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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.

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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.Electronic supplementary materialThe online version of this article (doi:10.1007/s00122-012-1953-0) contains supplementary material, which is available to authorized users.

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.

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.

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.Electronic supplementary materialThe online version of this article (doi:10.1007/s00122-013-2161-2) contains supplementary material, which is available to authorized users.

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.