Developing genomic resources in two Linum species via 454 pyrosequencing and genomic reduction

Recent advances in next-generation DNA sequencing (NGS) have enhanced the development of genomic resources such as contigs or single-nucleotide polymorphisms (SNPs) for evolutionary studies of a nonmodel species with a complex and unsequenced genome. This study presents an application of a NGS technique in combination with genomic reduction and advanced bioinformatics tools to identify contigs and SNPs from multiple samples of two Linum species. A full Roche 454 GS FLX run of 16 diverse Linum samples representing cultivated flax (Linum usitatissimum L.) and its wild progenitor (Linum bienne Mill.) generated approximately 1.6 million sequence reads with a total length of 498 Mbp. Application of the computational pipeline de novo identification of alleles identified 713 contigs and 1067 SNPs. A blast search revealed alignments of all 713 contigs with 491 existing Linum scaffolds and gene annotations associated with 512 contigs. Sanger sequencing confirmed 95% of 79 selected contigs and 94% of 272 SNPs and identified 211 new SNPs and 19 new indels. The scored 454 SNP data were highly imbalanced for assayed samples. These findings not only are useful for evolutionary studies of Linum species but also help to illustrate the utility of NGS technologies in SNP discovery for nonmodel organisms.     

亚麻根化学成分的研究

采用硅胶柱色谱法从亚麻根中分得5个化合物,通过波谱分析和甲醇酸水解鉴定了它们的结构,分别为Linum cerebrosideA（1）,1-O-β-D-glueopyranosyl-（2S,3R,4E,8Z）-2[（2（R）-hydroxyhexadecanoyl）amido]-4,8-octadecadiene-1,3-diol（2）,胡萝卜苷（4）,花生酸（5）和ent-kaurane-3-oxo-16α-17-diol（6）。其中化合物1为新化合物,化合物2、5、6均为首次从该植物中分离鉴定。     

Improved podophyllotoxin production by transformed cultures of Linum album

Various cell and hairy root cultures of L. album were developed and analyzed for podophyllotoxin content. Transformed callus and hairy root cultures developed from infection of stem portions of in vitro-germinated L. album plant with Agrobacterium rhizogenes NCIM 5140 strain were selected on the basis of high podophyllotoxin content and growth. Based on the integration of Ri T(L)-DNA and T(R)-DNA, integration of only the ags and not the rol gene in transformed cell culture indicated fragmented integration pattern. The effect of different cultivation media and carbon source on growth and podophyllotoxin production were studied in shake-flask suspension cultures. Detailed batch growth and production kinetics with sugar consumption profile were also established. Maximum volumetric productivity of 4.40 and 2.75 mg/L per day was obtained in cell suspension and hairy root cultures, respectively.     

亚麻立枯病拮抗菌的筛选、生防效果及发酵条件优化

【目的】从健康亚麻植株的根际土壤中筛选对亚麻立枯病菌具有较强抑菌作用的拮抗菌,优化其产生抑菌活性物质的发酵条件,为其生防利用奠定基础。【方法】采用稀释平板涂布法和对峙培养法进行拮抗菌的筛选;根据菌株形态学特征、生理生化特性以及16S r RNA基因序列分析对其进行鉴定;利用温室抗病实验确定其生防效果;通过单因素实验和均匀设计实验优化其发酵条件。【结果】分离筛选到一株对亚麻立枯病菌具有显著拮抗作用的细菌HXP-5,且其对另外7种植物病菌真菌均有拮抗作用;鉴定菌株HXP-5为枯草芽孢杆菌;温室抗病实验结果表明其生防效果可达71.22%;其产生抑菌活性物质的最佳发酵条件为:葡萄糖为2.3%,胰蛋白胨+酵母粉(3:1)为0.25%,Na Cl为0.18%,发酵时间为72 h,发酵温度为27°C,转速为210 r/min,250 m L摇瓶装液100 m L,接种量为1.7%。【结论】经鉴定,对亚麻立枯病病菌具拮抗作用的菌株HXP-5为枯草芽孢杆菌,且对亚麻立枯病具有较强的防治效果,发酵条件进行优化后其对亚麻立枯病病原菌显示出更强的拮抗作用。     

The Effect of a Submersion Pretreatment on In Vitro Explant Growth and Shoot Regeneration from Hypocotyls of Flax (Linum Usitatissimum)

This study was carried out to determine the effect of temporary submersion of hypocotyl segments in water on in vitro explant growth and shoot regeneration on MS (Murashige and Skoog, 1962) medium supplemented with 1 mg l⁻¹ BAP (6-benzylaminopurine) and 0.02 mg l⁻¹ NAA (naphthaleneacetic acid) in three flax cultivars. It was observed that water-treated hypocotyl explants gave rise to the highest values with respect to shoot regeneration percentage, shoot number per hypocotyl, shoot length and total shoot number per Petri dish, successful rooting and plantlet establishment. This procedure may be applicable for other species cultured in vitro.     

Building flax fibres: more than one brick in the walls

The objective of the review is to provide fundamental knowledge on the chemical composition and structural characteristics of flax fibres. These are long and multinucleate cells without septum or partition (average length 2–5 cm) and have a secondary wall of very large thickness (5–15 μm). Fibres are gathered in bundles of one to three dozen cells that encircle the vascular cylinder. The bundle cohesion is insured by pectins, accumulating in the primary wall and cell junctions. In contrast, lignin, which is present in very low amount, does not seem to play a major role in bundle cohesion. At maturity, secondary wall is characterised by (i) a high level of cellulose with microfibrils locked into an almost axial direction and (ii) 5–15% non-cellulosic polysaccharides (NCPs). The chemical composition of NCPs depends on growth stage, indicating important cell wall remodelling, fibre position and variety. Despite the large disparity of the results reported in the literature, galactose appears to be the predominant sugar of NCPs, and β-1-4-galactan together with rhamnogalacturonan of type I (RG-I) and polygalacturonic acid (PGA) become, with fibre maturity, the most abundant tightly bound NCPs. Glycine-rich proteins (GRPs) and arabinogalactan-proteins (AGPs), also present in flax fibres, are both characterised by appreciable levels of glycine and acidic amino acid and are deficient in hydroxyproline, and may contribute to the cross-linking of pectins. (Galacto)glucomanans/glucans rather than xylans consist of cross-linking polymers in fibre secondary wall. A model is proposed where cellulose microfibrils, tethered by cross-linking (galacto)glucomanans/glucans, are embedded in a pectic matrix.     

几丁质酶基因转化亚麻、红豆草、骆驼刺的同工酶研究

以亚麻、红豆草、骆驼刺的下胚轴为外植体,用含几丁质酶ＲＣ２４基因的根瘤农杆菌ＬＢＡ４４０４进行转化。对３种植物转化植株叶片及对照植株叶片的愈伤组织的过氧化物酶及酯酶同工酶分析表明,转基因植株叶片愈伤组织的同工酶谱与对照系相比具有明显的差异,由此推测转化系愈伤组织中可能存在变化了的基因产物,并认为导入的外源几丁质酶基因可能引起了转化材料的遗传变异。     

Host resistance and pathogen virulence across a plant hybrid zone

Hybridization between locally adapted plant populations has been postulated to have significant evolutionary consequences, and, in particular, may influence host-pathogen interactions with respect to resistance and virulence structure. This study investigated patterns of resistance and virulence in a hybrid zone between ”bog” and ”hill” ecotypes of the native Australian flax, Linum marginale, where the host is subject to attack by the rust pathogen, Melampsora lini. Analysis of the resistance structure of adjoining bog, hill and hybrid populations found that bog plants were generally susceptible to pathogen isolates taken from all these sites, but that hybrids exhibited resistance levels similar to the more resistant hill plants. Similarly, the virulence structure of rust isolates collected from the hybrid population was more similar to that of the hill isolates than the bog. Controlled crosses between bog and hill plants showed that crosses in one direction (bog females×hill males) were much more successful than the other. A multi-year reciprocal transplant study further indicated that bog plants had significantly higher survivorship than hill plants, regardless of site. It is suggested that likelihood of differential gene flow and survivorship for bog and hill plants may at least partially explain the maintenance of a relatively narrow hybrid zone. Received: 5 October 1999 / Accepted: 1 July 1999     

Cadmium uptake and bioaccumulation in selected cultivars of durum wheat and flax as affected by soil type

Accumulation of cadmium (Cd) in crop plants is of great concern due to the potential for food chain contamination through the soil-root interface. Although Cd uptake varies considerably with plant species, the processes which determine the accumulation of Cd in plant tissues are affected by soil factors. The influence of soil type on Cd uptake by durum wheat (Triticum turgidum var. durum L.) and flax (Linum usitatissimum L.) was studied in a pot experiment under environmentally controlled growth chamber conditions. Four cultivars/lines of durum wheat (Kyle, Sceptre, DT 627, and DT 637) and three cultivars/lines of flax (Flanders, AC Emerson, and YSED 2) were grown in two Saskatchewan soils: an Orthic Gray Luvisol (low background Cd concentration; total/ABDTPA extractable Cd: 0.12/0.03 mg kg^-1, respectively) and a Dark Brown Chernozem (relatively high background Cd concentration; total/ABDTPA Cd: 0.34/0.17 mg kg^-1 respectively). Plant roots, stems, newly developed heads, and grain/seeds were analyzed for Cd concentration at three stages of plant growth: two and seven weeks after germination, and at plant maturity. The results showed that Cd bioaccumulation and distribution within the plants were strongly affected by both soil type and plant cultivar/line. The Cd concentration in roots leaves and stems varied at different stages of plant growth. However, all cultivars of both plant species grown in the Chernozemic soil accumulated more Cd in grain/seeds than plants grown in the Orthic Gray Luvisol soil. The different Cd accumulation pattern also corresponded to the levels of ABDTPA extractable and metal-organic complex bound soil Cd found in both soils. Large differences were found in grain Cd among the durum wheat cultivars grown in the same soil type, suggesting the importance of rhizosphere processes in Cd bioaccumulation and/or Cd transport processes within the plant. Distribution of Cd in parts of mature plants showed that durum grain contained up to 21 and 36% of the total amount of Cd taken up by the plants for the Orthic Gray Luvisol and Chernozemic soils, respectively. These results indicate the importance of studying Cd speciation, bioaccumulation and cycling in the environment for the management of agricultural soils and crops.     

Distribution of selected elements within flax plants as affected by FeEDDHA

Summary Flax growing on a calcareous soil in the greenhouse developed Mn toxicity symptoms. The toxicity was eliminated by application of 2 ppm FeEDDHA-Fe. FeEDDHA had major effects on distribution of Mn, Zn, Fe and P among selected plant parts. Application of the chelate reduced Mn concentration in older leaves, the tissue most susceptible to Mn toxicity, associated stem tissue, plant tops, and roots from 2295 to 133 ppm, 62 to 7 ppm, 550 to 34 ppm, and 42 to 34 ppm, respectively. Analysis of older leaves is recommended for diagnosing Mn toxicity in flax.FeEDDHA reduced Zn concentration in plant tops and this was chiefly reflected in greatly reduced leaf concentrations, especially in older leaves. FeEDDHA increased plant Fe concentration and the effect was greatest in root and older leaf tissues. The overall effect of FeEDDHA on P concentration was small but large increases occurred in younger leaf tissue due to application of the chelate. Relative distributions of K, Na, Ca, and Mg among plant parts were only slightly affected by FeEDDHA.