Peter B. E. McVetty

Increasing the flow of carbon into seed oil

The demand for vegetable oils for food, fuel (bio-diesel) and bio-product applications is increasing rapidly. In Canada alone, it is estimated that a 50 to 75% increase in canola oil production will be required to meet the demand for seed oil in the next 7-10years. Plant breeding and genetics have demonstrated that seed oil content is a quantitative trait based on a number of contributing factors including embryo genetic effects, cytoplasmic effects, maternal genetic effects, and genotype-environment interactions. Despite the involvement of numerous quantitative trait loci in determining seed oil content, genetic engineering to over-express/repress specific genes encoding enzymes and other proteins involved in the flow of carbon into seed oil has led to the development of transgenic lines with significant increases in seed oil content. Proteins encoded by these genes include enzymes catalyzing the production of building blocks for oil assembly, enzymes involved in oil assembly, enzymes regulating metabolic carbon partitioning between oil, carbohydrate and secondary metabolite fractions, and transcription factors which orchestrate metabolism at a more general level.

Map-based cloning and characterization of a gene controlling hairiness and seed coat color traits in Brassica rapa

A glabrous, yellow-seeded doubled haploid (DH) line and a hairy, black-seeded DH line in Chinese cabbage (B. rapa) were used as parents to develop a DH line population that segregated for both hairiness and seed coat color traits. The data showed that both traits completely co-segregated each other, suggesting that one Mendelian locus controlled both hairiness and seed coat color in this population. A fine genetic map was constructed and a SNP marker that was located inside a Brassica ortholog of TRANSPARENT TESTA GLABRA 1 (TTG1) in Arabidopsis showed complete linkage to both the hairiness and seed coat color gene, suggesting that the BrassicaTTG1 ortholog shared the same gene function as its Arabidopsis counterpart. Further sequence analysis of the alleles from hairless, yellow-seeded and hairy, black-seeded DH lines in B. rapa showed that a 94-base deletion was found in the hairless, yellow-seeded DH lines. A nonfunctional truncated protein in the hairless, yellow-seeded DH lines in B. rapa was suggested by the coding sequence of the TTG1 ortholog. Both of the TTG1 homologs from the black and yellow seeded B. rapa lines were used to transform an Arabidopsis ttg1 mutant and the results showed that the TTG1 homolog from the black seeded B. rapa recovered the Arabidopsis ttg1 mutant, while the yellow seeded homolog did not, suggesting that the deletion in the Brassica TTG1 homolog had led to the yellow seeded natural mutant. This was the first identified gene in Brassica species that simultaneously controlled both hairiness and seed coat color traits.

Effectiveness of border areas in confining the spread of transgenic Brassica napus pollen

The development of transgenic Brassica napus L. cultivars requires field trials in agricultural settings. For field testing of transgenic constructs that have not been granted full environmental release, current Canadian Government regulations require either large isolation zones (200 m) or 10 m wide borders of synchronously flowering, non-transgenic B. napus to contain transgenic pollen. To investigate the effectiveness of border areas in containing transgenic B. napus pollen, border areas 15 to 30 m wide were planted around 30 m × 60 m central plots of bromoxynil-herbicide-resistant transgenic B. napus strains. Four field trials were conducted at Carman and Winnipeg, Manitoba, Canada in 1994 and 1995. Seed samples were harvested from the border area at 0, 2.5, 5, 10, and 15 m for the four cardinal directions and additionally at 20, 25, and 30 m for two cardinal directions. These seed samples were planted in the field in 1995 and 1996 and the seedlings screened for the presence of bromoxynil-resistant pl...

Development of SRAP, SNP and Multiplexed SCAR molecular markers for the major seed coat color gene in Brassica rapa L.

Seed coat color inheritance in B. rapa was studied in F1, F2, F3, and BC1 progenies from a cross of a Canadian brown-seeded variety ‘SPAN’ and a Bangladeshi yellow sarson variety ‘BARI-6’. A pollen effect was found when the yellow sarson line was used as the maternal parent. Seed coat color segregated into brown, yellow-brown and bright yellow classes. Segregation was under digenic control where the brown or yellow-brown color was dominant over bright yellow seed coat color. A sequence related amplified polymorphism (SRAP) marker linked closely to a major seed coat color gene (Br1/br1) was developed. This dominant SRAP molecular marker was successfully converted into single nucleotide polymorphism (SNP) markers and sequence characterized amplification region (SCAR) markers after the extended flanking sequence of the SRAP was obtained with chromosome walking. In total, 24 SNPs were identified with more than 2-kb sequence. A 12-bp deletion allowed the development of a SCAR marker linked closely to the Br1 gene. Using the five-fluorescence dye set supplied by ABI, four labeled M13 primers were integrated with different SCAR primers to increase the throughput of SCAR marker detection. Using multiplexed SCAR markers targeting insertions and deletions in a genome shows great potential for marker assisted selection in plant breeding.

Stereospecific analyses of seed triacylglycerols from high-erucic acid brassicaceae: Detection of erucic acid at thesn-2 position inBrassica oleracea L. Genotypes

Stereospecific analyses of triacylglycerols from selected high-erucic acid breeding lines or cultivars ofBrassica napus L. andB. oleracea L. have been performed. Initial lipase screening revealed that while allB. napus lines contained little or no erucic acid at thesn-2 position, several of theB. oleracea lines had significant proportions of erucic acid at this position. Detailed stereospecific analyses were performed on the triacylglycerols from these lines by using a Grignard-based deacylation, conversion of thesn-1,sn-2 andsn-3 monoacylglycerols to their di-dinitrophenyl urethane (DNPU) derivatives, resolution of the di-DNPU-monoacylglycerols (MAGs) by high-performance liquid chromatography on a chiral column, transmethylation of eachsn-di-DNPU MAG fraction and analysis of the resulting fatty acid methyl esters by gas chromatography. The findings unequivocally demonstrate for the first time that, within the Brassicaceae, there existsB. oleracea germplasm containing seed oils with substantial erucic acid (30–35 mol%) at thesn-2 position. This has important implications for biotechnology and breeding efforts designed to increase the levels of erucic acid in rapeseed beyond 66 mol% to supply strategic industrial feedstocks. In the first instance, the germplasm will be of direct use in retrieving a gene encoding aBrassica lyso-phosphatidic acid acyltransferase with an affinity for erucoyl-CoA. In a breeding program, the germplasm offers promise for the introduction of this trait intoB. napus by interspecific hybridization and embryo rescue.

High throughput genome-specific and gene-specific molecular markers for erucic acid genes in Brassica napus (L.) for marker-assisted selection in plant breeding

A single base change in the Bn-FAE1.1 gene in the A genome and a two-base deletion in the Bn-FAE1.2 gene in the C genome produce the nearly zero content of erucic acid observed in canola. A BAC clone anchoring Bn-FAE1.1 from a B. rapa BAC library and a BAC clone anchoring Bn-FAE1.2 from a B. oleracea BAC library were used in this research. After sequencing the gene flanking regions, it was found that the dissimilarity of the flanking sequences of these two FAE1 homologs facilitated the design of genome-specific primers that could amplify the corresponding genome in allotetraploid B. napus. The two-base deletion in the C genome gene was detected as a sequence-characterized amplified region (SCAR) marker. To increase the throughput, one genome-specific primer was labeled with four fluorescence dyes and combined with 20 different primers to produce PCR products with different fragment sizes. Eventually, a super pool of 80 samples was detected simultaneously. This dramatically reduces the cost of marker detection. The single base change in the Bn-FAE1.1 gene was detected as single nucleotide polymorphic (SNP) marker with an ABI SNaPshot kit. A multiplexing primer set was designed by adding a polyT to the 5′ primer end to increase SNP detection throughput through sample pooling. Furthermore, the Bn-FAE1.1 and Bn-FAE1.2 were integrated into the N8 and N13 linkage groups of our previously reported high-density sequence-related amplified polymorphism (SRAP) map, respectively. There were 124 SRAP markers in a N8 bin in which the Bn-FAE1.1 gene-specific SCAR marker was located and 46 SRAP markers in a N13 bin into which the Bn-FAE1.2 SNP marker was integrated. These three kinds of high throughput molecular markers have been successfully implemented in our canola/rapeseed breeding programs.

Inheritance of seed coat color genes in Brassica napus (L.) and tagging the genes using SRAP, SCAR and SNP molecular markers

Seed coat color inheritance in Brassica napus was studied in F1, F2, F3 and backcross progenies from crosses of five black seeded varieties/lines to three pure breeding yellow seeded lines. Maternal inheritance was observed for seed coat color in B. napus, but a pollen effect was also found when yellow seeded lines were used as the female parent. Seed coat color segregated from black to dark brown, light brown, dark yellow, light yellow, and yellow. Seed coat color was found to be controlled by three genes, the first two genes were responsible for black/brown seed coat color and the third gene was responsible for dark/light yellow seed coat color in B. napus. All three seed coat color alleles were dominant over yellow color alleles at all three loci. Sequence related amplified polymorphism (SRAP) was used for the development of molecular markers co-segregating with the seed coat color genes. A SRAP marker (SA12BG18388) tightly linked to one of the black/brown seed coat color genes was identified in the F2 and backcross populations. This marker was found to be anchored on linkage group A9/N9 of the A-genome of B. napus. This SRAP marker was converted into sequence-characterized amplification region (SCAR) markers using chromosome-walking technology. A second SRAP marker (SA7BG29245), very close to another black/brown seed coat color gene, was identified from a high density genetic map developed in our laboratory using primer walking from an anchoring marker. The marker was located on linkage group C3/N13 of the C-genome of B. napus. This marker also co-segregated with the black/brown seed coat color gene in B. rapa. Based on the sequence information of the flanking sequences, 24 single nucleotide polymorphisms (SNPs) were identified between the yellow seeded and black/brown seeded lines. SNP detection and genotyping clearly differentiated the black/brown seeded plants from dark/light/yellow-seeded plants and also differentiated between homozygous (Y2Y2) and heterozygous (Y2y2) black/brown seeded plants. A total of 768 SRAP primer pair combinations were screened in dark/light yellow seed coat color plants and a close marker (DC1GA27197) linked to the dark/light yellow seed coat color gene was developed. These three markers linked to the three different yellow seed coat color genes in B. napus can be used to screen for yellow seeded lines in canola/rapeseed breeding programs.

A review of Brassica seed color

Rahman, M. and McVetty, P. B. E. 2011. A review of Brassica seed color. Can. J. Plant Sci. 91: 437-446. Canola oil has excellent fatty acid composition and low saturated fat levels, and canola meal has protein with excellent amino acid composition. Canola seed quality can be further improved by the development of higher oil, higher protein and lower fiber content germplasm through the development of yellow seeded lines. While there is no naturally occurring yellow seeded B. napus, yellow seeded mutants that have arisen in nature can be readily indentified in Brassica rapa, B. juncea and B. carinata species. Brassica napus is widely cultivated in Asia, Australia, Europe and North America. Yellow seed in Brassica species is associated with seed that has higher oil and protein content and lower fiber content. Because of these seed quality advantages of yellow seeded lines, plant breeders around the world have been attempting to develop yellow seeded B. napus genotypes using crosses involving naturally occurring yellow seeded Brassica species. Seed color in B. rapa is controlled by two genes. Two duplicate genes are responsible for seed color in B. juncea. In B. carinata, one repressor gene represses the seed color gene resulting in yellow seed, while the absence of the repressor gene results in brown seed. Several yellow seeded B. napus genotypes have been developed and in most cases three genes are reported as being are responsible for seed color. Numerous different molecular markers for seed color genes in B. rapa, B. juncea and B. napus have been developed for use in marker-assisted selection in plant-breeding programs. These molecular markers can also be used to clone the Brassica seed color gene(s) and then create transgenic yellow seeded B. napus genotypes. This review summarizes past and current research on Brassica seed color breeding, genetics and genomics/biotechnology.

Integration of Solexa sequences on an ultradense genetic map in Brassica rapa L.

BackgroundSequence related amplified polymorphism (SRAP) is commonly used to construct high density genetic maps, map genes and QTL of important agronomic traits in crops and perform genetic diversity analysis without knowing sequence information. To combine next generation sequencing technology with SRAP, Illuminas Solexa sequencing was used to sequence tagged SRAP PCR products.ResultsThree sets of SRAP primers and three sets of tagging primers were used in 77,568 SRAP PCR reactions and the same number of tagging PCR reactions respectively to produce a pooled sample for Illuminas Solexa sequencing. After sequencing, 1.28 GB of sequence with over 13 million paired-end sequences was obtained and used to match Solexa sequences with their corresponding SRAP markers and to integrate Solexa sequences on an ultradense genetic map. The ultradense genetic bin map with 465 bins was constructed using a recombinant inbred (RI) line mapping population in B. rapa. For this ultradense genetic bin map, 9,177 SRAP markers, 1,737 integrated unique Solexa paired-end sequences and 46 SSR markers representing 10,960 independent genetic loci were assembled and 141 unique Solexa paired-end sequences were matched with their corresponding SRAP markers. The genetic map in B. rapa was aligned with the previous ultradense genetic map in B. napus through common SRAP markers in these two species. Additionally, SSR markers were used to perform alignment of the current genetic map with other five genetic maps in B. rapa and B. napus.ConclusionWe used SRAP to construct an ultradense genetic map with 10,960 independent genetic loci in B. rapa that is the most saturated genetic map ever constructed in this species. Using next generation sequencing, we integrated 1,878 Solexa sequences on the genetic map. These integrated sequences will be used to assemble the scaffolds in the B. rapa genome. Additionally, this genetic map may be used for gene cloning and marker development in B. rapa and B. napus.

Comparison of the performance of bromoxynil-resistant and susceptible near-isogenic populations of oilseed rape

Bromoxynil herbicide resistance is the newest type of broad-spectrum, non-selective herbicide resistance to be introduced into oilseed rape (Brassica napus L.). This herbicide resistance is conferred by a single transgene (the oxy gene), taken from a soil bacterium, which confers the ability to metabolize hydroxybenzonitrile herbicides such as bromoxynil. The level of resistance to bromoxynil herbicide in oilseed rape is high, but it is not known whether there are any performance changes associated with the oxy transgene or with the derived herbicide resistance. To determine if there are changes in performance related to the oxy transgene, or the derived herbicide resistance, two near-isogenic transgenic bromoxynil-resistant populations, with two different forms of the oxy gene, Westar 235 and Westar 237, were developed at the University of Manitoba, grown in the field in Manitoba for several years and evaluated for comparative performance. Westar 235 and Westar 237 near-isogenic populations, either spray...