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.

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.

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.

Gene expression analysis of flax seed development.

BACKGROUND: Flax, 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. RESULTS: We 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. CONCLUSIONS: We 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.

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

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Induced Mutagenesis in UGT74S1 Gene Leads to Stable New Flax Lines with Altered Secoisolariciresinol Diglucoside (SDG) Profiles.

Flax secoisolariciresinol (SECO) diglucoside (SDG) lignan is an emerging natural product purported to prevent chronic diseases in humans. SECO, the aglycone form of SDG, has shown higher intestinal cell absorption but it is not accumulated naturally in planta. Recently, we have identified and characterized a UDP-glucosyltransferase gene, UGT74S1, that glucosylates SECO into its monoglucoside (SMG) and SDG forms when expressed in yeast. However, whether this gene is unique in controlling SECO glucosylation into SDG in planta is unclear. Here, we report on the use of UGT74S1 in reverse and forward genetics to characterize an ethyl methane sulfonate (EMS) mutagenized flax population from cultivar CDC Bethune and consisting of 1996 M2 families. EMS mutagenesis generated 73 SNP variants causing 79 mutational events in the UGT74S1 exonic regions of 93 M2 families. The mutation frequency in the exonic regions was determined to be one per 28 Kb. Of these mutations, 13 homozygous missense mutations and two homozygous nonsense mutations were observed and all were transmitted into the M3 and M4 generations. Forward genetics screening of the population showed homozygous nonsense mutants completely lacking SDG biosynthesis while the production of SMG was observed only in a subset of the M4 lines. Heterozygous or homozygous M4 missense mutants displayed a wide range of SDG levels, some being greater than those of CDC Bethune. No additional deleterious mutations were detected in these mutant lines using a panel of 10 other genes potentially involved in the lignan biosynthesis. This study provides further evidence that UGT74S1 is unique in controlling SDG formation from SECO and this is the first report of non-transgenic flax germplasm with simultaneous knockout of SDG and presence of SMG in planta.

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.

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.

Two FAD3 desaturase genes control the level of linolenic acid in flax seed.

Industrial oil flax (Linum usitatissimum) and edible oil or solin flax differ markedly in seed linolenic acid levels. Despite the economic importance of low-linolenic-acid or solin flax, the molecular mechanism underlying this trait has not been established. Two independently inherited genes control the low-linolenic-acid trait in flax. Here, we identified two genes, LuFAD3A and LuFAD3B that encode microsomal desaturases capable of desaturating linoleic acid. The deduced proteins encoded by these genes shared 95.4% identity. In the low-linolenic-acid line solin 593-708, both LuFAD3A and LuFAD3B carry point mutations that produce premature stop codons. Expression of these genes in yeast (Saccharomyces cerevisiae) demonstrated that, while the wild-type proteins were capable of desaturating linoleic acid, the truncated proteins were inactive. Furthermore, the low-linolenic-acid phenotype in flax was complemented by transformation with a wild-type gene. Codominant DNA markers were developed to distinguish between null and wild-type alleles of both genes, and linolenic acid levels cosegregated with genotypes, providing further proof that LuFAD3A and LuFAD3B are the major genes controlling linolenic acid levels in flax. The level of LuFAD3 transcripts in seeds peaked at about 20 d after flowering, and transcripts were not detectable in leaf, root, or stem tissue. A dramatic reduction in transcript levels of both genes occurred in the low-linolenic-acid solin line, which was likely due to nonsense-mediated decay.