Gordon Rowland

Gene expression analysis of flax seed development

BackgroundFlax, Linum usitatissimum L., is an important crop whose seed oil and stem fiber have multiple industrial applications. Flax seeds are also well-known for their nutritional attributes, viz., omega-3 fatty acids in the oil and lignans and mucilage from the seed coat. In spite of the importance of this crop, there are few molecular resources that can be utilized toward improving seed traits. Here, we describe flax embryo and seed development and generation of comprehensive genomic resources for the flax seed.ResultsWe describe a large-scale generation and analysis of expressed sequences in various tissues. Collectively, the 13 libraries we have used provide a broad representation of genes active in developing embryos (globular, heart, torpedo, cotyledon and mature stages) seed coats (globular and torpedo stages) and endosperm (pooled globular to torpedo stages) and genes expressed in flowers, etiolated seedlings, leaves, and stem tissue. A total of 261,272 expressed sequence tags (EST) (GenBank accessions LIBEST_026995 to LIBEST_027011) were generated. These EST libraries included transcription factor genes that are typically expressed at low levels, indicating that the depth is adequate for in silico expression analysis. Assembly of the ESTs resulted in 30,640 unigenes and 82% of these could be identified on the basis of homology to known and hypothetical genes from other plants. When compared with fully sequenced plant genomes, the flax unigenes resembled poplar and castor bean more than grape, sorghum, rice or Arabidopsis. Nearly one-fifth of these (5,152) had no homologs in sequences reported for any organism, suggesting that this category represents genes that are likely unique to flax. Digital analyses revealed gene expression dynamics for the biosynthesis of a number of important seed constituents during seed development.ConclusionsWe have developed a foundational database of expressed sequences and collection of plasmid clones that comprise even low-expressed genes such as those encoding transcription factors. This has allowed us to delineate the spatio-temporal aspects of gene expression underlying the biosynthesis of a number of important seed constituents in flax. Flax belongs to a taxonomic group of diverse plants and the large sequence database will allow for evolutionary studies as well.

Present status and future strategy in breeding faba beans (Vicia faba L.) for resistance to biotic and abiotic stresses

Progress is being made, mainly by ICARDA but also elsewhere, in breeding for resistance to Botrytis, AScochyta, Uromyces, and Orobanche; and some lines have resistance to more than one pathogen. The strategy is to extend multiple resistance but also to seek new and durable forms of resistance. Internationally coordinated programs are needed to maintain the momentum of this work.

The application of chemical mutagenesis and biotechnology to the modification of linseed (Linum usitatissimum L.)

In the early 1980s the phenomenon of somaclonal variation induced by cell culture was exploited to produce genetic variation in linseed. The linseed variety Andro, derived from the widely grown Canadian variety McGregor, was selected in saline culture and was released for production in Canada. ‘Andro’ possesses traits very different from its parent, such as increased seedling vigour and tolerance to heat stress. Additional stable somaclonal variation in characters such as yield, days to maturity, seed weight and oil content were subsequently induced in ‘McGregor’. However, despite extensive screening of the somaclonal variants, no significant variation in the fatty acid profile was found.

CDC Triffid transgenic flax

CDC Triffid is a transgenic sulfonylurea herbicide resistant cultivar with agronomic features similar to NorLin. It is intended to provide a sustainable, broadleaf cropping option to summerfallowing or continuous cropping to cereals in soils previously treated with residual sulfonylurea herbicides. CDC Triffid flax was developed at the Crop Development Centre, University of Saskatchewan. Key words: Flax, linseed, Linum usitatissimum L., cultivar description, transgenic, biotechnology, sulfonylurea, herbicide resistance, herbicide residue, germplasm

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.

The effect of T-DNA on the agronomic performance of transgenic flax plants

SummaryRegistration and commercial release of crop cultivars improved using recombinant DNA technologies will require extensive field testing of the transgenic lines. In addition to testing the efficacy of the transferred gene(s), regulatory bodies and farmers need data from large scale agronomic studies to provide assurance that the foreign DNA does not adversely affect normal agronomic productivity or quality of the crop. Here, five transgenic sulfonylurea resistant flax (Linum usitatissimum) lines were included in a multiple location cultivar registration trial, evaluated and compared with four standard commercial cultivars and 24 other (conventionally produced) breeding lines. The transgenic lines did not appear to be affected by T-DNA for any quality or agronomic parameter tested, including yield.

QTL mapping and molecular characterization of the classical D locus controlling seed and flower color in Linum usitatissimum (flax)

The flowers of flax (linseed) are blue-hued, ephemeral and self-pollinating, and the seeds are typically brown. A century-old interest in natural yellow seed variants and a historical model point to recessive alleles in B1, D and G loci being responsible, but the functional aspects had remained unknown. Here, we characterized the “D” locus by quantitative trait loci (QTL) mapping and identified a FLAVONOID 3′5′ HYDROXYLASE (F3′5′H) gene therein. It does not belong to the F3′5′H clade, but resembles biochemically characterized F3′Hs (flavonoid 3′ hydroxylase) but without F3′H activity. The genome lacks other F3′H or F3′H-like genes. The apparent neo-functionalization from F3′H is associated with a Thr498 → Ser498 substitution in a substrate recognition site (SRS). The yellow seed and white flower phenotypes of the classical d mutation was found to be due to one nucleotide deletion that would truncate the deduced product and remove three of the six potential SRS, negatively impacting delphinidin synthesis. Delphinidin is sporadic in angiosperms, and flax has no known pollination syndrome(s) with functional pollinator group(s) that are attracted to blue flowers, raising questions on the acquisition of F3′5′H. The appearance of d allele is suggestive of the beginning of the loss of F3′5′H in this species.

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.

Effects of diurnal temperature range and seasonal temperature pattern on the agronomic traits of fibre flax (Linum usitatissimum L.)

Yang, F., Liu, F.-h. and Rowland, G. 2013. Effects of diurnal temperature range and seasonal temperature pattern on the agronomic traits of fibre flax ( Linum usitatissimum L.). Can. J. Plant Sci. 93: 1249-1255. Three cultivars of fibre flax (Linum usitatissimum L.), Viking, Argos and Ariane, were used to study the effect on various agronomic traits of diurnal temperature ranges (DIF) and seasonal temperature patterns (STP) applied throughout the crop growth period. Diurnal temperature ranges were set at 5, 10 and 15°C with the same daily mean temperature and accumulated growing degree days (GDDa), under a parabolic STP. Seasonal temperature patterns were set following parabolic, anti-parabolic and quasi-horizontal configurations with DIF set at 10°C. The results showed that DIF or cultivar had a significant influence on the biomass, plant height (PH), stem diameter (SD), length of technical stem (LTS), weight of technical stem (WTS), fibre weight (FW), fibre content (FC) and seed weight per pot (SWp). Seasonal temperature pattern also had a significant influence on these traits except for WTS and FW. Significant interactions were observed between DIF and cultivar, affecting all traits with the exception of FW, and between STP and cultivar, affecting biomass, PH, SD and WTS. Diurnal temperature range set at 10°C was optimal for biomass, PH, SD, LTS, WTS, and FW, while DIF set at 5°C benefited FC and SWp. Among the different STP configurations, anti-parabolic pattern was optimal for biomass, PH, SD, LTS and FC, and the quasi-horizontal pattern best fit SWp. In conclusion, the combination of DIF set at 10°C and anti-parabollic STP provided the most favorable temperature conditions for fibre flax growth, which is the situation with winter flax production in Yunnan, China.