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.     

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.     

Shoot regeneration from cotyledon explants of fibre flax (Linum usitatissimum) and their subsequent rooting

In this study, the morphogenic ability of cotyledon explants of fibre flax (Linum usitatissimum L.) was investigated in relation to their physiological age and genotype and the effect of growth regulators in the medium. Among four investigated genotypes, the explants of cvs. A-29 and Alexim manifested the highest and the lowest morphogenic ability, respectively. The optimum ratio of growth regulators for shoot regeneration was shown to depend on the age of donor plantlets from which the explants were obtained. For explants excised from younger plantlets, the lower concentration of benzyladenine (BA) and the absence of auxin were preferable. For explants from older plantlets, the higher BA concentration and NAA presence in the medium were required for shoot regeneration. The addition of ABA to the regeneration medium usually reduced shoot regeneration frequency; however, the inhibitory effect of ABA depended on other growth regulators.     

Cloning and expression of (R)-hydroxynitrile lyase from Linum usitatissimum (flax)

The gene encoding for (R)-hydroxynitrile lyase ((R)-HNL) from Linum usitatissimum has been cloned by polymerase chain reaction using 3′,5′-RACE (rapid amplification of cDNA ends). The resulting clone contained an open reading frame of 1266 bp corresponding to a protein of 422 amino acids (45.8 kDa), which shows significant homologies to zinc-dependent formaldehyde dehydrogenases and alcohol dehydrogenases from various organisms. The dimeric active enzyme was expressed in Escherichia coli as N-terminal hexa-histidine fusion protein allowing the purification of homogeneous protein in one step. The formation of inclusion bodies could be reduced using a thioreductase deficient E. coli strain as a host and performing expression of (R)-HNL at 28°C. Under these conditions recombinant (R)-HNL was obtained with a specific activity of 76 U/mg.     

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.     

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.     

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.     

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.     

Heat-stress effects on reproduction and seed set in Linum usitatissimum L. (flax)

Heat stress can detrimentally affect the reproductive capacity of many plants. The effect of a 7 or 14 d heat stress on flowering, seed set, pollen viability and germinability of flax (Linum usistatissimum L.) was assessed under growth chamber conditions. An incremental (2 °C/h), cyclical (daytime high 40 °C and night‐time low 18 °C) heat stress was applied 12 d after the initiation of flowering. Although flower formation in flax was not affected by heat stress, boll formation and seed set were reduced with onset of the heat stress. On removal of heat stress the stressed plants showed a compensatory response, flowering and producing bolls at a greater rate than the control plants. Heat stress significantly prolonged flowering by 17 d. Boll weight and seed weight were reduced with heat stress and the number of malformed, sterile seed increased three‐fold after 14 d of heat stress. Pollen viability and appearance were negatively affected after 6 and 10 d of heat stress, respectively. Pollen germinability decreased by the sixth day of heat stress, with no pollen germinating by the tenth day. Effects of heat stress on pollen viability and germinability alone, which did not occur until after the sixth day of the stress, could not account for the decreased boll formation due to heat stress in flax. These observations suggest that a combined effect of heat stress on both pollen and ovules contributes to decreased boll formation and seed set in flax.     

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.     

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.     

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.