen lncRNAs were identified as target mimics of the microRNAs (miRNAs) and eight were identified as the precursors of miRNAs. KEGG pathway analysis of the DE lncRNAs showed that the cis-regulated target genes mostly belong to the phenylpropanoid biosynthetic pathway (15%) and plant-pathogen interactions (15%) while the transregulated target genes mostly belong to carbon (18%) and amino acid biosynthesis pathway (19%). In all, 24 DE lncRNAs were identified from chromosome A08, which is known to harbor a quantitative trait locus conferring resistance to different P. brassicae pathotypes; however, eight of these lncRNAs showed expression only in the resistant plants. These results could form the basis for future studies aimed at delineating the roles of lncRNAs in plant-microbe interactions.

The GH3 gene family plays a vital role in the phytohormone-related growth and developmental processes. The effects of allopolyploidization on GH3 gene structures and expression levels have not been reported. In this study, a total of 38, 25, and 66 GH3 genes were identified in Brassica rapa (A r A r ), Brassica oleracea (C o C o ), and Brassica napus (A n AC n C n ), respectively. BnaGH3 genes were unevenly distributed on chromosomes with 39 on A n and 27 on C n , in which six BnaGH3 genes may appear as new genes. The whole genome triplication allowed the GH3 gene family to expand in diploid ancestors, and allopolyploidization made the GH3 gene family re-expand in B. napus . For most BnaGH3 genes, the exon-intron compositions were similar to diploid ancestors, while the cis-element distributions were obviously different from its ancestors. After allopolyploidization, the expression patterns of GH3 genes from ancestor species changed greatly in B. napus , and the orthologous gene pairs between A n /A r and C n /C o had diverged expression patterns across four tissues. Our study provides a comprehensive analysis of the GH3 gene family in B. napus , and these results could contribute to identifying genes with vital roles in phytohormone-related growth and developmental processes.

Identification of superior alleles for agronomic traits in genetic resources of oilseed rape ( Brassica napus L.) would be useful for improving the performance of locally adapted cultivars in Iran. The objective of the present work was to analyse the genetic variation and inheritance of important agronomic traits in a doubled haploid population derived from a cross between two German oilseed rape cultivars, Sansibar and Oase. Field experiments were performed in 2016-2017 with 200 doubled haploid lines and the parental genotypes applying an alpha-lattice design with two replicates. Phenological traits were recorded during the cultivation period and at maturity, seed yield, yield components and seed quality traits were determined. Significant genetic variation was found in most of the traits and heritabilities ranged from medium (48.5%) for days to end of flowering to high (92.6%) for oil content. A molecular marker linkage map was used to map 36 QTL for different traits on 17 linkage groups. Between three and four QTL were identified for each seed yield, seed weight, oil and protein content. Some of the plant material and positive QTL alleles identified for agronomic traits may be useful for improving those characters in locally adapted cultivars in Iran.

Background: Very few near-infrared reflectance spectroscopy (NIRS) calibration models are available for non-destructive estimation of seed quality traits in Brassica juncea. Those that are available also fail to adequately discern variation for oleic acid (C 18:1 ) , linolenic (C 18:3 ) fatty acids, meal glucosinolates and phenols. We report the development of a new NIRS calibration equation that is expected to fill the gaps in the existing NIRS equations., Results: Calibrations were based on the reference values of important quality traits estimated from a purposely selected germplasm set comprising 240 genotypes of B. juncea and 193 of B. napus. We were able to develop optimal NIRS-based calibration models for oil, phenols, glucosinolates, oleic acid, linoleic acid and erucic acid for B. juncea and B. napus. Correlation coefficients (RSQ) of the external validations appeared greater than 0.7 for the majority of traits, such as oil (0.766, 0.865), phenols (0.821, 0.915), glucosinolates (0.951, 0.986), oleic acid (0.814. 0.810), linoleic acid (0.974, 0.781) and erucic acid (0.963, 0.943) for B. juncea and B. napus, respectively., Conclusion: The results demonstrate the robust predictive power of the developed calibration models for rapid estimation of many quality traits in intact rapeseed-mustard seeds which will assist plant breeders in effective screening and selection of lines in quality improvement breeding programmes. © 2018 Society of Chemical Industry., (© 2018 Society of Chemical Industry.)

MADS-box transcription factors are important for plant growth and development, and hundreds of MADS-box genes have been functionally characterized in plants. However, less is known about the functions of these genes in the economically important allopolyploid oil crop, Brassica napus. We identified 307 potential MADS-box genes (BnMADSs) in the B. napus genome and categorized them into type I (Mα, Mβ, and Mγ) and type II (MADS DNA-binding domain, intervening domain, keratin-like domain, and C-terminal domain [MIKC]c and MIKC*) based on phylogeny, protein motif structure, and exon-intron organization. We identified one conserved intron pattern in the MADS-box domain and seven conserved intron patterns in the K-box domain of the MIKCc genes that were previously ignored and may be associated with function. Chromosome distribution and synteny analysis revealed that hybridization between Brassica rapa and Brassica oleracea, segmental duplication, and homologous exchange (HE) in B. napus were the main BnMADSs expansion mechanisms. Promoter cis-element analyses indicated that BnMADSs may respond to various stressors (drought, heat, hormones) and light. Expression analyses showed that homologous genes in a given subfamily or sister pair are highly conserved, indicating widespread functional conservation and redundancy. Analyses of BnMADSs provide a basis for understanding their functional roles in plant development., Competing Interests: The authors have declared that no competing interests exist.

Background: The BES1 gene family, an important class of plant-specific transcription factors, play key roles in the BR signal pathway in plants, regulating various development processes. Until now, there has been no comprehensive analysis of the BES1 gene family in Brassica napus, and a cross-genome exploration of their origin, copy number changes, and functional innovation in plants was also not available., Results: We identified 28 BES1 genes in B. napus from its two subgenomes (AA and CC). We found that 71.43% of them were duplicated in the tetraploidization, and their gene expression showed a prominent subgenome bias in the roots. Additionally, we identified 104 BES1 genes in another 18 representative angiosperms and performed a comparative analysis with B. napus, including evolutionary trajectory, gene duplication, positive selection, and expression pattern. Exploiting the available genome datasets, we performed a large-scale analysis across plants and algae suggested that the BES1 gene family could have originated from group F, expanding to form other groups (A to E) by duplicating or alternatively deleting some domains. We detected an additional domain containing M4 to M8 in exclusively groups F1 and F2. We found evidence that whole-genome duplication (WGD) contributed the most to the expansion of this gene family among examined dicots, while dispersed duplication contributed the most to expansion in certain monocots. Moreover, we inferred that positive selection might have occurred on major phylogenetic nodes during the evolution of plants., Conclusions: Grossly, a cross-genome comparative analysis of the BES1 genes in B. napus and other species sheds light on understanding its copy number expansion, natural selection, and functional innovation.

In higher plants, heavy metal transporters are responsible for metal uptake, translocation and homeostasis. These metals include essential metals such as zinc (Zn) or manganese (Mn) and non-essential metals like cadmium (Cd) or lead (Pb). Although a few heavy metal transporters have been well identified in model plants (e.g. Arabidopsis and rice), little is known about their functionality in rapeseed (Brassica napus). B. napus is an important oil crop ranking the third largest sources of vegetable oil over the world. Importantly, B. napus has long been considered as a desirable candidate for phytoremediation owning to its massive dry weight productivity and moderate to high Cd accumulation. In this study, 270 metal transporter genes (MTGs) from B. napus genome were identified and annotated using bioinformatics and high-throughput sequencing. Most of the MTGs (74.8%, 202/270) were validated by RNA-sequencing (RNA-seq) the seedling libraries. Based on the sequence identity, nine superfamilies including YSL, OPT, NRAMP, COPT, ZIP, CDF/MTP, HMA, MRP and PDR have been classified. RNA-sequencing profiled 202 non-redundant MTGs from B. napus seedlings, of which, 108 MTGs were differentially expressed and 62 genes were significantly induced under Cd stress. These differentially expressed genes (DEGs) are dispersed in the rapeseed genome. Some of the genes were well confirmed by qRT-PCR. Analysis of the genomic distribution of MTGs on B. napus chromosomes revealed that their evolutional expansion was probably through localized allele duplications.

Selection breeding has played a key role in the improvement of seed yield and quality in oilseed rape (Brassica napus L.). We genotyped Tapidor (European), Ningyou7 (Chinese) and their progenitors with the Brassica 60 K Illumina Infinium SNP array and mapped a total of 29,347 SNP markers onto the reference genome of Darmor-bzh. Identity by descent (IBD) refers to a haplotype segment of a chromosome inherited from a shared common ancestor. IBDs identified on the C subgenome were larger than those on the A subgenome within both the Tapidor and Ningyou7 pedigrees. IBD number and length were greater in the Ningyou7 pedigree than in the Tapidor pedigree. Seventy nine QTLs for flowering time, seed quality and root morphology traits were identified in the IBDs of Tapidor and Ningyou7. Many more candidate genes had been selected within the Ningyou7 pedigree than within the Tapidor pedigree. These results highlight differences in the transfer of favorable gene clusters controlling key traits during selection breeding in Europe and China.

Diacylglycerol acyltransferase (DGAT) catalyzes the acyl-CoA-dependent acylation of sn-1, 2-diacylglycerol to produce triacylglycerol, which is the main component of the seed oil of Brassica oilseed species. Phylogenetic analysis of the amino acid sequences encoded by four transcriptionally active DGAT1 genes from Brassica napus suggests that the gene forms diverged over time into two clades (I and II), with representative members in each genome (A and C). The majority of the amino acid sequence differences in these forms of DGAT1, however, reside outside of motifs suggested to be involved in catalysis. Despite this, the clade II enzymes displayed a significantly enhanced preference for linoleoyl-CoA when assessed using in-vitro enzyme assays with yeast microsomes containing recombinant enzyme forms. These findings contribute to our understanding of triacylglycerol biosynthesis in B. napus, and may advance our ability to engineer DGAT1s with desired substrate selectivity properties.

Enhancing nitrogen use efficiency (NUE) in crop plants is an important breeding target to reduce excessive use of chemical fertilizers, with substantial benefits to farmers and the environment. In Arabidopsis (Arabidopsis thaliana), allocation of more NO3 (-) to shoots was associated with higher NUE; however, the commonality of this process across plant species have not been sufficiently studied. Two Brassica napus genotypes were identified with high and low NUE. We found that activities of V-ATPase and V-PPase, the two tonoplast proton-pumps, were significantly lower in roots of the high-NUE genotype (Xiangyou15) than in the low-NUE genotype (814); and consequently, less vacuolar NO3 (-) was retained in roots of Xiangyou15. Moreover, NO3 (-) concentration in xylem sap, [(15)N] shoot:root (S:R) and [NO3 (-)] S:R ratios were significantly higher in Xiangyou15. BnNRT1.5 expression was higher in roots of Xiangyou15 compared with 814, while BnNRT1.8 expression was lower. In both B. napus treated with proton pump inhibitors or Arabidopsis mutants impaired in proton pump activity, vacuolar sequestration capacity (VSC) of NO3 (-) in roots substantially decreased. Expression of NRT1.5 was up-regulated, but NRT1.8 was down-regulated, driving greater NO3 (-) long-distance transport from roots to shoots. NUE in Arabidopsis mutants impaired in proton pumps was also significantly higher than in the wild type col-0. Taken together, these data suggest that decrease in VSC of NO3 (-) in roots will enhance transport to shoot and essentially contribute to higher NUE by promoting NO3 (-) allocation to aerial parts, likely through coordinated regulation of NRT1.5 and NRT1.8., (© 2016 American Society of Plant Biologists. All Rights Reserved.)

High-density genetic markers are the prerequisite for understanding linkage disequilibrium (LD) and genome-wide association studies (GWASs) of complex traits in crops. To evaluate the LD pattern in oilseed rape, we sequenced a previous association panel containing 189 B. napus inbred lines using double-digested restriction-site associated DNA (ddRAD) and genotyped 19,327 RAD tags. A total of 15,921 RAD tags were assigned to a published genetic linkage map and the majority (71.1%) of these tags was uniquely mapped to the draft reference genome "Darmor-bzh." The distance of LD decay was 1,214 kb across the genome at the background level (r2 = 0.26), with the distances of LD decay being 405 kb and 2,111 kb in the A and C subgenomes, respectively. A total of 361 haplotype blocks with length > 100 kb were identified in the entire genome. The association panel could be classified into two groups, P1 and P2, which are essentially consistent with the geographical origins of varieties. A large number of group-specific haplotypes were identified, reflecting that varieties in the P1 and P2 groups experienced distinct selection in breeding programs to adapt their different growth habitats. GWAS repeatedly detected two loci significantly associated with oil content of seeds based on the developed SNPs, suggesting that the high-density SNPs were useful for understanding the genetic determinants of complex traits in GWAS.

Crop plant architecture plays a highly important role in its agronomic performance. Plant height (PH) and primary branch number (PB) are two major factors that affect the plant architecture of rapeseed (Brassica napus). Previous studies have shown that these two traits are controlled by multiple quantitative trait loci (QTL); however, QTLs have not been delimited to regions less than 10cM. Genome-wide association study (GWAS) is a highly efficient approach for identifying genetic loci controlling traits at relatively high resolution. In this study, variations in PH and PB of a panel of 472 rapeseed accessions that had previously been analyzed by a 60k SNP array were investigated for three consecutive years and studied by GWAS. Eight QTLs on chromosome A03, A05, A07 and C07 were identified for PH, and five QTLs on A01, A03, A07 and C07 were identified for PB. Although most QTLs have been detected in previous studies based on linkage analyses, the two QTLs of PH on A05 and the QTL of PB on C07 were novel. In the genomic regions close to the GWAS peaks, orthologs of the genes involved in flower development, phytohormone biosynthesis, metabolism and signaling in Arabidopsis were identified., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Rapeseed is one of the most important edible oil crops in the world and the seed yield has lagged behind the increasing demand driven by population growth. Winter oilseed rape (Brassica napus L.) is widely cultivated with relatively low yield in China, so it is necessary to find the strategies to improve the expression of yield potential. Planting density has great effects on seed yield of crops. Hence, field experiments were conducted in Wuhan in the Yangtze River basin with one conventional variety (Zhongshuang 11, ZS11) and one hybrid variety (Huayouza 9, HYZ9) at five planting densities (27.0×10(4), 37.5×10(4), 48.0×10(4), 58.5×10(4), 69.0×10(4) plants ha(-1)) during 2010-2012 to investigate the yield components. The physiological traits for high-yield and normal-yield populations were measured during 2011-2013. Our results indicated that planting densities of 58.5×10(4) plants ha(-1) in ZS11 and 48.0×10(4) plants ha(-1) in HYZ9 have significantly higher yield compared with the density of 27.0×10(4) plants ha(-1) for both varieties. The ideal silique numbers for ZS11 and HYZ9 were ∼0.9×10(4) (n m(-2)) and ∼1×10(4) (n m(-2)), respectively, and ideal primary branches for ZS11 and HYZ9 were ∼250 (n m(-2)) and ∼300 (n m(-2)), respectively. The highest leaf area index (LAI) and silique wall area index (SAI) was ∼5.0 and 7.0, respectively. Moreover, higher leaf net photosynthetic rate (Pn) and water use efficiency (WUE) were observed in the high-yield populations. A significantly higher level of silique wall photosynthesis and rapid dry matter accumulation were supposed to result in the maximum seed yield. Our results suggest that increasing the planting density within certain range is a feasible approach for higher seed yield in winter rapeseed in China.

Background: Canola (Brassica napus L.) is one of the most important oil-producing crops in China and worldwide. The yield and quality of canola is frequently threatened by environmental stresses including drought, cold and high salinity. Calcium is a ubiquitous intracellular secondary messenger in plants. Calcineurin B-like proteins (CBLs) are Ca2+ sensors and regulate a group of Ser/Thr protein kinases called CBL-interacting protein kinases (CIPKs). Although the CBL-CIPK network has been demonstrated to play crucial roles in plant development and responses to various environmental stresses in Arabidopsis, little is known about their function in canola., Results: In the present study, we identified seven CBL and 23 CIPK genes from canola by database mining and cloning of cDNA sequences of six CBLs and 17 CIPKs. Phylogenetic analysis of CBL and CIPK gene families across a variety of species suggested genome duplication and diversification. The subcellular localization of three BnaCBLs and two BnaCIPKs were determined using green fluorescence protein (GFP) as the reporter. We also demonstrated interactions between six BnaCBLs and 17 BnaCIPKs using yeast two-hybrid assay, and a subset of interactions were further confirmed by bimolecular fluorescence complementation (BiFC). Furthermore, the expression levels of six selected BnaCBL and 12 BnaCIPK genes in response to salt, drought, cold, heat, ABA, methyl viologen (MV) and low potassium were examined by quantitative RT-PCR and these CBL or CIPK genes were found to respond to multiple stimuli, suggesting that the canola CBL-CIPK network may be a point of convergence for several different signaling pathways. We also performed a comparison of interaction patterns and expression profiles of CBL and CIPK in Arabidospsis, canola and rice, to examine the differences between orthologs, highlighting the importance of studying CBL-CIPK in canola as a prerequisite for improvement of this crop., Conclusions: Our findings indicate that CBL and CIPK family members may form a dynamic complex to respond to different abiotic or hormone signaling. Our comparative analyses of the CBL-CIPK network between canola, Arabidopsis and rice highlight functional differences and the necessity to study CBL-CIPK gene functions in canola. Our data constitute a valuable resource for CBL and CPK genomics.

Chloroplast molecular markers can provide useful information for high-resolution analysis of inter- and intra-specific variation in Brassicaceae and for differentiation between its species. Combining data generated from nuclear and chloroplast markers enables the study of seed and pollen movement, and assists in the assessment of gene-flow from genetically modified (GM) plants through hybridization studies. To develop chloroplast DNA markers for monitoring of transgene introgression in Brassica napus L., we searched for sequence variations in the chloroplast (cp) genome, and developed a simple cpDNA marker that is reliable, time-saving, and easily discriminates among 4 species (B. napus, B. rapa, Raphanus sativus, and Sinapis alba) based on PCR-product length polymorphism. This marker will be useful to identify maternal lineages and to estimate transgene movement of GM canola.

Developing yellow-seeded Brassica napus (rapeseed) with improved qualities is a major breeding goal. The intermediate and final metabolites of the phenylpropanoid and flavonoid pathways affect not only oil quality but also seed coat colour of B. napus. Here, the accumulation of phenolic compounds was analysed in the seed coats of black-seeded (ZY821) and yellow-seeded (GH06) B. napus. Using toluidine blue O staining and liquid chromatography-mass spectrometry, histochemical and biochemical differences were identified in the accumulation of phenolic compounds between ZY821 and GH06. Two and 13 unique flavonol derivatives were detected in ZY821 and GH06, respectively. Quantitative real-time PCR analysis revealed significant differences between ZY821 and GH06 in the expression of common phenylpropanoid biosynthetic genes (BnPAL and BnC4H), common flavonoid biosynthetic genes (BnTT4 and BnTT6), anthocyanin- and proanthocyandin-specific genes (BnTT3 and BnTT18), proanthocyandin-specific genes (BnTT12, BnTT10, and BnUGT2) and three transcription factor genes (BnTTG1, BnTTG2, and BnTT8) that function in the flavonoid biosynthetic pathway. These data provide insight into pigment accumulation in B. napus, and serve as a useful resource for researchers analysing the formation of seed coat colour and the underlying regulatory mechanisms in B. napus.

Introduction: Brassica napus L. is a crop widely grown for its oil production and other nutritional components in the seed. In addition to the seed, other organs contain a wide range of phenolic metabolites although they have not been investigated to the same extent as in seeds., Objective: To define and compare the phytochemical composition of B. napus L. organs, namely the root, stem, leaf, inflorescence and seeds., Method: Non-targeted metabolomic analysis via UPLC-QTOF-MS was utilised in order to localise compounds belonging to various chemical classes (i.e. oxygenated fatty acids, flavonols, phenolic acids and sinapoyl choline derivatives)., Results: The vast majority of identified metabolites were flavonol glycosides that accumulated in most of the plant organs. Whereas other classes were detected predominantly in specific organs, i.e. sinapoyl cholines were present uniquely in seeds. Furthermore, variation in the accumulation pattern of metabolites from the same class was observed, particularly in the case of quercetin, kaempferol and isorhamnetin flavonols. Anti-oxidant activity, based on 2,2-diphenyl-1-picrylhdrazyl analysis was observed for all extracts, and correlated to some extent with total flavonoid content., Conclusion: This study provides the most complete map for polyphenol composition in B. napus L. organs. By describing the metabolites profile in B. napus L., this study provides the basis for future investigations of seeds for potential health and/or medicinal use., (Copyright © 2012 John Wiley & Sons, Ltd.)

Brassica napus L. is the leading European oilseed crop and has therefore a high economical importance. The objectives of our study were to examine (1) the patterns of phenotypic diversity in a species-wide B. napus germplasm set of 518 inbreds with respect to various seedling development, agronomic, and seed quality traits as well as (2) the interrelationship of the examined traits and their use in selection on correlated traits. The B. napus germplasm set was evaluated in greenhouse and field trials for several seedling development, agronomic, and seed quality traits. The traits were highly correlated within the individual trait categories and moderately correlated between the different trait categories. We observed differences in phenotypic diversity among the examined eight germplasm types. The reduction of phenotypic diversity was on average more pronounced for the seedling development traits than for the agronomic and seed quality traits, suggesting that plant breeders need to introgress new genetic variation with respect to the former.

Crosstalk between intracellular signalling pathways is a functionally important and widespread phenomenon in cell physiology across phyla. In the salivary gland of the blowfly, serotonin induces fluid secretion via parallel activation of both the InsP(3)/Ca(2+) and the cAMP/PKA signalling pathways, which interact on multiple levels. We have determined the molecular identity of a link between both pathways that mediates a Ca(2+)-dependent rise of intracellular cAMP. Whereas hydrolysis of cAMP via phosphodiesterases is largely independent of Ca(2+), cAMP synthesis by adenylyl cyclases (AC) is potentiated in a Ca(2+)/calmodulin (Ca(2+)/CaM)-dependent manner. The existence of a Ca(2+)/CaM-dependent AC is supported by physiological data and a molecular approach. We have cloned Cv rutabaga cDNA, encoding the first blowfly AC, and confirmed its expression in the salivary gland via reverse transcription followed by polymerase chain reaction. The putative gene product of Cv rutabaga is a Ca(2+)/CaM-dependent type I AC and shows highest homology to Rutabaga from Drosophila. Thus, a Ca(2+)/CaM-dependent AC serves as a link between the InsP(3)/Ca(2+) and the cAMP/PKA signalling pathways in the salivary gland of the blowfly and might be important for the amplification and optimization of the secretory response., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The putative role of cytokinins in processes leading to reproductive development of plants was investigated by analysing the shoot apical parts of a winter cultivar of oilseed rape (Brassica napus L. var. oleifera, cv. Górczański). The endogenous cytokinin levels were measured by liquid chromatography-tandem mass spectrometry (LC-MS) in the shoot apices of vegetative plants (grown at 20/17°C with a 16/8h day/night regime) and vernalized plants (56 days at 5/2°C with a 16/8h photoperiod) at different times during floral transition. During vernalization, the content of all isoprenoid cytokinins increased significantly, coinciding well with the onset of the early stages of reproductive development. Cytokinin levels reached their maxima when most of the plants became irreversibly reproductive (after 42 days of cold treatment). cis-Zeatin riboside (unequivocally identified by quadrupole-time-of-flight MS) accounted for ca. 87-89% of the total isoprenoid cytokinin content in control and vernalized plants, whilst N(6)-isopentenyladenosine ( approximately 6% in control and approximately 8% in vernalized plants) and cis-zeatin (approxiamtely 2% in control and approximately 1% in vernalized plants) were the next most abundant cytokinins. In the post-vernalization period, endogenous cytokinin levels decreased, but remained significantly higher in the reproductive plants than in the vegetative controls. These results suggest that cytokinins, especially those of the cis-zeatin type, are involved in vernalization-induced reproductive development of B. napus., (© 2012 Elsevier Ireland Ltd. All rights reserved.)

Chromosome rearrangements are common, but their dynamics over time, mechanisms of occurrence and the genomic features that shape their distribution and rate are still poorly understood. We used allohaploid Brassica napus (AC, n = 19) as a model to analyze the effect of genomic features on the formation and diversity of meiotically driven chromosome rearrangements. We showed that allohaploid B. napus meiosis leads to extensive new structural diversity. Almost every allohaploid offspring carried a unique combination of multiple rearrangements throughout the genome, and was thus structurally differentiated from both its haploid parent and its sister plants. This large amount of genome reshuffling was remarkably well-tolerated in the heterozygous state, as neither male nor female fertility were strongly reduced, and meiosis behavior was normal in most cases. We also used a quantitative statistical model, which accounted for 75% of the observed variation in rearrangement rates, to show that the distribution of meiotically driven chromosome rearrangements was not random but was shaped by three principal genomic features. In descending order of importance, the rate of marker loss increased strongly with genetic distance from the centromere, the degree of collinearity between chromosomes, and the genome of origin (A < C). Overall, our results demonstrate that B. napus accumulates a large number of genetic changes, but these rearrangements are not randomly distributed in the genome. The structural genetic diversity produced by the allohaploid pathway and its role in the evolution of polyploid species compared to diploid meiosis are discussed., (© 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd.)

We developed Diversity Array Technology (DArT) markers for application in genetic studies of Brassica napus and other Brassica species with A or C genomes. Genomic representation from 107 diverse genotypes of B. napus L. var. oleifera (rapeseed, AACC genomes) and B. rapa (AA genome) was used to develop a DArT array comprising 11 520 clones generated using PstI/BanII and PstI/BstN1 complexity reduction methods. In total, 1547 polymorphic DArT markers of high technical quality were identified and used to assess molecular diversity among 89 accessions of B. napus, B. rapa, B. juncea, and B. carinata collected from different parts of the world. Hierarchical cluster and principal component analyses based on genetic distance matrices identified distinct populations clustering mainly according to their origin/pedigrees. DArT markers were also mapped in a new doubled haploid population comprising 131 lines from a cross between spring rapeseed lines 'Lynx-037DH' and 'Monty-028DH'. Linkage groups were assigned on the basis of previously mapped simple sequence repeat (SSRs), intron polymorphism (IP), and gene-based markers. The map consisted of 437 DArT, 135 SSR, 6 IP, and 6 gene-based markers and spanned 2288 cM. Our results demonstrate that DArT markers are suitable for genetic diversity analysis and linkage map construction in rapeseed.

Three coding region cDNAs of duplicated PISTILLATA-like (PI-like) MADS-box genes, BnPI-1, BnPI-2 and BnPI-3, were isolated from B. napus by RT-PCR. The sequence analysis showed that the three PI cDNAs possessed 627, 627 and 625 nucleotides, respectively, and their nucleotide sequences had 96.49-98.72% similarity. Due to a deletion of two nucleotides, the protein sequence in the downstream of the frameshift site was altered in BnPI-3. Therefore, there were only 171 amino acids coded by BnPI-3, while there were 208 ones coded by BnPI-1 or BnPI-2. The deduced amino acid identity between BnPI-1 and BnPI-2 was 97.6% and the amino acid sequence of BnPI-1 and BnPI-2 shared 72.6% identity with BnPI-3. The deduced amino acid sequences of the coded proteins indicated high homology with the members of the PI family of MADS-box proteins. RT-PCR analysis showed that BnPI transcription was only detectable in petals and stamens. The yeast two-hybrid assays results showed that the three BnPI proteins exhibited different dimerization affinities with three BnAP3. BnPI-1 and BnPI-2 could form strong heterodimers with BnAP3. The dimerization affinity of BnPI-1 with BnAP3-4 is the strongest in all the combinations, while the affinity of BnPI-3 with BnAP3-4 is the weakest. The dimerization affinity to BnAP3-4 of BnPI-1 is 3.5 times of that of BnPI-3. The distinguished weak interaction to AP3 of BnPI-3 is probably due to the loss of the PI motif. The divergences of sequence and affinity of protein interaction might reflect some functional divergence of the three PI genes in B. napus.

In an attempt to determine the adaptation strategy to phosphorous (Pi) deficiency in oilseed rape, comparative proteome analyses were conducted to investigate the differences of metabolic changes in two oilseed rape genotypes with different tolerance to low phosphorus (LP). Generally in either roots or leaves, there existed few low phosphorus (LP)-induced proteins shared in the two lines. The LP-tolerant genotype 102 maintained higher Pi concentrations than LP-sensitive genotype 105 when growing hydroponically under the 5-μM phosphorus condition. In 102 we observed the downregulation of the proteins related to gene transcription, protein translation, carbon metabolism, and energy transfer in leaves and roots, and the downregulation of proteins related to leaf growth and root cellular organization. But the proteins related to the formation of lateral root were upregulated, such as the auxin-responsive family proteins in roots and the sucrose-phosphate synthase-like protein in roots and leaves. On the other hand, the LP-sensitive genotype 105 maintained the low level of Pi concentrations and suffered high oxidative pressure under the LP condition, and stress-shocking proteins were pronouncedly upregulated such as the proteins for signal transduction, gene transcription, secondary metabolism, universal stress family proteins, as well as the proteins involved in lipid oxygenation and the disease resistance in both leaves and roots. Although the leaf proteins for growth in 105 were downregulated, the protein expressions in roots related to glycolysis and tricarboxylic acid (TCA) cycle were enhanced to satisfy the requirement of organic acid secretion.

A new full-length beta-carbonic anhydrase cDNA was obtained from Brassica napus by homologous cloning. The cDNA has an open-reading frame of 996 nucleotides, encoding 331 amino acids with a calculated molecular weight of 35,692 Da and an estimated pI value of 5.459. The deduced amino acid sequence of beta-carbonic anhydrase from Brassica napus shared significant identity with beta-carbonic anhydrases from Brassica carinata, Arabidopsis thaliana, and Thlaspi caerulescens (97.9%, 94%, and 93.5% identity, respectively). This cDNA was expressed in Escherichia coli BL21 (DE3) using the expression vector pET-32a(+). The expression band corresponded to the calculated mass plus the N-terminal fusion protein derived from the vector.

A rapid method was developed for concurrent screening of transgenic elements in GM canola. This method utilizes a single multiplex PCR coupled with an oligonucleotide DNA array capable of simultaneously detecting the 12 approved GM canola lines in Canada. The assay includes construct-specific elements for identification of approved lines, common elements (e.g., CaMV 35S promoter, Agrobacterium tumefaciens nos terminator, or nptII gene) for screening of approved or unapproved lines, a canola-specific endogenous gene, and endogenous genes from heterologous crops to serve as additional controls. Oligonucleotide probes were validated individually for functionality and specificity by amplification of specific transgene sequences from appropriate GM canola lines corresponding to each probe sequence, and hybridization of amplicons to the array. Each target sequence hybridized to its corresponding oligonucleotide probe and no significant cross-hybridization was observed. The limit of detection was examined for the GM lines GT73, T45, and MS8/RF3, and was determined to be 0.1%, 0.1%, and 0.5%, respectively, well within the European food and feed labeling threshold level of 0.9% for approved GM product. Practically, the method was demonstrated to be effective for the detection of GM canola in several types of animal feed, as well as in commercial canola meal.

Ability of shoot regeneration from cotyledon explants of rapeseed (Brassica napus) winter genotypes and the effect of silver nitrate and naphthalene acetic acid in shoot regeneration were investigated. The optimum medium for regeneration was the medium supplemented with 3 mg L(-1) 6-Benzylaminopurine and 0.15 mg L(-1) 1-naphthaleneacetic acid. The addition of 5 mg L(-1) silver nitrate significantly improved shoot regeneration. Shoot regeneration response was strongly different between genotypes with a range of variation from 79% in spring genotype PF and 7% in Okpi. The highest root production was recorded on medium containing indol-3-butyric acid. The rooted plants successfully transferred to soil and adapted to greenhouse conditions. No abnormally was observed and the regenerated plants were morphologically similar to the field grown parental plants.

AP2/ERF is a large family of transcription factors in plant. Genes in the AP2/ERF family encode transcriptional regulators with a variety of functions involved in the developmental and physiological processes in plants. Two AP2/ERF family transcriptional regulators (BnaERFB3-1 and BnaERFB3-2) were isolated from B. napus by in silico cloning method using the conserved domain amino acid sequence of A. thaliana AP2/ERF-B3 subfamily as probe. Based on the sequences of BnaERFB3-1 and BnaERFB3-2, we isolated the BnaERFB3-1-Hy15 gene and BnaERFB3-2-Hy15 gene from winter and spring type B. napus L. cv Huyou15 by RT-PCR and PCR using cDNA and DNA as template. DNA sequencing and analyzing indicated that there was only one amino acid residue difference between BnaERFB3-1 and BnaERFB3-1-Hy15, BnaERFB3-2 and BnaERFB3-2-Hy15, respectively. No intron localized on the two genes from Huyou15. Then, deduced amino acid sequence, composition, hydrophobicty and hydrophilicity, physical and chemical characterization, phylogenetic tree, conserved domain sequences, function domain, molecular modeling, and folding state were predicted and analyzed. BnaERFB3-1-Hy15 and BnaERFB3-2-Hy15 were hydrophilic protein. The two proteins and AtERF5 have similar three-dimension structure. The disordered residues of protein BnaERFB3-1-Hy15 and BnaERFB3-2-Hy15 were higher than that of AtERF5. BnaERFB3-1 was mainly expressed in seed, while BnaERFB3-2 was mainly expressed in root. Moreover, those genes were successfully constructed into the recombinant plasmids of plant expression vector and yeast expression vector, which established a base for transformation of oilseed and studies of those genes function in abiotic stresses of B. napus.

Brassica species represent several important crops including canola (Brassica napus). Understanding of genetic elements that contribute to seed-associated functions will impact future improvements in the canola crop. Brassica species share a very close taxonomic and molecular relationship with Arabidopsis thaliana. However, there are several subtle but distinct seed-associated agronomic characteristics that differ among the oil seed crop species. To address these, we have generated 67,535 ESTs predominately from Brassica seeds, analyzed these sequences, and identified 10,642 unigenes for the preparation of a targeted seed cDNA array. A set of 10,642 PCR primer pairs was designed and corresponding amplicons were produced for spotting, along with relevant controls. Critical quality control tests produced satisfactory results for use of this microarray in biological experiments. The microarray was also tested with specific RNA targets from embryos, germinating seeds, and leaf tissues. The hybridizations, signal intensities, and overall quality of these slides were consistent and reproducible. Additionally, there are 429 ESTs represented on the array that show no homology with any A. thaliana annotated gene or any gene in the Brassica genome databases or other plant databases; however, all of these probes hybridized to B. napus transcripts, indicating that the array also will be useful in defining expression patterns for genes so far unique to Brassica species.

A genetic marker system based on the S1 Short Interspersed Elements (SINEs) in the important commercial crop, oilseed rape ( Brassica napus L.) has been developed. SINEs provided a successful multilocus, dominant marker system that was capable of clearly delineating winter- and spring-type crop varieties. Sixteen of 20 varieties tested showed unique profiles from the 17 polymorphic SINE markers generated. The 3' or 5' flank region of nine SINE markers were cloned, and DNA was sequenced. In addition, one putative pre-transposition SINE allele was cloned and sequenced. Two SINE flanking sequences were used to design real-time PCR assays. These quantitative SINE assays were applied to study the genetic structure of eight fields of oilseed rape crops. Studied fields were more genetically diverse than expected for the chosen loci (mean H T = 0.23). The spatial distribution of SINE marker frequencies was highly structured in some fields, suggesting locations of volunteer impurities within the crop. In one case, the assay identified a mislabeling of the crop variety. SINE markers were a useful tool for crop genetics, phylogenetics, variety identification, and purity analysis. The use and further application of quantitative, real-time PCR markers are discussed.

Oilseed rape (Brassica napus) in Australia and China have similar origins, with introductions from Europe, Canada, and Japan in the mid 20th century, and there has been some interchange of germplasm between China and Australia since that time. Allelic diversity of 72 B. napus genotypes representing contemporary germplasm in Australia and China, including samples from India, Europe, and Canada, was characterized by 55 polymorphic simple sequence repeat (SSR) markers spanning the entire B. napus genome. Hierarchical clustering and two-dimensional multidimensional scaling identified a Chinese group (China-1) that was separated from "mixed group" of Australian, Chinese (China-2), European, and Canadian lines. A small group from India was distinctly separated from all other B. napus genotypes. Chinese genotypes, especially in the China-1 group, have inherited unique alleles from interspecific crossing, primarily with B. rapa, and the China-2 group has many alleles in common with Australian genotypes. The concept of "private alleles" is introduced to describe both the greater genetic diversity and the genetic distinctiveness of Chinese germplasm, compared with Australian germplasm, after 50 years of breeding from similar origins.

Field experiments were conducted at Cereal Crops Research Institute, Pirsabak, Nowshera, Pakistan, during winter 2003-2004 and 2004-2005 to evaluate the effect of nitrogen and sulfur levels and methods of nitrogen application on canola (Brassica napus L. cv. Bulbul-98) under rainfed conditions. Four levels of S (0, 10, 20, and 30 kg/ha) and three levels of N (40, 60, and 80 kg/ha) and a control treatment with both nutrients at zero level were included in the experiments. Sulfur levels were applied at sowing while N levels were applied by three methods (100% soil application, 90% soil+10% foliar application, and 80% soil +20% foliar application). The experiments were laid out in randomized complete block (RCB) design having four replications. Oil content increased significantly up to 20 kg S/ha but further increase in S level did not enhance oil content. Glucosinolate content increased from 13.6 to 24.6 micromol/g as S rate was increased from 0 to 30 kg/ha. Protein content increased from 22.4% to 23.2% as S rate was increased from 0 to 20 kg/ha. Oil content responded negatively to the increasing N levels. The highest N level resulted in the highest values for protein (23.5%) and glucosinolate (19.9 micromol/g) contents. Methods of N application had no significant impact on any parameters under study.

Estimation of genetic diversity of Brassica germplasm provides the basis for rapeseed/mustard genetic improvement. Studies were undertaken to estimate the genetic diversity of 30 lines of Brassica napus using Randomly Amplified Polymorphic DNA (RAPD) primers. A total of 30 B. napus genotypes of local and exotic origin were characterized using molecular markers. Four RAPD primers were used to estimate the genetic distances among the genotypes in all the possible combinations. The genetic diversity study revealed different levels of genetic polymorphism for RAPD primers GLA05, GLA07, GLA09 and GLA 10, resulting in amplification of 5.7, 3.5, 3.1 and 5.4 scorable bands (loci) per genotype, respectively. Individual genetic distances observed among B. napus genotypes ranged from 6.5 to 51%. Bivariate data matrix was generated and genetic distances were calculated using Unweighted Pair Group of Arithmetic Mean (UPGMA) procedure. The UPGMA cluster analyses revealed maximum genetic dissimilarity for 8966-1 and 8969-1 genotypes, closely followed by Ganyou-5, 89127-1, 89111-2 and Mlep-048. It is recommended that among the thirty B. napus genotypes, genetically distinct lines pointed out in the present study, should be used in future breeding programs for improvement of Brassica napus.

A breeding strategy for widening the germplasm of Brassica napus was proposed by introgression of the A(r) subgenome of B. rapa (A(r)A(r)) and C(c) of B. carinata (B(c)B(c)C(c)C(c)) into natural B. napus (A(n)A(n)C(n)C(n)). The progenies with 38 chromosomes that were derived from the self-pollinated seeds of pentaploid hybrids (A(r)A(n)B(c)C(c)C(n)) were used for further research. Some of the partial new-typed B. napus showed normal meiotic behavior, high portion of germinated pollen and normal embryological development. This indicates that the selected new-typed B. napus had a balanced genetic base. Molecular analysis showed that about 50% of the genome in the new-typed B. napus was replaced by A(r) and C(c) subgenome from B. rapa and B. carinata. Considering the genetic diversity among different lines of new-typed B. napus it was deduced that the introgression of the genomic components from B. rapa and B. carinata could widen the genetic diversity of rapeseed.