André Laroche

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.

Genotyping-by-sequencing (GBS), an ultimate marker-assisted selection (MAS) tool to accelerate plant breeding

Marker-assisted selection (MAS) refers to the use of molecular markers to assist phenotypic selections in crop improvement. Several types of molecular markers, such as single nucleotide polymorphism (SNP), have been identified and effectively used in plant breeding. The application of next-generation sequencing (NGS) technologies has led to remarkable advances in whole genome sequencing, which provides ultra-throughput sequences to revolutionize plant genotyping and breeding. To further broaden NGS usages to large crop genomes such as maize and wheat, genotyping-by-sequencing (GBS) has been developed and applied in sequencing multiplexed samples that combine molecular marker discovery and genotyping. GBS is a novel application of NGS protocols for discovering and genotyping SNPs in crop genomes and populations. The GBS approach includes the digestion of genomic DNA with restriction enzymes followed by the ligation of barcode adapter, PCR amplification and sequencing of the amplified DNA pool on a single lane of flow cells. Bioinformatic pipelines are needed to analyze and interpret GBS datasets. As an ultimate MAS tool and a cost-effective technique, GBS has been successfully used in implementing genome-wide association study (GWAS), genomic diversity study, genetic linkage analysis, molecular marker discovery and genomic selection under a large scale of plant breeding programs.

Metabolic control analysis is helpful for informed genetic manipulation of oilseed rape (Brassica napus) to increase seed oil content

Top–down control analysis (TDCA) is a useful tool for quantifying constraints on metabolic pathways that might be overcome by biotechnological approaches. Previous studies on lipid accumulation in oilseed rape have suggested that diacylglycerol acyltransferase (DGAT), which catalyses the final step in seed oil biosynthesis, might be an effective target for enhancing seed oil content. Here, increased seed oil content, increased DGAT activity, and reduced substrate:product ratio are demonstrated, as well as reduced flux control by complex lipid assembly, as determined by TDCA in Brassica napus (canola) lines which overexpress the gene encoding type-1 DGAT. Lines overexpressing DGAT1 also exhibited considerably enhanced seed oil content under drought conditions. These results support the use of TDCA in guiding the rational selection of molecular targets for oilseed modification. The most effective lines had a seed oil increase of 14%. Moreover, overexpression of DGAT1 under drought conditions reduced this environmental penalty on seed oil content.

Molecular modification of triacylglycerol accumulation by over-expression of DGAT1 to produce canola with increased seed oil content under field conditions

The final step in the Kennedy pathway for seed oil synthesis is catalyzed by an acyl-CoA-dependent diacylglycerol acyltransferase, DGAT1 (EC. 2.3.1.20). We have cloned DGAT1 genes from both Arabido...

Differential expression of proteins in response to the interaction between the pathogen Fusarium graminearum and its host, Hordeum vulgare

Using proteomic techniques, a study aimed at isolating and identifying proteins associated with resistance to fusarium head blight (FHB) was conducted on six barley genotypes of varying resistance. At anthesis, barley spikelets were point inoculated with Fusarium graminearum macroconidial suspensions or mock inoculum. In total, 43 acidic protein spots out of 600 were detected 3 days postinoculation to be differentially expressed due to FHB and were identified. Identification of proteins responsive to FHB included those associated with oxidative burst and oxidative stress response, such as malate dehydrogenase and peroxidases, and pathogenesis‐related (PR). An increase in abundance of PR‐3 or PR‐5 could be associated with the resistant genotypes CI4196, Svansota, and Harbin, as well as the intermediate resistant genotype CDC Bold. On the contrary, the susceptible genotype Stander showed a decrease in abundance of these acidic PR‐proteins. In the susceptible and intermediate resistant genotypes Stander and CDC Bold, as well as CI4196, the increased abundance of proteins associated with an oxidative response might have prepared the terrain for saprophytic fungal invasion. On the contrary, in the resistant sources Harbin and Svansota we did not observed change in abundance of these proteins. Not a single significant change in acidic protein abundance could be detected in Chevron. Three distinct response patterns are reported from these six barley genotypes.

Characterization of cDNAs encoding diacylglycerol acyltransferase from cultures of Brassica napus and sucrose-mediated induction of enzyme biosynthesis.

cDNAs encoding acyl-CoA:diacylglycerol acyltransferase (DGAT, EC 2.3.1.20), designated BnDGAT1 and BnDGAT2, were obtained from a microspore-derived cell suspension culture of oilseed rape (Brassica napus L. cv Jet Neuf). BnDGAT2 shares a very high level of identity with BnDGAT1, but is a smaller protein lacking the relatively hydrophilic N-terminal segment found in BnDGAT1. Both transcripts were produced in the cell suspension cultures and the cDNAs were functionally expressed in transformed yeast (Pichia pastoris) cells. Sucrose-mediated changes in triacylglycerol (TAG) metabolism and expression of BnDGAT1 were examined in the cell suspension cultures following transfer of cells from media containing 6% (w/v) sucrose to media containing 14% sucrose. TAG content and DGAT activity of the cells increased transiently within the first 12 h after transfer (HAT). The rapid decline in TAG content observed at 12 HAT was inversely related to an increase in TAG lipase (EC 3.1.1.3) activity. The transient increases in TAG content and DGAT activity correlated with the elevated amounts of BnDGAT1 polypeptide. Transcript levels were also induced, but levels of mRNA encoding BnDGAT1 were not tightly correlated with DGAT activity and amount of polypeptide suggesting some control of expression at the post-transcriptional level. In general, the rapid changes in TAG content were closely associated with the changes in the activity of TAG-metabolizing enzymes and expression of BnDGAT1.

Molecular characterization of the genome composition of partial amphiploids derived from Triticum aestivum×Thinopyrum ponticum and T. aestivum×Th. intermedium as sources of resistance to wheat streak mosaic virus and its vector, Aceria tosichella

Abstract Wheat streak mosaic virus (WSMV), vectored by the wheat curl mite (WCM), is one of the most important viral diseases of wheat (Triticum aestivum) in the world. Genetic resistance to WSMV and the WCM does not exist in wheat. Resistance to WSMV and the WCM was evaluated in five different partial amphiploids namely Agrotana, OK7211542, ORRPX, Zhong 5 and TAF 46, which were derived from hybrids of wheat with decaploid Thinopyrum ponticum or with hexaploid Th. intermedium. Agrotana was shown to be immune to WSMV and the WCM; the other four partial amphiploids were susceptible to the WCM. Genomic in situ hybridization (GISH) using genomic DNA probes from Th. elongatum (EE, 2n=14), Th. bessarabicum (JJ, 2n=14), Pseudoroegneria strigosa (SS, 2n=14) and T. aestivum (AABBDD, 2n=42) demonstrated that three of the partial amphiploids, Agrotana, OK7211542 and ORRPX, have almost identical alien genome constitutions: all have 16 alien chromosomes, with 8 chromosomes being closely related to the Js genome and 8 chromosomes belonging to the E or J genomes and no evidence of any S-genome chromosomes. GISH confirmed that the alien genomes of Agrotana and OK7211542, like ORRPX, were all derived from Th. ponticum, and not from Th. intermedium. However, in contrast to Agrotana, ORRPX and OK7211542 were susceptible to the WCM and WSMV. The partial amphiploid Zhong 5 had a reconstituted alien genome composed of 4 S-and 4 Js-genome chromosomes of Th. intermedium with 6 translocated chromosomes between the S and Js genomes. This line was highly resistant to WSMV, but was susceptible to the WCM. TAF 46, which contained a synthetic genome consisting of 3 pairs of S-genome chromosomes and 4 pairs of E- or J-genome chromosomes in addition to the 21 pairs of wheat chromosomes, was susceptible to the WCM, but moderately resistant to WSMV. Agrotana offers great potential for transferring WSMV and WCM resistance into wheat.

Characterization and Antifungal Properties of Wheat Nonspecific Lipid Transfer Proteins

This study simultaneously considered the phylogeny, fatty acid binding ability, and fungal toxicity of a large number of monocot nonspecific lipid transfer proteins (ns-LTP). Nine novel full-length wheat ns-LTP1 clones, all possessing coding sequences of 348 bp, isolated from abiotic- and biotic-stressed cDNA libraries from aerial tissues, exhibited highly conserved coding regions with 78 to 99 and 71 to 100% identity at the nucleotide and amino acid levels, respectively. Phylogenetic analyses revealed two major ns-LTP families in wheat. Eight wheat ns-LTP genes from different clades were cloned into the expression vector pPICZalpha and transformed into Pichia pastoris. Sodium dodecyl sulfate polyacrylamide gel electrophoresis, Western blotting, and in vitro lipid binding activity assay confirmed that the eight ns-LTP were all successfully expressed and capable of in vitro binding fatty acid molecules. A comparative in vitro study on the toxicity of eight wheat ns-LTP to mycelium growth or spore germination of eight wheat pathogens and three nonwheat pathogens revealed differential toxicities among different ns-LTP. Values indicating 50% inhibition of fungal growth or spore germination of three selected ns-LTP against six fungi ranged from 1 to 7 microM. In vitro lipid-binding activity of ns-LTP was not correlated with their antifungal activity. Using the fluorescent probe SYTOX Green as an indicator of fungal membrane integrity, the in vitro toxicity of wheat ns-LTP was associated with alteration in permeability of fungal membranes.

NBS-LRR sequence family is associated with leaf and stripe rust resistance on the end of homoeologous chromosome group 1S of wheat

Abstract A detailed RFLP map was constructed of the distal end of the short arm of chromosome 1D of Aegilops tauschii and wheat. At least two unrelated resistance-gene analogs (RGAs) mapped close to known leaf rust resistance genes (Lr21 and Lr40) located distal to seed storage protein genes on chromosome 1DS. One of the two RGA clones, which was previously shown to be part of a candidate gene for stripe rust resistance (Yr10) located within the homoeologous region on 1BS, identified at least three gene family members on chromosome 1DS of Ae. tauschii. One of the gene members co-segregated with the leaf rust resistance genes, Lr21 and Lr40, in Ae. tauschii and wheat segregating families. Hence, a RGA clone derived from a candidate gene for stripe rust resistance located on chromosome 1BS detected candidate genes for leaf rust resistance located in the corresponding region on 1DS of wheat.

Molecular cytogenetic characterization of Thinopyrum intermedium-derived wheat germplasm specifying resistance to wheat streak mosaic virus

Abstract  Thinopyrum intermedium is a promising source of resistance to wheat streak mosaic virus (WSMV), a devastating disease of wheat. Three wheat germplasm lines possessing resistance to WSMV, derived from Triticum aestivum×Th. intermedium crosses, are analyzed by C-banding and genomic in situ hybridization (GISH) to determine the amount and location of alien chromatin in the transfer lines. Line CI15092 was confirmed as a disomic substitution line in which wheat chromosome 4A was replaced by Th. intermedium chromosome 4Ai?2. The other two lines, CI17766 and A29-13-3, carry an identical Robertsonian translocation chromosome in which the complete short arm of chromosome 4Ai?2 was transferred to the long arm of wheat chromosome 4A. Fluorescence in situ hybridization (FISH) using ABD genomic DNA from wheat as a probe and S genomic DNA from Pseudoroegneria stipifolia as the blocker, and vice versa, revealed that the entire short arm of the translocation was derived from the short arm of chromosome 4Ai?2 and the breakpoint was located at the centromere. Chromosomal arm ratios (L/S) of 2.12 in CI17766 and 2.15 in A29-13-3 showed that the translocated chromosome is submetacentric. This translocated chromosome is designated as T4AL ⋅ 4Ai?2S as suggested by Friebe et al. (1991).