Isolation and characterisation of RNA from flax (Linum usitatissimum L.)

A method for isolation of flax RNA is described; properties of the isolated RNA are given. RNA degradation was held to a minimum through a high pH (9.5) extraction buffer, diethylpyrocarbonate (4%) as a nuclease inhibitor, a high concentration (1.5%) of sodium dodecylsulphate, 2 mm Mg²⁺, and separation of the RNA from contaminating materials on Sephadex G-50.     

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. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Identification of microspore-derived plants in anther culture of flax (Linum usitatissimum L.) using molecular markers

The microspore origin of anther-culture-derived plants of flax was determined using inter-simple sequence repeat (ISSR) and randomly amplified polymorphic DNA (RAPD) markers. Polymorphic fragments between the two parents of the F₁ donor plants were identified and their segregation patterns in anther-culture-derived plants were used to elucidate the origin of those plants and to determine the degree of independence of plants regenerated from the same callus. Using one ISSR primer (UBC 889) and two RAPD primers (UBC 556 and 561), 12 out of 16 plants were unequivocally identified as being derived from microspores. Plants derived from the same callus had identical PCR patterns at five polymorphic loci and thus were likely derived from the same microspore. Therefore, it is proposed that the number of calli forming shoots be used to describe the anther culture efficiency in flax. Received: 3 February 1998 / Revision received: 8 June 1998 / Accepted: 8 July 1998     

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.     

A lipoxygenase-divinyl ether synthase pathway in flax (Linum usitatissimum L.) leaves

Incubation of linoleic acid with an enzyme preparation from leaves of flax (Linum usitatissimum L.) led to the formation of a divinyl ether fatty acid, i.e. (9Z,11E,1'Z)-12-(1'-hexenyloxy)-9,11-dodecadienoic [(omega5Z)-etheroleic] acid, as well as smaller amounts of 13-hydroxy-9(Z),11(E)-octadecadienoic acid. The 13-hydroperoxide of linoleic acid afforded the same set of products, whereas incubations of alpha-linolenic acid and its 13-hydroperoxide afforded the divinyl ether (9Z,11E,1'Z,3'Z)-12-(1',3'-hexadienyloxy)-9,11-dodecadienoic [(omega5Z)-etherolenic] as the main product. Identification of both divinyl ethers was substantiated by their UV, mass-, (1)H NMR and COSY spectral data. In addition to the 13-lipoxygenase and divinyl ether synthase activities demonstrated by these results, flax leaves also contained allene oxide synthase activity as judged by the presence of endogenously formed (15Z)-cis-12-oxo-10,15-phytodienoic acid in all incubations.     

Genetic polymorphism of flax Linum usitatissimum based on use of molecular cytogenetic markers

Using a set of approaches based on the use of molecular cytogenetic markers (DAPI/C-banding, estimation of the total area of DAPI-positive regions in prophase nuclei, FISH with 26S and 5S rDNA probes) and the microsatellite (SSR-PCR) assay, we studied genomic polymorphism in 15 flax (Linum usitatissimum L.) varieties from different geographic regions belonging to three directions of selection (oil, fiber, and intermediate flaxes) and in the k-37 x Viking hybrid. All individual chromosomes have been identified in the karyotypes of these varieties on the basis of the patterns of differential DAPI/C-banding and the distribution of 26S and 5S rDNA, and idiograms of the chromosomes have been generated. Unlike the oil flax varieties, the chromosomes in the karyotypes of the fiber flax varieties have, as a rule, pericentromeric and telomeric DAPI-positive bands of smaller size, but contain larger intercalary regions. Two chromosomal rearrangements (chromosome 3 inversions) were discovered in the variety Luna and in the k-37 x Viking hybrid. In both these forms, no colocalization of 26S rDNA and 5S rDNA on the satellite chromosome was detected. The SSR assay with the use of 20 polymorphic pairs of primers revealed 22 polymorphic loci. Based on the SSR data, we analyzed genetic similarity of the flax forms studied and constructed a genetic similarity dendrogram. The genotypes studied here form three clusters. The oil varieties comprise an independent cluster. The genetically related fiber flax varieties Vita and Luna, as well as the landrace Lipinska XIII belonging to the intermediate type, proved to be closer to the oil varieties than the remaining fiber flax varieties. The results of the molecular chromosomal analysis in the fiber and oil flaxes confirm their very close genetic similarity. In spite of this, the combined use of the chromosomal and molecular markers has opened up unique possibilities for describing the genotypes of flax varieties and creating their genetic passports.     

Sensitivity of different flax (Linum usitatissimum L.) genotypes to salinity determined by GE biplot

Plants respond differently to salt stress depending on their genetic structure and the severity of the stress. Salinity reduces seed germination, delays plant emergence, and inhibits seedling growth. The selection of the tolerant genotypes, however, plays a vital role in increasing agricultural output since various genotypes greatly vary for their tolerance to salinity. Therefore, this study determined the impact of five different NaCl levels (i.e., 0, 50, 100, 150 and 200 mM) on seed germination and growth attributes of 10 flax (Linum usitatissimum L.) genotypes. The germination and growth characteristics of the genotypes under study were examined using the biplot approach at varied salt levels. The results indicated that individual and interactive effects of genotypes and salinity levels significantly (p ≤ 0.01 or p ≤ 0.05) affected several seed germination traits. The relations of genotype × germination traits indicated that ‘G4′ and ‘G6′ were the most stable genotypes with the highest performance regarding seed germination characteristics. The genotype ‘G2′ was associated with shoot length, while ‘G7′ was linked with salinity tolerance index. The biplot divided the germination characteristics into five different groups according to sector analysis. Most of the germination parameters had higher values under 100 mM, while some of the parameters had better values under 0, 50 and 200 mM NaCl levels. The tested genotypes varied for their seed germination and growth response depending on the NaCl levels. The genotypes ‘G4′, ‘G5′ and ‘G6′ proved more tolerant to high NaCl levels. Therefore, these genotypes can be used to improve flax productivity under saline soils.     

Genetic polymorphism of flax Linum usitatissimum based on the use of molecular cytogenetic markers

Using a set of approaches based on the use of molecular cytogenetic markers (DAPI/C-banding, estimation of the total area of DAPI-positive regions in prophase nuclei, FISH with 26S and 5S rDNA probes) and the microsatellite (SSR-PCR) assay, we studied genomic polymorphism in 15 flax (Linum usitatissimum L.) varieties from different geographic regions belonging to three directions of selection (oil, fiber, and intermediate flax) and in the k-37 × Viking hybrid. All individual chromosomes have been identified in the karyotypes of these varieties on the basis of the patterns of differential DAPI/C-banding and the distribution of 26S and 5S rDNA, and idiograms of the chromosomes have been generated. Unlike the oil flax varieties, the chromosomes in the karyotypes of the fiber flax varieties have, as a rule, pericentromeric and telomeric DAPI-positive bands of smaller size, but contain larger intercalary regions. Two chromosome rearrangements (chromosome 3 inversions) were detected in the variety Luna and in the k-37 × Viking hybrid. In both these forms, no colocalization of 26S rDNA and 5S rDNA on the satellite chromosome was detected. The SSR assay with the use of 20 polymorphic pairs of primers revealed 22 polymorphic loci. Based on the SSR data, we analyzed genetic similarity of the flax forms studied and constructed a genetic similarity dendrogram. The genotypes studied here form three clusters. The oil varieties comprise an independent cluster. The genetically related fiber flax varieties Vita and Luna, as well as the landrace Lipinska XIII belonging to the intermediate type, proved to be closer to the oil varieties than the remaining fiber flax varieties. The results of the molecular chromosome analysis in the fiber and oil flax confirm their very close genetic similarity. In spite of this, the combined use of the chromosome and molecular markers has opened up unique possibilities for describing the genotypes of flax varieties and creating their genetic passports.     

Evidence of the domestication history of flax (Linum usitatissimum L.) from genetic diversity of the sad2 locus

A phylogenetic analysis was conducted on 34 alleles of 2.5 kb sized stearoyl-ACP desaturase II (sad2), obtained from 30 accessions of cultivated and pale flax (Linum spp.), to elucidate the history of flax domestication. The analysis supports a single domestication origin for extant cultivated flax. The phylogenetic evidence indicates that flax was first domesticated for oil, rather than fibre. The genetic diversity of the sad2 locus in cultivated flax is low when compared to that of the pale flax assayed. An absolute archaeological date could be applied to the synonymous substitution rate of sad2 in cultivated flax, yielding a high estimate of 1.60–1.71×10⁻⁷ substitutions/site/year. The occurrence of nonsynonymous substitutions at conserved positions of the third exon in alleles from cultivated flax suggests that the locus may have been subjected to an artificial selection pressure. The elevated synonymous substitution rate is also compatible with a population expansion of flax since domestication, followed by a population decline in historic times. These findings provide new insight into flax domestication and are significant for the continuous exploration of the flax germplasm for utilization.     

Cellulose synthase genes that control the fiber formation of flax (Linum usitatissimum L.)

Four cellulose synthase genes were identified by analysis of their class-specific regions (CSRII) in plants of fiber flax during the “rapid growth” stage. These genes were designated as LusCesA1, LusCesA4, LusCesA7 and LusCesA9. LusCesA4, LusCesA7, and LusCesA9 genes were expressed in the stem; LusCesA1 and LusCesA4 genes were expressed in the apex part of plants; and the LusCesA4 gene was expressed in the leaves of fiber flax. The expression of the LusCesA7 and LusCesA9 genes was specific to the stems of fiber flax. These genes may influence the quality of the flax fiber.     

Notes on Thysanoptera found on flax (Linum usitatissimum L.) in the British Isles

The following eighteen species of Thysanoptera Terebrantia have been found on flax in the British Isles: Melanthrips fuscus (Sulzer), Aeolothrips fasciatus (L.), Anaphothrips obscurus (Müler), Aptinothrips rufus (Gmelin), Chirothrips manicatus Hal., Limothrips cerealium Hal., L. denticornis Hal., Stenothrips graminum Uzel, Taeniothrips atratus (Hal.), T. vulgatissimus (Hal.), Thrips angusticeps Uzel, T. discolor Hal., T. flavus Schrank, T. fuscipennis Hal., T. major Uzel, T. minutissimus L., T. physapus L., T. tabaci Lindeman. Each species is described briefly with notes on habits of adults and larvae, place of pupation, number of generations in the year, hibernation, time of occurrence on plants, plants and objects on which found, host plants of larvae and adults, importance to flax, record of locality and collector on flax, distribution, including altitudes, in the British Isles. More species occur in the south than in the north of Great Britain, and species common to both regions usually occur in greater numbers in the south. The insects breed on certain species of crop plants, weeds or trees of arable land. No damage of economic importance to flax by Thysanoptera has been proven in the British Isles, and the flax thrips, Thrips lini Ladureau, has not been found. Taeniothrips vulgatissimus (Hal.) may breed on flax and its adults, and those of T. atratus (Hal.) may cause superficial damage to petals of flowers. Thrips angusticeps Uzel and T. tabaci Lindeman will probably breed on flax.     

RAPD analysis of the flax (Linum usitatissimum L.) varieties and hybrids of various productivity

Genetic polymorphism in varieties and hybrids of cultivated flax (Linum usitatissimum L.) has been investigated by RAPD-PCR. Analysis with 15 primers has revealed varietal specificity and hybrid inheritance of RAPD alleles. This allows genetic certification of the original varieties and their hybrids for breeding purposes. Polymorphic amplification products were obtained in RAPD analysis of DNA from two cultivated flax varieties with the use of 10-11 nucleotide primers.     

Histological study of embryo-like structures initiated from hypocotyl segments of flax (Linum usitatissimum L.)

Cultivation of flax hypocotyl segments on MS medium supplemented with auxin (2,4-d, NAA) and combination of auxin (NAA) and cytokinin (BAP, zeatin) resulted in production of callus on the cut ends of segments and prolonged cultivation in globular structures resembling early stages of somatic embryos. Embryo-like structures protruded on the surface directly from the subepidermal layers of hypocotyl segments. Despite these globular structures closely resembling somatic embryos, histological observations did not reveal their embryogenic character–organogenesis was the predominant developmental morphogenic pathway. Based on our experiments, as well as on critical revision of existing reports on flax somatic embryogenesis, we conclude, that there has not yet been convincing histological proof of somatic embyogenesis from flax hypocotyl segments.     

Purification of several pectin methyltransferases from cell suspension cultures of flax (Linum usitatissimum L.)

Three pectin methyltransferases (PMT5, PMT7, PMT18; EC 2.1.1.6.x) were solubilized from the endo-membrane complex of flax cells, with 0.05% Triton X-100. After a 3 step-chromatography procedure, PMT7 and PMT5 were purified to apparent homogeneity. PMT5 and PMT7 differed regarding their optimum pH (5 or 7), the methyl acceptor (low or highly methylesterified pectin), their focusing pH range (6-7 or 8-9) and relative molecular mass (40 +/- 5 or 110 +/- 10 kDa). SDS-PAGE of PMT5 and PMT7 did not reveal bands at 40 or 110 kDa but only a silver stained band of about 18 kDa. Two independent methods (photo labelling and enzymatic activity) showed that this silverstained band corresponded to a methyltransferase with affinity for pectins. This polypeptide was of the same size as the enzyme designed PMT18 (18 +/- 3 kDa; pl 4-4.5) recovered during size exclusion chromatography of either PMT7 or PMT5, suggesting that PMT18 bears the catalytic site of PMT5 and PMT7.     

Xylem changes in the correlative inhibition of lateral bud growth in flax (Linum usitatissimum L.)

Xylem or tracheary changes at the base of the cotyledonary buds of flax seedlings (Linum usitatissimum L.), released from inhibition by decapitation of the main apex were studied. The differentiation of xylem strands and/or tracheary elements was correlated with the growth in length of the lateral buds, especially 48–72 hr after the removal of the main apex. The xylem strands, connected to the hypocotylary stele or not, and the tracheary elements increased with age within and outside the strands of both non-decapitated and decapitated seedlings. In the latter, the differentiation of these structures, however, occurred much earlier and in greater abundance in the same regions. The early growth in length of lateral buds, 1 or 2 hr after decapitation, was correlated with the early development of tracheary perforations in the xylem strands. The xylary strands with perforated elements are known to be more efficient than those without them. Therefore, it is suggested that the inhibition of lateral-bud growth was due, in fact, to a lack of appropriate tracheary perforations in the bud xylem strands that were connected with the hypocotylary stele of flax seedlings.