Scott Duguid

Gene expression of stearoyl-ACP desaturase and Δ12 fatty acid desaturase 2 is modulated during seed development of flax (Linum usitatissimum)

Flaxs 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 Δ12 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∶1 cisΔ9) into linoleic acid (18∶2 cisΔ9, 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∶2 cis Δ9, 12) were not observed. However, linolenic acid (ALA; α-18∶3; 18∶3 cisΔ9, 12, 15) levels increased steadily, meaning that linoleic acid (18∶2 cisΔ9, 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∶2 cisΔ9, 12 and 18∶3 cisΔ9, 12, 15, and higher levels of 18∶1 cisΔ9. FAD2 expression and/or activity may therefore be affected by the environment.

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.

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.

Effect of sucrose concentration on elongation of shoots from flax anther culture

Anther culture is considered as the most successful method of producing doubled haploid plants in flax. The efficiency of shoot regeneration from anther culture has been improved dramatically by optimizing culture temperature and callus induction medium. However, shoot elongation has become increasingly the limiting factor for further improvement of the overall efficiency of doubled haploid production. The effect of sucrose con- 21 centration on shoot elongation was investigated in this study. The medium containing 10 g l sucrose produced longer and more vigorous shoots than the same medium containing other concentrations of sucrose. The possible physiological basis of sucrose on shoot elongation in flax was discussed.

Prairie Blue flax

Prairie Blue, a medium- late maturing oilseed flax (Linum usitatissimum L.), was released in 2003 by Agriculture and Agri-Food Canada, Morden Research Station, Morden, Manitoba. This cultivar has high oil content, high oil quality, small seed size, very good lodging resistance and high yield in all soil zones of the prairies. It is immune to current North American races of rust [Melampsora lini (Ehrenb.) Desmaz], and moderately resistant to wilt caused by Fusarium oxysporum Schlecht. f. sp. lini (Bolley) Snyder & Hansen. Key words: Flax, oilseed, Linum usitatissimum L., cultivar description

Pedigrees and genetic base of flax cultivars registered in Canada

Abstract: Flax is an important oilseed crop with industrial, animal, and human nutrition uses. Breeding programs for linseed and fibre flax were initiated in Canada in the early 1900s. A total of 82 flax cultivars have been registered in Canada since 1910, including 24 cultivars introduced from foreign countries and 58 cultivars developed by Canadian breeders. This study collated pedigree data of Canadian flax cultivars and quantified their genetic base via pedigree analysis and coefficient of parentage (CP). A fairly high mean CP of 0.14 was observed between all registered cultivars; this value was even higher (0.23) when only the 46 cultivars released from 1981–2015 were considered. The registered cultivars traced back to 46 ancestors; 72% originated from foreign countries and contributed 83% of the genetic base of all cultivars, illustrating the dominant role played by foreign germplasm in the genetic improvement of Canadian flax. The top 11 ancestors contributed 70%–93% of the genetic base of modern flax cultivars released in the last three decades and formed the core gene pool of Canadian flax cultivars. The genetic base of Canadian cultivars is relatively narrow, although it has gradually expanded, especially in the last two decades. Broadening the genetic base through the introduction of new exotic germplasm is needed to invigorate the gene pool of Canadian flax breeding programs.

Inheritance of resistance to powdery mildew in flax

Powdery mildew, caused by Oidium lini, is an important disease of flax (Linum usitatissimum) in western Canada. Investigations on the genetics of resistance to powdery mildew in flax resulted in the identification of several resistant Canadian and introduced cultivars. A single dominant gene for resistance to powdery mildew, designated PM1, was identified in Canadian flax cultivars (‘AC Watson’, ‘AC McDuff’, and ‘AC Emerson’) and introduced flax cultivars (‘Atalante’ and ‘Linda’). In addition, two putative dominant genes for resistance to powdery mildew were identified in ‘Linda’.

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.

Structural equation modeling of the Canadian flax (Linum usitatissimum L.) core collection for multiple phenotypic traits

Zhang, T., Lamb, E. G., Soto-Cerda, B., Duguid, S., Cloutier, S., Rowland, G., Diederichsen, A. and Booker, H. M. 2014. Structural equation modeling of the Canadian flax (Linum usitatissimum L.) core collection for multiple phenotypic traits. Can. J. Plant Sci. 94: 1325-1332. Flax seed yield is a complex trait that results from the inter-relationship between many crop characteristics. Structural equation modeling (SEM) is a statistical method used to determine the relationship between measured variables such as crop characteristics. Crop phenology, canopy traits, yield, and its components were included in structural equation models to determine how these crop characteristics relate to seed yield in a phenotypically diverse collection of flax germplasm. Early season vigor (scored as greater plant stand) was positively associated with canopy light interception and higher seed yield. Plant height also had a significant positive effect on seed yield. Moreover, yield components such as 1000-seed weight, number of bolls per unit area, and boll weight were strongly and positively correlated with seed yield. Focusing on yield-related traits, canopy architecture and expansion, and seed weight may be advantageous over yield per se in breeding for yield improvement.