RFLP and RAPD mapping in flax (Linum usitatissimum)

A map of flax (Linum usitatissimum) using restriction fragment length polymorphisms (RFLPs) and random amplified polymorphic DNAs (RAPDs), and comprising 15 linkage groups containing 94 markers, has been developed covering about 1000 cM. The mapping populations were the F₂ populations from two crosses between diverse cultivars. From one cross, CI1303 and Stormont Cirrus, 20 RFLP and 520 RAPD markers were analyzed. Thirteen RFLP and 80 RAPD markers were on the 15 linkage groups, in addition to one sequence-tagged site (STS). Seven polymorphic RAPD markers were found to have unusual segregation patterns. RAPDs were expressed as dominant markers, but for these markers a prevalence of the progeny lacked a band rather than the expected one-fourth ratio. However, these exceptions may be related to the instability of the genome of Stormont Cirrus in which stable and heritable genomic changes can be induced by environmental factors. The current map could be used for the identification of markers linked to loci controlling the ability to generate heritable changes in response to environmental growth conditions, and to develop anchor loci with STSs for a more general application. Received: 20 March 1999 / Accepted: 16 December 1999     

Characterization of transgenic sulfonylurea-resistant flax (Linum usitatissimum)

Summary Fourteen transgenic flax (Linum usitatissimum) lines, carrying a mutant Arabidopsis acetolactate synthase (ALS) gene selected for resistance to chlorsulfuron, were characterized for resistance to two sulfonylurea herbicides. Progeny of 10 of the 14 lines segregated in a ratio of 3 resistant to 1 susceptible, indicating a single insertion. Progeny of 1 line segregated in a 151 ratio, indicating two insertions of the ALS gene at independent loci. Progeny from 3 lines did not segregate in a Mendelian fashion and were likely the products of chimeric shoots. Resistance to chlorsulfuron was stably inherited in all lines. At the enzyme level, the transgenic lines were 2.5 to more than 60 times more resistant to chlorsulfuron than the parental lines. The transgenic lines were 25–260 times more resistant to chlorsulfuron than the parental lines in root growth experiments and demonstrated resistance when grown in soil treated with 20 g ha^-1 chlorsulfuron. The lines demonstrated less resistance to metsulfuron methyl; in root growth experiments, the transgenic lines were only 1.6–4.8 times more resistant to metsulfuron methyl than the parental lines. Resistance was demonstrated in the field at half (2.25 g ha^-1) and full (4.5 g ha^-1) rates of metsulfuron methyl.     

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.     

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.     

Variation in the isozymes of flax (Linum usitatissimum) genotrophs

Peroxidase, esterase, and acid phosphatase isozymes of environmentally induced L and S genotrophs, nuclear DNA reversion types, and the orginal plastic (Pl) type of the flax variety Stormont Cirrus have been compared by polyacrylamide gel electrophoresis. Differences were observed in particular line was not correlated with the nuclear DNA amount. The relationship between the isozyme pattern and the phenotypes of the lines in which they are expressed is discussed.     

Effects of genetic modifications to flax (Linum usitatissimum) on arbuscular mycorrhiza and plant performance

Although arbuscular mycorrhizal fungi (AMF) are known for their positive effect on flax growth, the impact of genetic manipulation in this crop on arbuscular mycorrhiza and plant performance was assessed for the first time. Five types of transgenic flax that were generated to improve fiber quality and resistance to pathogens, through increased levels of either phenylpropanoids (W92.40), glycosyltransferase (GT4, GT5), or PR2 beta-1,3-glucanase (B14) or produce polyhydroxybutyrate (M50), were used. Introduced genetic modifications did not change the degree of mycorrhizal colonization as compared to parent cultivars Linola and Nike. Arbuscules were well developed in each tested transgenic type (except M50). In two lines (W92.40 and B14), a higher abundance of arbuscules was observed when compared to control, untransformed flax plants. However, in some cases (W92.40, GT4, GT5, and B14 Md), the mycorrhizal dependency for biomass production of transgenic plants was slightly lower when compared to the original cultivars. No significant influence of mycorrhiza on the photosynthetic activity of transformed lines was found, but in most cases P concentration in mycorrhizal plants remained higher than in nonmycorrhizal ones. The transformed flax lines meet the demands for better quality of fiber and higher resistance to pathogens, without significantly influencing the interaction with AMF.     

Differential accumulation of monolignol-derived compounds in elicited flax (Linum usitatissimum) cell suspension cultures

Lignin and lignans share monolignols as common precursors and are both potentially involved in plant defence against pathogens. In this study, we investigated the effects of fungal elicitors on lignin and lignan metabolism in flax (Linum usitatissimum) cell suspensions. Cell suspension cultures of flax were treated with elicitor preparations made from mycelium extracts of Botrytis cinerea, Phoma exigua and Fusarium oxysporum F ssp lini. Elicitors induced a rapid stimulation of the monolignol pathway, as confirmed by the increase in PAL (phenylalanine ammonia-lyase, EC 4.1.3.5), CCR (cinnamoyl-CoA reductase EC 1.2.1.44) and CAD (cinnamyl alcohol dehydrogenase EC 1.1.1.195) gene expression and PAL activity. At the same time, CCR activity only increased significantly in F. oxysporum-treated cells 24 h post elicitation. On the other hand, CAD activity measured for coniferyl alcohol formation was transiently decreased but a substrate-specific activation of CAD activity was observed in F. oxysporum-treated cells when using sinapyl alcohol as substrate. The accumulation of monolignol-derived products varied according to the elicitor used. B. cinerea or P. exigua-elicited cell cultures were characterised by a reinforcement of the cell wall by a deposit of 8-O-4′-linked non-condensed lignin structures and phenolic monomers, while at the same time no stimulation of 8-8′-linked lignan or 8-5′-linked phenylcoumaran lignan accumulation was observed. Additionally, elicitation of cell cultures with F. oxysporum extracts even triggered a strong incorporation of monolignols in the non condensed labile ether-linked lignin fraction concomitantly with a decrease in lignan and phenylcoumaran lignan accumulation. Several hypotheses are proposed to explain the putative role of these compounds in the defence response of flax cells against pathogens. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users. C. Hano and M. Addi contributed equally to this work.     

Isolation of a flax (Linum usitatissimum) gene induced during susceptible infection by flax rust (Melampsora lini)

In a susceptible infection of flax ( Linum usitatissimum ), the obligate rust pathogen ( Melampsora lini ) can grow in the leaf without triggering the hypersensitive resistance response. The rust establishes specialized structures (haustoria) in plant mesophyll cells and induces changes in plant subcellular organization. Subtraction hybridization methods were used to isolate cDNA clones of mRNAs that have altered expression in infected leaves. Most of the cDNAs recovered were of fungal origin, but one clone, pFIS1 ( f lax i nducible s equence No. 1), recovered from several independent experiments, was a plant-specified mRNA that showed a 10-fold increase in steady-state levels during susceptible growth. The increase in fis1 mRNA levels was not seen in the resistant reaction (hypersensitive reaction) and the predicted protein sequence (551 amino acids with a predicted molecular weight of 61 kDa) has no similarity to known pathogenesis-related proteins. Searches of sequence data bases showed that fis1 encodes a protein which contains amino acid sequence motifs that are conserved in all previously characterized aldehyde dehydrogenases.     

Genetic control of seed weight in flax (Linum usitatissimum) and possible implications

Summary Mean seed weight data were obtained from the F₁ and F₂ of a six-by-six diallel cross with flax (Linum usita-tissimum L.). Pronounced reciprocal differences appeared in the F₁, but had largely disappeared by the F₂. The genetic control of mean seed weight was examined using two types of analysis of variance. The models underlying both analyses were fitted to the data by matrix methods supplying weighted least-squares estimates of the parameters in the models. Weights, the use of which dealt with the problem of variation in the reliability of means, were the reciprocals of the variances of individual cell (cross/self) means in the diallel data table. Elimination of redundant parameters supplied the minimum adequate models for each analysis type. Dominance was apparently masked by the large transient maternal effects in the F₁, but surfaced in the F₂, where dominance was towards larger mean seed weight. This may be coupled with findings elsewhere of possible advantages for larger seed weights to speculate on a role in preserving infrequent hybrid progeny among inbreeding (flax) species. Maternal effects producing larger seed size, plus dominance with the same result might be valuable, in conjunction with growth and competitive advantages conferred by larger seed, in preventing early elimination of rare hybrids.     

亚麻原生质体培养及植株再生

Shoot protoplasts of four fiber flax (Linum usitatissimum) varieties (7309, 948, Belinka and Viking) were isolated and cultured. The optimal condition for higher protoplast yield 1.8 x 10(6)/gFW and activity 85.5% (c.v. 948) were from 10 day old seedings. Culture in V-KM Agroase-island medium led to first divisions after 3 days (c. v. 948), and after twenty days with an efficiency of 36% of divided cells and 5.2% in plating efficiency. Plant regeneration was obtained in 7309 and Belinka on agar media B5-2, MS3 containing 0.6 mg/L 6-BA and 0.1 mg/L NAA. Roots and leaves regeneration were observed in Viking and 948 respectively.     

The biosynthesis of cyanogenic glucosides in Linum usitatissimum (linen flax) in Vitro

Microsomal preparations from dark-grown Linum usitatissimum (linen flax) seedlings synthesize acetone cyanohydrin, the precursor of the cyanogenic glucoside linamarin, from valine in the presence of NADPH. N-Hydroxyvaline and isobutyraldoxime, which are predicted intermediates in the pathway, are also converted into products. These microsomal preparations also convert isoleucine into 2-butanone cyanohydrin the precursor of lotaustralin. The biosynthetic activity is located exclusively in the developing cotyledons.     

Intraspecific 5S rRNA gene variation in flax,Linum usitatissimum (Linaceae)

A relatively large proportion of the flax genome (~ 3%) is comprised of 5S rRNA genes. This study focuses on the intraspecific sequence variation among five distinct groups of 5S rRNA genes. The results indicate that group 1 and 2 5S rRNA genes most closely resemble other angiosperm 5S genes, while groups 3–5 are highly divergent. Sequence variation is higher in the spacer region compared to the transcribed region for all pairwise comparisons. The large degree of sequence variation observed in this study is discussed with respect to genome organization and proposed models for repetitive sequence maintenance.     

A possible role of prolyl oligopeptidase during Linum usitatissimum (flax) seed development

Involvement of prolyl oligopeptidases (POPs) in the control of several mammalian peptide hormone signalling pathways has been studied extensively in recent years. POPs are ubiquitous enzymes, but little attention has been paid to understanding their function in plants. Using a cDNA-AFLP approach, two flax (Linum usitatissimum) POP ESTs were identified as being specifically expressed in the early stages of flax seed development. This specific expression was confirmed using real time RT-PCR and in situ hybridisation approaches. Seed expression of Arabidopsis POP genes was measured and showed no specificity. Comparison between results obtained with flax and Arabidopsis is discussed in order to address a hypothetic function for POPs during seed formation. These results provide the first insights into POP gene expression and hypothetical function in plants.     

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.     

Genetic control of polyunsaturated fatty acid biosynthesis in flax (Linum usitatissimum) seed oil

Summary The inheritance of two mutants of flax (Linum usitatissimum), having altered proportions of the C18 polyunsaturated fatty acids, linoleic and linolenic, was examined. Both lines, M1589 and M1722, are homozygous for a single gene mutation which reduces linolenic acid content from 34% to 22% and raises linoleic acid from 15% to 27%. Genetic analysis of crosses involving M1589, M1722 and their parental cultivar Glenelg revealed that these mutations are in different unlinked genes and exhibit additive (codominant) gene action. The symbolsLn1 andLn2 are proposed for the mutated genes in M1589 and M1722, respectively. Recombinant genotypes homozygous for the mutant alleles at both loci are very low in linolenic acid (2%) and high in linoleic acid (48%), with unaltered proportions of other fatty acids. The complete inverse correlation between linoleic and linolenic acids (r=-0.98) indicates that the mutations block the synthesis of linolenic acid at the linoleic desaturation step.     

Variation within flax (Linum usitatissimum) and barley (Hordeum vulgare) in response to allelopathic chemicals

Summary A possible method of manipulating allelopathy would be to develop crop varieties showing an increased tolerance to allelopathic chemicals. We therefore examined four flax (Linum usitatissimum) varieties and two wild Linum species in the presence of p-coumaric acid and four barley (Hordeum vulgare) varieties in the presence of p-coumaric acid, scopoletin and wild oat (Avena fatua) extract. Analysis of variance indicates significant interaction between variety and treatment for shoot and root growth for seedling flax, shoot growth for older flax, and root growth for seedling barley. These differences in tolerance between varieties could be exploited to develop-varieties with greater tolerances to the allelochemicals produced by weeds or in crop residues and therefore potentially more tolerant of the presence of weeds.