Loci harboring genes with important role in drought and related abiotic stress responses in flax revealed by multiple GWAS models.

KEY MESSAGE: QTNs associated with drought tolerance traits and indices were identified in a flax mini-core collection through multiple GWAS models and phenotyping at multiple locations under irrigated and non-irrigated field conditions. Drought is a critical phenomenon challenging today's agricultural sector. Crop varieties adapted to moisture deficit are becoming vital. Flax can be greatly affected by limiting moisture conditions, especially during the early development and reproductive stages. Here, a mini-core collection comprising genotypes from more than 20 major growing countries was evaluated for 11 drought-related traits in irrigated and non-irrigated fields for 3 years. Heritability of the traits ranged from 44.7 to 86%. Six of the 11 traits showed significant phenotypic difference between irrigated and non-irrigated conditions. A genome-wide association study (GWAS) was performed for these six traits and their corresponding stress indices based on 106 genotypes and 12,316 single nucleotide polymorphisms (SNPs) using six multi-locus and one single-locus models. The SNPs were then assigned to 8050 linkage disequilibrium (LD) blocks to which a restricted two-stage multi-locus multi-allele GWAS was applied. A total of 144 quantitative trait nucleotides (QTNs) and 13 LD blocks were associated with at least one trait or stress index. Of these, 16 explained more than 15% of the genetic variance. Most large-effect QTN loci harbored gene(s) previously predicted to play role(s) in the associated traits. Genes mediating responses to abiotic stresses resided at loci associated with stress indices. Flax genes Lus10009480 and Lus10030150 that are predicted to encode WAX INDUCER1 and STRESS-ASSOCIATED PROTEIN (SAP), respectively, are among the important candidates detected. Accessions with multiple favorable alleles outperformed others for grain yield, thousand seed weight and fiber/biomass in non-irrigated conditions, suggesting their potential usefulness in breeding and genomic selection.

Genomic Prediction Accuracy of Seven Breeding Selection Traits Improved by QTL Identification in Flax.

Molecular markers are one of the major factors affecting genomic prediction accuracy and the cost of genomic selection (GS). Previous studies have indicated that the use of quantitative trait loci (QTL) as markers in GS significantly increases prediction accuracy compared with genome-wide random single nucleotide polymorphism (SNP) markers. To optimize the selection of QTL markers in GS, a set of 260 lines from bi-parental populations with 17,277 genome-wide SNPs were used to evaluate the prediction accuracy for seed yield (YLD), days to maturity (DTM), iodine value (IOD), protein (PRO), oil (OIL), linoleic acid (LIO), and linolenic acid (LIN) contents. These seven traits were phenotyped over four years at two locations. Identification of quantitative trait nucleotides (QTNs) for the seven traits was performed using three types of statistical models for genome-wide association study: two SNP-based single-locus (SS), seven SNP-based multi-locus (SM), and one haplotype-block-based multi-locus (BM) models. The identified QTNs were then grouped into QTL based on haplotype blocks. For all seven traits, 133, 355, and 1,208 unique QTL were identified by SS, SM, and BM, respectively. A total of 1420 unique QTL were obtained by SS+SM+BM, ranging from 254 (OIL, LIO) to 361 (YLD) for individual traits, whereas a total of 427 unique QTL were achieved by SS+SM, ranging from 56 (YLD) to 128 (LIO). SS models alone did not identify sufficient QTL for GS. The highest prediction accuracies were obtained using single-trait QTL identified by SS+SM+BM for OIL (0.929 ± 0.016), PRO (0.893 ± 0.023), YLD (0.892 ± 0.030), and DTM (0.730 ± 0.062), and by SS+SM for LIN (0.837 ± 0.053), LIO (0.835 ± 0.049), and IOD (0.835 ± 0.041). In terms of the number of QTL markers and prediction accuracy, SS+SM outperformed other models or combinations thereof. The use of all SNPs or QTL of all seven traits significantly reduced the prediction accuracy of traits. The results further validated that QTL outperformed high-density genome-wide random markers, and demonstrated that the combined use of single and multi-locus models can effectively identify a comprehensive set of QTL that improve prediction accuracy, but further studies on detection and removal of redundant or false-positive QTL to maximize prediction accuracy and minimize the number of QTL markers in GS are warranted.

Genome-Wide Association Study and Selection Signatures Detect Genomic Regions Associated with Seed Yield and Oil Quality in Flax.

A genome-wide association study (GWAS) was performed on a set of 260 lines which belong to three different bi-parental flax mapping populations. These lines were sequenced to an averaged genome coverage of 19× using the Illumina Hi-Seq platform. Phenotypic data for 11 seed yield and oil quality traits were collected in eight year/location environments. A total of 17,288 single nucleotide polymorphisms were identified, which explained more than 80% of the phenotypic variation for days to maturity (DTM), iodine value (IOD), palmitic (PAL), stearic, linoleic (LIO) and linolenic (LIN) acid contents. Twenty-three unique genomic regions associated with 33 quantitative trait loci (QTL) for the studied traits were detected, thereby validating four genomic regions previously identified. The 33 QTL explained 48⁻73% of the phenotypic variation for oil content, IOD, PAL, LIO and LIN but only 8⁻14% for plant height, DTM and seed yield. A genome-wide selective sweep scan for selection signatures detected 114 genomic regions that accounted for 7.82% of the flax pseudomolecule and overlapped with the 11 GWAS-detected genomic regions associated with 18 QTL for 11 traits. The results demonstrate the utility of GWAS combined with selection signatures for dissection of the genetic structure of traits and for pinpointing genomic regions for breeding improvement.

Genetic Variability of 27 Traits in a Core Collection of Flax (Linum usitatissimum L.).

Assessment of genetic variability of plant core germplasm is needed for efficient germplasm utilization in breeding improvement. A total of 391 accessions of a flax core collection, which preserves the variation present in the world collection of 3,378 accessions maintained by Plant Gene Resources of Canada (PGRC) and represents a broad range of geographical origins, different improvement statuses and two morphotypes, was evaluated in field trials in up to 8 year-location environments for 10 agronomic, eight seed quality, six fiber and three disease resistance traits. The large phenotypic variation in this subset was explained by morphotypes (22%), geographical origins (11%), and other variance components (67%). Both divergence and similarity between two basic morphotypes, namely oil or linseed and fiber types, were observed, whereby linseed accessions had greater thousand seed weight, seeds m-2, oil content, branching capability and resistance to powdery mildew while fiber accessions had greater straw weight, plant height, protein content and resistance to pasmo and fusarium wilt diseases, but they had similar performance in many traits and some of them shared common characteristics of fiber and linseed types. Weak geographical patterns within either fiber or linseed accessions were confirmed, but specific trait performance was identified in East Asia for fiber type, and South Asia and North America for linseed type. Relatively high broad-sense heritability was obtained for seed quality traits, followed by agronomic traits and resistance to powdery mildew and fusarium wilt. Diverse phenotypic and genetic variability in the flax core collection constitutes a useful resource for breeding.

Litter decay controlled by temperature, not soil properties, affecting future soil carbon.

Widespread global changes, including rising atmospheric CO2 concentrations, climate warming and loss of biodiversity, are predicted for this century; all of these will affect terrestrial ecosystem processes like plant litter decomposition. Conversely, increased plant litter decomposition can have potential carbon-cycle feedbacks on atmospheric CO2 levels, climate warming and biodiversity. But predicting litter decomposition is difficult because of many interacting factors related to the chemical, physical and biological properties of soil, as well as to climate and agricultural management practices. We applied 13 C-labelled plant litter to soil at ten sites spanning a 3500-km transect across the agricultural regions of Canada and measured its decomposition over five years. Despite large differences in soil type and climatic conditions, we found that the kinetics of litter decomposition were similar once the effect of temperature had been removed, indicating no measurable effect of soil properties. A two-pool exponential decay model expressing undecomposed carbon simply as a function of thermal time accurately described kinetics of decomposition. (R2  = 0.94; RMSE = 0.0508). Soil properties such as texture, cation exchange capacity, pH and moisture, although very different among sites, had minimal discernible influence on decomposition kinetics. Using this kinetic model under different climate change scenarios, we projected that the time required to decompose 50% of the litter (i.e. the labile fractions) would be reduced by 1-4 months, whereas time required to decompose 90% of the litter (including recalcitrant fractions) would be reduced by 1 year in cooler sites to as much as 2 years in warmer sites. These findings confirm quantitatively the sensitivity of litter decomposition to temperature increases and demonstrate how climate change may constrain future soil carbon storage, an effect apparently not influenced by soil properties.

QTL for fatty acid composition and yield in linseed (Linum usitatissimum L.).

KEY MESSAGE: The combined SSR-SNP map and 20 QTL for agronomic and quality traits will assist in marker assisted breeding as well as map-based cloning of key genes in linseed. Flax is an important nutraceutical crop mostly because it is a rich source of omega-3 fatty acids and antioxidant compounds. Canada is the largest producer and exporter of oilseed flax (or linseed), creating a growing need to improve crop productivity and quality. In this study, a genetic map was constructed based on selected 329 single nucleotide polymorphic markers and 362 simple sequence repeat markers using a recombinant inbred line population of 243 individuals from a cross between the Canadian varieties CDC Bethune and Macbeth. The genetic map consisted of 15 linkage groups comprising 691 markers with an average marker density of one marker every 1.9 cM. A total of 20 quantitative trait loci (QTL) were identified corresponding to 14 traits. Three QTL each for oleic acid and stearic acid, two QTL each for linoleic acid and iodine value and one each for palmitic acid, linolenic acid, oil content, seed protein, cell wall, straw weight, thousand seed weight, seeds per boll, yield and days to maturity were identified. The QTL for cell wall, straw weight, seeds per boll, yield and days to maturity all co-located on linkage group 4. Analysis of the candidate gene regions underlying the QTL identified proteins involved in cell wall and fibre synthesis, fatty acid biosynthesis as well as their metabolism and yield component traits. This study provides the foundation for assisting in map-based cloning of the QTL and marker assisted selection of a wide range of quality and agronomic traits in linseed and potentially fibre flax.

Functional characterization of flax fatty acid desaturase FAD2 and FAD3 isoforms expressed in yeast reveals a broad diversity in activity.

With 45 % or more oil content that contains more than 55 % alpha linolenic (LIN) acid, linseed (Linum usitatissimum L.) is one of the richest plant sources of this essential fatty acid. Fatty acid desaturases 2 (FAD2) and 3 (FAD3) are the main enzymes responsible for the Δ12 and Δ15 desaturation in planta. In linseed, the oilseed morphotype of flax, two paralogous copies, and several alleles exist for each gene. Here, we cloned three alleles of FAD2A, four of FAD2B, six of FAD3A, and seven of FAD3B into a pYES vector and transformed all 20 constructs and an empty construct in yeast. The transformants were induced in the presence of oleic (OLE) acid substrate for FAD2 constructs and linoleic (LIO) acid for FAD3. Conversion rates of OLE acid into LIO acid and LIO acid into LIN acid were measured by gas chromatography. Conversion rate of FAD2 exceeded that of FAD3 enzymes with FAD2B having a conversion rate approximately 10 % higher than FAD2A. All FAD2 isoforms were active, but significant differences existed between isoforms of both FAD2 enzymes. Two FAD3A and three FAD3B isoforms were not functional. Some nonfunctional enzymes resulted from the presence of nonsense mutations causing premature stop codons, but FAD3B-C and FAD3B-F seem to be associated with single amino acid changes. The activity of FAD3A-C was more than fivefold greater than the most common isoform FAD3A-A, while FAD3A-F was fourfold greater. Such isoforms could be incorporated into breeding lines to possibly further increase the proportion of LIN acid in linseed.

Association mapping of seed quality traits using the Canadian flax (Linum usitatissimum L.) core collection.

KEY MESSAGE: The identification of stable QTL for seed quality traits by association mapping of a diverse panel of linseed accessions establishes the foundation for assisted breeding and future fine mapping in linseed. Linseed oil is valued for its food and non-food applications. Modifying its oil content and fatty acid (FA) profiles to meet market needs in a timely manner requires clear understanding of their quantitative trait loci (QTL) architectures, which have received little attention to date. Association mapping is an efficient approach to identify QTL in germplasm collections. In this study, we explored the quantitative nature of seed quality traits including oil content (OIL), palmitic acid, stearic acid, oleic acid, linoleic acid (LIO) linolenic acid (LIN) and iodine value in a flax core collection of 390 accessions assayed with 460 microsatellite markers. The core collection was grown in a modified augmented design at two locations over 3 years and phenotypic data for all seven traits were obtained from all six environments. Significant phenotypic diversity and moderate to high heritability for each trait (0.73-0.99) were observed. Most of the candidate QTL were stable as revealed by multivariate analyses. Nine candidate QTL were identified, varying from one for OIL to three for LIO and LIN. Candidate QTL for LIO and LIN co-localized with QTL previously identified in bi-parental populations and some mapped nearby genes known to be involved in the FA biosynthesis pathway. Fifty-eight percent of the QTL alleles were absent (private) in the Canadian cultivars suggesting that the core collection possesses QTL alleles potentially useful to improve seed quality traits. The candidate QTL identified herein will establish the foundation for future marker-assisted breeding in linseed.

The potential of pale flax as a source of useful genetic variation for cultivated flax revealed through molecular diversity and association analyses.

Pale flax (Linum bienne Mill.) is the wild progenitor of cultivated flax (Linum usitatissimum L.) and represents the primary gene pool to broaden its genetic base. Here, a collection of 125 pale flax accessions and the Canadian flax core collection of 407 accessions were genotyped using 112 genome-wide simple sequence repeat markers and phenotyped for nine traits with the aim of conducting population structure, molecular diversity and association mapping analyses. The combined population structure analysis identified two well-supported major groups corresponding to pale and cultivated flax. The L. usitatissimum convar. crepitans accessions most closely resembled its wild progenitor, both having dehiscent capsules. The unbiased Nei's genetic distance (0.65) confirmed the strong genetic differentiation between cultivated and pale flax. Similar levels of genetic diversity were observed in both species, albeit 430 (48 %) of pale flax alleles were unique, in agreement with their high genetic differentiation. Significant associations were identified for seven and four traits in pale and cultivated flax, respectively. Favorable alleles with potentially positive effect to improve yield through yield components were identified in pale flax. The allelic frequencies of markers associated with domestication-related traits such as capsular dehiscence indicated directional selection with the most common alleles in pale flax being absent or rare in cultivated flax and vice versa. Our results demonstrated that pale flax is a potential source of novel variation to improve multiple traits in cultivated flax and that association mapping is a suitable approach to screening pale flax germplasm to identify favorable quantitative trait locus alleles.

Genomic regions underlying agronomic traits in linseed (Linum usitatissimum L.) as revealed by association mapping.

The extreme climate of the Canadian Prairies poses a major challenge to improve yield. Although it is possible to breed for yield per se, focusing on yield-related traits could be advantageous because of their simpler genetic architecture. The Canadian flax core collection of 390 accessions was genotyped with 464 simple sequence repeat markers, and phenotypic data for nine agronomic traits including yield, bolls per area, 1,000 seed weight, seeds per boll, start of flowering, end of flowering, plant height, plant branching, and lodging collected from up to eight environments was used for association mapping. Based on a mixed model (principal component analysis (PCA) + kinship matrix (K)), 12 significant marker-trait associations for six agronomic traits were identified. Most of the associations were stable across environments as revealed by multivariate analyses. Statistical simulation for five markers associated with 1000 seed weight indicated that the favorable alleles have additive effects. None of the modern cultivars carried the five favorable alleles and the maximum number of four observed in any accessions was mostly in breeding lines. Our results confirmed the complex genetic architecture of yield-related traits and the inherent difficulties associated with their identification while illustrating the potential for improvement through marker-assisted selection.

Genetic variation of six desaturase genes in flax and their impact on fatty acid composition.

Flax (Linum usitatissimum L.) is one of the richest plant sources of omega-3 fatty acids praised for their health benefits. In this study, the extent of the genetic variability of genes encoding stearoyl-ACP desaturase (SAD), and fatty acid desaturase 2 (FAD2) and 3 (FAD3) was determined by sequencing the six paralogous genes from 120 flax accessions representing a broad range of germplasm including some EMS mutant lines. A total of 6 alleles for sad1 and sad2, 21 for fad2a, 5 for fad2b, 15 for fad3a and 18 for fad3b were identified. Deduced amino acid sequences of the alleles predicted 4, 2, 3, 4, 6 and 7 isoforms, respectively. Allele frequencies varied greatly across genes. Fad3a, with 110 SNPs and 19 indels, and fad3b, with 50 SNPs and 5 indels, showed the highest levels of genetic variations. While most of the SNPs and all the indels were silent mutations, both genes carried nonsense SNP mutations resulting in premature stop codons, a feature not observed in sad and fad2 genes. Some alleles and isoforms discovered in induced mutant lines were absent in the natural germplasm. Correlation of these genotypic data with fatty acid composition data of 120 flax accessions phenotyped in six field experiments revealed statistically significant effects of some of the SAD and FAD isoforms on fatty acid composition, oil content and iodine value. The novel allelic variants and isoforms identified for the six desaturases will be a resource for the development of oilseed flax with unique and useful fatty acid profiles.

Integrated consensus genetic and physical maps of flax (Linum usitatissimum L.).

Three linkage maps of flax (Linum usitatissimum L.) were constructed from populations CDC Bethune/Macbeth, E1747/Viking and SP2047/UGG5-5 containing between 385 and 469 mapped markers each. The first consensus map of flax was constructed incorporating 770 markers based on 371 shared markers including 114 that were shared by all three populations and 257 shared between any two populations. The 15 linkage group map corresponds to the haploid number of chromosomes of this species. The marker order of the consensus map was largely collinear in all three individual maps but a few local inversions and marker rearrangements spanning short intervals were observed. Segregation distortion was present in all linkage groups which contained 1-52 markers displaying non-Mendelian segregation. The total length of the consensus genetic map is 1,551 cM with a mean marker density of 2.0 cM. A total of 670 markers were anchored to 204 of the 416 fingerprinted contigs of the physical map corresponding to ~274 Mb or 74 % of the estimated flax genome size of 370 Mb. This high resolution consensus map will be a resource for comparative genomics, genome organization, evolution studies and anchoring of the whole genome shotgun sequence.

Simple sequence repeat marker development from bacterial artificial chromosome end sequences and expressed sequence tags of flax (Linum usitatissimum L.).

Flax is an important oilseed crop in North America and is mostly grown as a fibre crop in Europe. As a self-pollinated diploid with a small estimated genome size of ~370 Mb, flax is well suited for fast progress in genomics. In the last few years, important genetic resources have been developed for this crop. Here, we describe the assessment and comparative analyses of 1,506 putative simple sequence repeats (SSRs) of which, 1,164 were derived from BAC-end sequences (BESs) and 342 from expressed sequence tags (ESTs). The SSRs were assessed on a panel of 16 flax accessions with 673 (58 %) and 145 (42 %) primer pairs being polymorphic in the BESs and ESTs, respectively. With 818 novel polymorphic SSR primer pairs reported in this study, the repertoire of available SSRs in flax has more than doubled from the combined total of 508 of all previous reports. Among nucleotide motifs, trinucleotides were the most abundant irrespective of the class, but dinucleotides were the most polymorphic. SSR length was also positively correlated with polymorphism. Two dinucleotide (AT/TA and AG/GA) and two trinucleotide (AAT/ATA/TAA and GAA/AGA/AAG) motifs and their iterations, different from those reported in many other crops, accounted for more than half of all the SSRs and were also more polymorphic (63.4 %) than the rest of the markers (42.7 %). This improved resource promises to be useful in genetic, quantitative trait loci (QTL) and association mapping as well as for anchoring the physical/genetic map with the whole genome shotgun reference sequence of flax.

Transcript profiling and gene characterization of three fatty acid desaturase genes in high, moderate, and low linolenic acid genotypes of flax (Linum usitatissimum L.) and their role in linolenic acid accumulation.

Three genes encoding fatty acid desaturase 3 (fad3a, fad3b, and a novel fad3c) were cloned from four flax genotypes varying in linolenic acid content. Real-time PCR was used to quantify expression levels of the three fad3 genes during seed development. High amounts of both fad3a and fad3b transcripts were observed and reached their peak levels at 20 days after anthesis, except for fad3a from SP2047 where only low level expression was observed throughout seed development. Transcript accumulation of the novel fad3c gene was at similar background levels. The fatty acid composition was analysed for all genotypes and stages of development and compared with the fad3 gene expression patterns. α-Linolenic acid gradually accumulated during seed development, while linoleic acid was transient and decreased in M5791, UGG5-5, and AC McDuff. In contrast, the linolenic acid present in the early stages of development nearly completely disappeared in SP2047, while linoleic acid steadily accumulated. fad3a of the low linolenic acid line SP2047 encoded a truncated protein caused by a premature stop codon resulting from a single point mutation, and the low level of transcript accumulation in this genotype is likely due to nonsense-mediated mRNA decay caused by the premature termination of translation as a result of this early stop codon. Although substantial amounts of transcript accumulation occurred with fad3b of SP2047 genotype, cloning of the gene revealed a mutation in the first histidine box causing an amino acid change. Heterologous expression in yeast of the SP2047 and UGG5-5 fad3b genes showed that the mutation in the histidine box in SP2047 caused the enzyme inactivity. Taken together, these results showed that fad3a and fad3b are responsible for linolenic acid accumulation in flax seeds but did not support a major role for the novel fad3c. These observations were further supported by phenotypic and genotypic assessment of a doubled haploid population. Expression patterns of fad3a and fad3b were highly correlated with linolenic acid accumulation during seed development, with the exception of fad3b in SP2047 whose lack of activity was caused by the histidine box mutation despite its transcript accumulation being similar to that of the fad3b of the other genotypes.

Development and analysis of EST-SSRs for flax (Linum usitatissimum L.).

A set of 146,611 expressed sequence tags (ESTs) were generated from 10 flax cDNA libraries. After assembly, a total of 11,166 contigs and 11,896 singletons were mined for the presence of putative simple sequence repeats (SSRs) and yielded 806 (3.5%) non-redundant sequences which contained 851 putative SSRs. This is equivalent to one EST-SSR per 16.5 kb of sequence. Trinucleotide motifs were the most abundant (76.9%), followed by dinucleotides (13.9%). Tetra-, penta- and hexanucleotide motifs represented <10% of the SSRs identified. A total of 83 SSR motifs were identified. Motif (TTC/GAA)n was the most abundant (10.2%) followed by (CTT/AAG)n (8.7%), (TCT/AGA)n (8.6%), (CT/AG)n (6.7%) and (TC/GA)n (5.3%). A total of 662 primer pairs were designed, of which 610 primer pairs yielded amplicons in a set of 23 flax accessions. Polymorphism between the accessions was found for 248 primer pairs which detected a total of 275 EST-SSR loci. Two to seven alleles were detected per marker. The polymorphism information content value for these markers ranged from 0.08 to 0.82 and averaged 0.35. The 635 alleles detected by the 275 polymorphic EST-SSRs were used to study the genetic relationship of 23 flax accessions. Four major clusters and two singletons were observed. Sub-clusters within the main clusters correlated with the pedigree relationships amongst accessions. The EST-SSRs developed herein represent the first large-scale development of SSR markers in flax. They have potential to be used for the development of genetic and physical maps, quantitative trait loci mapping, genetic diversity studies, association mapping and fingerprinting cultivars for example.

Gene expression of stearoyl-ACP desaturase and delta12 fatty acid desaturase 2 is modulated during seed development of flax (Linum usitatissimum).

Flax's recent popularity in human and animal foods is mostly due to its desirable FA composition. Flax is an excellent source of omega-3 FA, which have been shown to have many health benefits. To date, little is known about the genetic and environmental factors that control the FA composition of flax seeds. To elucidate some of the important genetic components, reverse transcriptase (RT)-PCR and real-time PCR were used to determine the expression profiles of two key FA biosynthetic genes during seed development. Plants of flax cultivar AC McDuff were grown under field conditions, and RNA was extracted from ovaries and developing bolls collected from 2 d after anthesis (DAA) to maturity. Desaturation enzymes stearoyl-ACP desaturase (SAD) and delta12 FA desaturase 2 (FAD2) were both expressed in ovaries, and their expression was differentially modulated throughout seed development. SAD was most highly expressed in ovaries. Its expression quickly decreased until 4 DAA; this was followed by a slight peak at 8 DAA, only to return to relatively low levels of expression in maturing bolls, ranging from 2.1% to 4.5% relative to the level observed in ovaries. FAD2 expression displayed a different temporal pattern. While expression of FAD2 did decrease in the early stages of seed development, expression increased starting at 8 DAA, peaking at 16 DAA, when it was 158% relative to the level observed in ovaries. FAD2, which desaturates oleic acid (18:1cisdelta9) into linoleic acid (18:2cisdelta9,12), is therefore controlled at the transcription level. To relate enzyme expression with FA profile, GC was performed on the same subsamples used for RT-PCR and real-time PCR, and proportions of palmitic, stearic, oleic, linoleic, and linolenic acids were determined for the same developmental stages. Although FAD2 expression increased from 8 to 16 DAA, relative changes in linoleic acid (18:2cis delta9,12) were not observed. However, linolenic acid (ALA; alpha-18:3; 18:3cisdelta9,12,15) levels increased steadily, meaning that linoleic acid (18:2cisdelta9,12) is a transient substrate converted by FAD3 as quickly as it is produced by FAD2. Phenotypes are the result of genotypes, environment, and the interaction of the two. To evaluate the environmental impact on the production of FA in flax, FA profiles were assessed in a total of four environments (two locations, two years). Warm and dry environmental conditions resulted in lower levels of PUFA 18:2cisdelta9,12 and 18:3cisdelta9,12,15, and higher levels of 18:1 cisdelta9. FAD2 expression and/or activity may therefore be affected by the environment.