Identification of genome-wide single nucleotide polymorphisms in allopolyploid crop Brassica napus

Background

Single nucleotide polymorphisms (SNPs) are the most common type of genetic variation. Identification of large numbers of SNPs is helpful for genetic diversity analysis, map-based cloning, genome-wide association analyses and marker-assisted breeding. Recently, identifying genome-wide SNPs in allopolyploid Brassica napus (rapeseed, canola) by resequencing many accessions has become feasible, due to the availability of reference genomes of Brassica rapa (2n = AA) and Brassica oleracea (2n = CC), which are the progenitor species of B. napus (2n = AACC). Although many SNPs in B. napus have been released, the objective in the present study was to produce a larger, more informative set of SNPs for large-scale and efficient genotypic screening. Hence, short-read genome sequencing was conducted on ten elite B. napus accessions for SNP discovery. A subset of these SNPs was randomly selected for sequence validation and for genotyping efficiency testing using the Illumina GoldenGate assay.

Results

A total of 892,536 bi-allelic SNPs were discovered throughout the B. napus genome. A total of 36,458 putative amino acid variants were located in 13,552 protein-coding genes, which were predicted to have enriched binding and catalytic activity as a result. Using the GoldenGate genotyping platform, 94 of 96 SNPs sampled could effectively distinguish genotypes of 130 lines from two mapping populations, with an average call rate of 92%.

Conclusions

Despite the polyploid nature of B. napus, nearly 900,000 simple SNPs were identified by whole genome resequencing. These SNPs were predicted to be effective in high-throughput genotyping assays (51% polymorphic SNPs, 92% average call rate using the GoldenGate assay, leading to an estimated >450 000 useful SNPs). Hence, the development of a much larger genotyping array of informative SNPs is feasible. SNPs identified in this study to cause non-synonymous amino acid substitutions can also be utilized to directly identify causal genes in association studies.     

免耕稻田氮肥运筹对土壤NH3挥发及氮肥利用率的影响

通过大田试验,设置5种不同的施肥比例(基肥:分蘖肥:拔节肥:穗肥-2:2:3:3(R1)、3:2:2:3(R2)、4:2:2:2(R3)、4:3:1:2(R4)与0:0:0:0(CK)),研究氮肥运筹对稻田NH₃挥发和氮肥利用率的影响。结果表明,(1)相对于不施肥,施肥显著提高了稻田NH₃挥发量。氮肥施用后,NH₃挥发损失量占施氮量的6.2%-8.5%,其中,以分蘖期NH₃挥发损失量最大,齐穗期次之,苗期和拔节期最小。施肥处理间,处理R1稻田累积NH₃挥发量最小,显著低于其它施肥处理,比处理R2、R3和R4分别低9.1%(P<0.05)、10.9%(P<0.05)和17.7%(P<0.05)。(2)相关分析表明,田面水NH₄⁺、pH值和土壤NH₄⁺和pH值均与稻田土壤NH₃挥发通量呈显著或者极显著相关;(3)处理R1水稻氮肥利用率相对于处理R2、R3和R4增加了28.4%(P<0.05)、55.4%(P<0.05)和74.9%(P<0.05)。研究表明,氮肥后移能有效降低免耕稻田NH₃挥发,提高水稻的氮肥利用率。     

Unravelling the complex trait of harvest index in rapeseed (Brassica napus L.) with association mapping

Background

Harvest index (HI), the ratio of grain yield to total biomass, is considered as a measure of biological success in partitioning assimilated photosynthate to the harvestable product. While crop production can be dramatically improved by increasing HI, the underlying molecular genetic mechanism of HI in rapeseed remains to be shown.

Results

In this study, we examined the genetic architecture of HI using 35,791 high-throughput single nucleotide polymorphisms (SNPs) genotyped by the Illumina BrassicaSNP60 Bead Chip in an association panel with 155 accessions. Five traits including plant height (PH), branch number (BN), biomass yield per plant (BY), harvest index (HI) and seed yield per plant (SY), were phenotyped in four environments. HI was found to be strongly positively correlated with SY, but negatively or not strongly correlated with PH. Model comparisons revealed that the A–D test (ADGWAS model) could perfectly balance false positives and statistical power for HI and associated traits. A total of nine SNPs on the C genome were identified to be significantly associated with HI, and five of them were identified to be simultaneously associated with HI and SY. These nine SNPs explained 3.42 % of the phenotypic variance in HI.

Conclusions

Our results showed that HI is a complex polygenic phenomenon that is strongly influenced by both environmental and genotype factors. The implications of these results are that HI can be increased by decreasing PH or reducing inefficient transport from pods to seeds in rapeseed. The results from this association mapping study can contribute to a better understanding of natural variations of HI, and facilitate marker-based breeding for HI.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1607-0) contains supplementary material, which is available to authorized users.     

Genetic investigation of the origination of allopolyploid with virtually synthesized lines: application to the C subgenome of Brassica napus

Although there are a number of different allopolyploids in the plant kingdom, the exact ancestral parents of some allopolyploids have not been well characterized. We propose a strategy in which virtual allopolyploid lines derived from different types of parental species are used to investigate the progenitors of an allopolyploid. The genotypes of the parental lines and the natural allopolyploid were established using a set of DNA molecular markers. The genotypes of the virtual lines were then derived from those of the parental lines, and compared extensively with that of the natural allopolyploid. We applied this strategy to investigate the progenitors of the C subgenome of Brassica napus (rapeseed, AACC). A total of 39 accessions from 10 wild and 7 cultivated types of the B. oleracea cytodeme (CC), and 4 accessions of B. rapa (AA) were used to construct 156 virtual rapeseed lines. Genetic structure was compared among natural rapeseed, virtual rapeseed lines, and their parental lines by principal component analysis and analysis of ancestry. Our data showed that the C subgenome of natural rapeseed was related closely to the genome of cultivated B. oleracea and its related wild types, such as B. incana, B. bourgeaui, B. montana, B. oleracea ssp. oleracea and B. cretica. This finding indicated that these types or their progeny might be ancestral donors of the C subgenome of rapeseed. The successful application of the strategy of virtual allopolyploidy in rapeseed demonstrates that it can possibly be used to identify the progenitors of an allopolyploid species.     

Identification and characterization of LIM gene family in Brassica rapa

Background

LIM (Lin-11, Isl-1 and Mec-3 domains) genes have been reported to trigger the formation of actin bundles, a major higher-order cytoskeletal assembly, in higher plants; however, the stress resistance related functions of these genes are still not well known. In this study, we collected 22 LIM genes designated as Brassica rapa LIM (BrLIM) from the Brassica database, analyzed the sequences, compared them with LIM genes of other plants and analyzed their expression after applying biotic and abiotic stresses in Chinese cabbage.

Results

Upon sequence analysis these genes were confirmed as LIM genes and found to have a high degree of homology with LIM genes of other species. These genes showed distinct clusters when compared to other recognized LIM proteins upon phylogenetic analysis. Additionally, organ specific expression of these genes was observed in Chinese cabbage plants, with BrPLIM2a, b, c, BrDAR1, BrPLIM2e, f and g only being expressed in flower buds. Furthermore, the expression of these genes (except for BrDAR1 and BrPLIM2e) was high in the early flowering stages. The remaining genes were expressed in almost all organs tested. All BrDAR genes showed higher expression in flower buds compared to other organs. These organ specific expressions were clearly correlated with the phylogenetic grouping. In addition, BrWLIM2c and BrDAR4 responded to Fusarium oxysporum f. sp. conglutinans infection, while commonly two BrDARs and eight BrLIMs responded to cold, ABA and pH (pH5, pH7 and pH9) stress treatments in Chinese cabbage plants.

Conclusion

Taken together, the results of this study indicate that BrLIM and BrDAR genes may be involved in resistance against biotic and abiotic stresses in Brassica.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-641) contains supplementary material, which is available to authorized users.     

Cloning and characterization of microRNAs from Brassica napus

A library containing approximately 40,000 small RNA sequences was constructed for Brassica napus. Analysis of 3025 sequences obtained from this library resulted in the identification of 11 conserved miRNA families, which were validated by secondary structure prediction using surrounding sequences in the Brassica genome. Two 21 nt small RNA sequences reside within the arm of a pre-miRNA like stem-loop structure, making them likely candidates for novel non-conserved miRNAs in B. napus. Most of the conserved miRNAs were expressed at similar levels in a F1 hybrid B. napus line and its four double haploid progeny that showed marked variations in phenotypes, but many were differentially expressed between B. napus and Arabidopsis. The miR169 family was expressed at high levels in young leaves and stems, but was undetectable in roots and mature leaves, suggesting that miR169 expression is developmentally regulated in B. napus.     

A triallelic genetic male sterility locus in Brassica napus: an integrative strategy for its physical mapping and possible local chromosome evolution around it

Background and Aims

Spontaneous male sterility is an advantageous trait for both constructing efficient pollination control systems and for understanding the developmental process of the male reproductive unit in many crops. A triallelic genetic male-sterile locus (BnMs5) has been identified in Brassica napus; however, its complicated genome structure has greatly hampered the isolation of this locus. The aim of this study was to physically map BnMs5 through an integrated map-based cloning strategy and analyse the local chromosomal evolution around BnMs5.

Methods

A large F₂ population was used to integrate the existing genetic maps around BnMs5. A map-based cloning strategy in combination with comparative mapping among B. napus, Arabidopsis, Brassica rapa and Brassica oleracea was employed to facilitate the identification of a target bacterial artificial chromosome (BAC) clone covering the BnMs5 locus. The genomic sequences from the Brassica species were analysed to reveal the regional chromosomal evolution around BnMs5.

Key Results

BnMs5 was finally delimited to a 0·3-cM genetic fragment from an integrated local genetic map, and was anchored on the B. napus A8 chromosome. Screening of a B. napus BAC clone library and identification of the positive clones validated that JBnB034L06 was the target BAC clone. The closest flanking markers restrict BnMs5 to a 21-kb region on JBnB034L06 containing six predicted functional genes. Good collinearity relationship around BnMs5 between several Brassica species was observed, while violent chromosomal evolutionary events including insertions/deletions, duplications and single nucleotide mutations were also found to have extensively occurred during their divergence.

Conclusions

This work represents major progress towards the molecular cloning of BnMs5, as well as presenting a powerful, integrative method to mapping loci in plants with complex genomic architecture, such as the amphidiploid B. napus.     

Effects of sowing time and plant age on critical nitrogen concentrations in canola (Brassica napus L.)

Critical concentrations of NO₃-N in fresh petiole tissue and total N in the dried lamina were determined for the youngest mature leaf (YML) of field-grown canola. For dry matter yield of canola sown on 4 May, critical NO₃-N concentration in the YML petiole at the rosette stage (RS) was 1.46 mg/g fresh wt. At the flower-buds-visible stage (BV) it was 0.45 mg/g fresh wt. For seed yield the values were 1.72 and 0.53 mg/g fresh wt. Critical total N concentration in the YML lamina for dry matter yield were 69 mg/g dry wt. at RS and 57 at BV. For seed yield they were 71 and 59 mg/g dry wt. Critical NO₃-N concentrations in the YML petiole of canola sown on 30 May were reduced by 50%; critical total-N concentrations in the YML lamina were not reduced to the same extent. Despite the reductions in critical N concentrations in the YML, critical N fertilizer rates for vegetative growth and seed yield were unaffected by sowing date or plant growth stage.     

Nitrogen incorporation and remobilization in different shoot components of field-grown winter oilseed rape (Brassica napus L.) as affected by rate of nitrogen application and irrigation

The seasonal course of nitrogen uptake, incorporation and remobilization in different shoot components of winter oilseed rape (Brassica napus L.) was studied under field conditions including three rates of ¹⁵N labelled nitrogen application (0, 100 or 200 kg N ha^-1) and two irrigation treatments (rainfed or watered at a deficit of 20 mm). The total amount of irrigation water applied was 260 mm, split over 13 occasions in a 7-week-period ranging from 1 week before onset of flowering until 4 weeks after flowering.Nitrogen application and irrigation increased plant growth and nitrogen accumulation. Irrespective of N and irrigation treatment more than 50% of total shoot N was present in the stem when flowering started. At the end of flowering, pod walls were the main N store containing about 30–40% of shoot N. The quantities of N remobilized from stems and pod walls amounted in all treatments to about 70% of the N present in these organs at mid-flowering. At harvest, stem and pod walls each contained about 10% of total shoot N, the remaining 80% being incorporated into seeds. The main component contributing to the response of seed N accumulation to nitrogen application and irrigation was pods in axillary racemes. Up to 20 kg N ha^-1, corresponding to about 10% of final shoot N content, was lost from the plants by leaf drop.Irrigation increased the recovery at harvest of applied N from 30% to about 50%, while the level of N application did not affect the N recovery. ¹⁵N labelled (fertilizer derived) nitrogen constituted a greater proportion of the N content in old leaves than in young leaves and increased with age in the former, but not in the latter. Relative to soil N, fertilizer derived N also contributed more to the N content of vegetative than to that of reproductive shoot components. Small net changes in shoot N content after flowering reflected a balance between N import and export, leading to continuous dilution of ¹⁵N labelled N with unlabelled N.     

Computational identification of novel microRNAs and targets in Brassica napus

MicroRNAs (miRNAs) are a newly discovered class of non-protein-coding small RNAs with roughly 22 nucleotide-long. Increasing evidence has shown that miRNAs play multiple roles in biological processes, including development, cell proliferation and apoptosis and stress responses. In this research, several approaches were combined to make computational prediction of potential miRNAs and their targets in Brassica napus. We used previously known miRNAs from Arabidopsis, rice and other plant species against both expressed sequence tags (EST) and genomic survey sequence (GSS) databases to search for potential miRNAs in B. napus. A total of 21 potential miRNAs were detected following a range of strict filtering criteria. Using these potential miRNA sequences, we could further blast the mRNA database and found 67 potential targets in this species. According to the mRNA target information provided by NCBI (http://www.ncbi.nlm.nih.gov/), most of the target mRNAs appeared to be involved in plant growth, development and stress responses. To validate the prediction of miRNAs in B. napus, we performed a RT-PCR based assay of mature miRNA expression. Five miRNAs were identified in response to auxin, cadmium stress and phosphate starvation. So far, little is known about experimental or computational identification of miRNA in B. napus species. To improve efficiency for blast search, we developed an implementation (miRNAassist) that can identify homologs of miRNAs and their targets, with high sensitivity and specificity. The program is allowed to be run on Windows Operation System platform. miRNAassist is freely available if required.     

EST-based in silico identification and in vitro test of antimicrobial peptides in Brassica napus

Background

Brassica napus is the third leading source of vegetable oil in the world after soybean and oil palm. The accumulation of gene sequences, especially expressed sequence tags (ESTs) from plant cDNA libraries, has provided a rich resource for genes discovery including potential antimicrobial peptides (AMPs). In this study, we used ESTs including those generated from B. napus cDNA libraries of seeds, pathogen-challenged leaves and deposited in the public databases, as a model, to perform in silico identification and consequently in vitro confirmation of putative AMP activities through a highly efficient system of recombinant AMP prokaryotic expression.

Results

In total, 35,788 were generated from cDNA libraries of pathogen-challenged leaves and 187,272 ESTs from seeds of B. napus, and the 644,998 ESTs of B. napus were downloaded from the EST database of PlantGDB. They formed 201,200 unigenes. First, all the known AMPs from the AMP databank (APD2 database) were individually queried against all the unigenes using the BLASTX program. A total of 972 unigenes that matched the 27 known AMP sequences in APD2 database were extracted and annotated using Blast2GO program. Among these unigenes, 237 unigenes from B. napus pathogen-challenged leaves had the highest ratio (1.15 %) in this unigene dataset, which is 13 times that of the unigene datasets of B. napus seeds (0.09 %) and 2.3 times that of the public EST dataset. About 87 % of each EST library was lipid-transfer protein (LTP) (32 % of total unigenes), defensin, histone, endochitinase, and gibberellin-regulated proteins. The most abundant unigenes in the leaf library were endochitinase and defensin, and LTP and histone in the pub EST library. After masking of the repeat sequence, 606 peptides that were orthologous matched to different AMP families were found. The phylogeny and conserved structural motifs of seven AMPs families were also analysed. To investigate the antimicrobial activities of the predicted peptides, 31 potential AMP genes belonging to different AMP families were selected to test their antimicrobial activities after bioinformatics identification. The AMP genes were all optimized according to Escherichia coli codon usage and synthetized through one-step polymerase chain reaction method. The results showed that 28 recombinant AMPs displayed expected antimicrobial activities against E. coli and Micrococcus luteus and Sclerotinia sclerotiorum strains.

Conclusion

The study not only significantly expanded the number of known/predicted peptides, but also contributed to long-term plant genetic improvement for increased resistance to diverse pathogens of B.napus. These results proved that the high-throughput method developed that combined an in silico procedure with a recombinant AMP prokaryotic expression system is considerably efficient for identification of new AMPs from genome or EST sequence databases.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1849-x) contains supplementary material, which is available to authorized users.     

Reduced Susceptibility of Brassica napus to Pratylenchus neglectus in Plants with Elevated Root Levels of 2-Phenylethyl Glucosinolate

The effect of canola (Brassica napus) as a crop suppressive to Pratylenchus neglectus is in part due to the release of nematicidal isothiocyanates, particularly 2-phenylethyl isothiocyanate, from degrading root tissues. However, many cultivars of canola are relatively susceptible to P. neglectus and will fail to reduce soil populations of the nematode. A survey of B. napus accessions and closely related species revealed limited scope to decrease the susceptibility of canola through conventional intercrossing. Susceptibility to P. neglectus was not related to the total glucosinolate levels, but there were signifi- cant, negative correlations (r = -0.619, -0.517; P < 0.001) between root levels of 2-phenylethyl glucosinolate (isothiocyanate precursor) and plant susceptibility to P. neglectus: plants containing more than a certain threshold level of 2-phenylethyl glucosinolate showed reduced susceptibility to the nematode. Selection for high root levels of 2-phenylethyl glucosinolate should reduce the susceptibility of the plants during the growing season while also increasing the nematicidal impact of the degrading root tissues, thereby improving the suppressive benefits of the crop when used in rotation with cereals.     

Sources of N uptake by wheat (Triticum aestivum L.) and N transformations in soil treated with a nitrification inhibitor (nitrapyrin)

Rates of N uptake by spring wheat as ammonium and as nitrate, and rates of nitrification, gross N immobilization and gross N mineralization were measured in a pot experiment during 84 days of growth in a clay soil. Soil treatments included an unfertilized control and addition of ¹⁵NH₄NO₃ or NH₄ ¹⁵NO₃ in the absence and presence of N-serve 24E. Incorporation of ammonium into the soil organic N pool was considerably higher in the presence compared to the absence of nitrapyrin, but the processes contributing to this effect could not be positively identified. Both dry matter and grain yield as well as N uptake by wheat were enhanced in the presence of the inhibitor in N fertilized soil, despite the increased immobilization of N. On the other hand, inhibitor application had a detrimental effect on yield and N uptake by wheat in unfertilized soil. Both ammonium and nitrate forms of inorganic N were absorbed by wheat, but nitrate uptake was dominant in the absence of the inhibitor. The uptake of N as ammonium was higher and the uptake of N as nitrate was less, both in absolute and proportional terms, in the presence compared to the absence of inhibitor. In addition, the proportion of N taken up as ammonium was higher than the proportion of N as ammonium in the available N pool up to day 56 in the inhibitor treatment, which indicated a preference for ammonium uptake by wheat. Evidence was obtained which suggested that several factors may have contributed to the positive response of wheat to inhibitor application in N fertilized soil, including reduced N losses, higher gross N mineralization and a physiological response due to the proportional increase in uptake of inorganic N as ammonium.     

Quantitative trait loci affecting seed mineral concentrations in Brassica napus grown with contrasting phosphorus supplies

MethodsA population of 124 recombinant inbred lines derived from a cross between P-inefficient ‘B104-2’ and P-efficient ‘Eyou Changjia’ was used for phenotypic investigation and QTL analysis. Two-year field trials were conducted with two P treatments. Concentrations of mineral elements (P, Ca, Mg, Fe, Zn, Cu and Mn) in seeds were determined and QTLs were identified by composite interval mapping.ConclusionsThe accumulation of mineral elements in seeds is controlled by multiple genes. Common physiological and molecular mechanisms could be involved in the accumulation of several mineral elements, and genes involved in these processes in B. napus are suggested. These results offer insights to the genetic basis of seed mineral accumulation across different P levels in B. napus.     

Isolation and characterization of a polygalacturonase gene highly expressed in Brassica napus pollen

A cDNA clone, Sta 44-4, corresponding to a mRNA highly expressed in Brassica napus cv. Westar stamens, was isolated by differential screening and characterized. Northern blot and in situ analyses demonstrated that Sta 44-4 is synthesized in pollen beginning at the late uninucleate stage and reaches a maximum in trinucleate microspores. Sta 44-4 displayed significant sequence similarity to known pollen polygalacturonase genes. The B. napus pollen polygalacturonase gene was shown to be part of a small gene family and to display some polymorphism among different cultivars.     

The influence of nitrogen concentration and ammonium/nitrate ratio on N-uptake,mineral composition and yield of citrus

In short-term water culture experiments with different ¹⁵N labeled ammonium or nitrate concentrations, citrus seedlings absorbed NH₄ ⁺ at a higher rate than NO₃ ^–. Maximum NO₃ ^– uptake by the whole plant occurred at 120 mg L^–1 NO₃ ^–-N, whereas NH₄ ⁺ absorption was saturated at 240 mg L^–1 NH₄ ⁺-N. ¹⁵NH₄ ⁺ accumulated in roots and to a lesser degree in both leaves and stems. However, ¹⁵NO₃ ^– was mostly partitioned between leaves and roots.Adding increasing amounts of unlabeled NH₄ ⁺ (15–60 mg L^–1 N) to nutrient solutions containing 120 mg L^–1 N as ¹⁵N labeled nitrate reduced ¹⁵NO₃ ^– uptake. Maximum inhibition of ¹⁵NO₃ ^– uptake was about 55% at 2.14 mM NH₄ ⁺ (30 mg L^–1 NH₄ ⁺-N) and it did not increase any further at higher NH₄ ⁺ proportions.In a long-term experiment, the effects of concentration and source of added N (NO₃ ^– or NH₄ ⁺) on nutrient concentrations in leaves from plants grown in sand were evaluated. Leaf concentration of N, P, Mg, Fe and Cu were increased by NH₄ ⁺ versus NO₃ ^– nutrition, whereas the reverse was true for Ca, K, Zn and Mn.The effects of different NO₃ ^–-N:NH₄ ⁺-N ratios (100:0, 75:25, 50:50, 25:75 and 0:100) at 120 mg L^–1 total N on leaf nutrient concentrations, fruit yield and fruit characteristics were investigated in another long-term experiment with plants grown in sand cultures. Nitrogen concentrations in leaves were highest when plants were provided with either NO₃ ^– or NH₄ ⁺ as a sole source of N. Lowest N concentration in leaves was found with a 75:25 NO₃ ^–-N/NH₄ ⁺-N ratio. With increasing proportions of NH₄ ⁺ in the N supply, leaf nutrients such as P, Mg, Fe and Cu increased, whereas Ca, K, Mn and Zn decreased. Yield in number of fruits per tree was increased significantly by supplying all N as NH₄ ⁺, although fruit weight was reduced. The number of fruits per tree was lowest with the 75:25 NO₃ ^–-N:NH₄ ⁺-N ratio, but in this treatment fruits reached their highest weight. Rind thickness, juice acidity, and colour index of fruits decreased with increasing NH₄ ⁺ in the N supply, whereas the % pulp and maturity index increased. Percent of juice in fruits and total soluble solids were only slightly affected by NO₃ ^–:NH₄ ⁺ ratio.     

Changes in fitness-associated traits due to the stacking of transgenic glyphosate resistance and insect resistance in Brassica napus L

Increasingly, genetically modified crops are being developed to express multiple 'stacked' traits for different types of transgenes, for example, herbicide resistance, insect resistance, crop quality and tolerance to environmental stresses. The release of crops that express multiple traits could result in ecological changes in weedy environments if feral crop plants or hybrids formed with compatible weeds results in more competitive plants outside of agriculture. To examine the effects of combining transgenes, we developed a stacked line of canola (Brassica napus L.) from a segregating F(2) population that expresses both transgenic glyphosate resistance (CP4 EPSPS) and lepidopteran insect resistance (Cry1Ac). Fitness-associated traits were evaluated between this stacked genotype and five other Brassica genotypes in constructed mesocosm plant communities exposed to insect herbivores (Plutella xylostella L.) or glyphosate-drift. Vegetative biomass, seed production and relative fecundity were all reduced in stacked trait plants when compared with non-transgenic plants in control treatments, indicating potential costs of expressing multiple transgenes without selection pressure. Although costs of the transgenes were offset by selective treatment, the stacked genotype continued to produce fewer seeds than either single transgenic line. However, the increase in fitness of the stacked genotype under selective pressure contributed to an increased number of seeds within the mesocosm community carrying unselected, hitchhiking transgenes. These results demonstrate that the stacking of these transgenes in canola results in fitness costs and benefits that are dependent on the type and strength of selection pressure, and could also contribute to changes in plant communities through hitchhiking of unselected traits.