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.     

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.     

张掖市不同海拔地区5个胡麻品种品质特性比较分析

胡麻是甘肃省主要经济作物之一,海拔2 400 m以下的河西地区为甘肃高产区。为探明张掖不同海拔高度对胡麻品质特性的影响和不同胡麻品种的最佳种植区域,利用近红外品质分析仪,分析了张掖市4个不同海拔地区5个胡麻品种的常规品质指标、饱和脂肪酸和不饱和脂肪酸的含量差异。结果表明：蛋白质、粗脂肪、膳食纤维、棕榈酸、十七碳酸、豆蔻酸、二十四碳酸、油酸和α-亚麻酸含量的品种和海拔间互作效应极显著(P<0.01);山嵛酸、棕榈油酸、顺-11-二十碳一烯酸、亚油酸、γ-亚麻酸和顺-11,14,17-二十碳三烯酸含量随海拔高度不同,变化较小,稳定性好。在高含油量育种目标的基础上,兼顾高三大主要不饱和脂肪酸（α-亚麻酸、油酸和亚油酸）、低两大饱和脂肪酸（棕榈酸和硬脂酸）以及高蛋白质等其他品质育种目标的角度考虑,在张掖市甘州区、高台县巷道镇和民乐县三堡镇、民乐县六坝镇,最适宜种植的胡麻品种分别是5-9812-2-1、09-3-11、张亚2号。不同海拔高度对胡麻各品质指标具有重要影响,张掖市作为优质胡麻籽生产基地,可利用不同胡麻品种适宜种植区域不同,大力发展胡麻籽品质育种和胡麻籽综合开发等。     

Locus-specific view of flax domestication history

Crop domestication has been inferred genetically from neutral markers and increasingly from specific domestication-associated loci. However, some crops are utilized for multiple purposes that may or may not be reflected in a single domestication-associated locus. One such example is cultivated flax (Linum usitatissimum L.), the earliest oil and fiber crop, for which domestication history remains poorly understood. Oil composition of cultivated flax and pale flax (L. bienne Mill.) indicates that the sad2 locus is a candidate domestication locus associated with increased unsaturated fatty acid production in cultivated flax. A phylogenetic analysis of the sad2 locus in 43 pale and 70 cultivated flax accessions established a complex domestication history for flax that has not been observed previously. The analysis supports an early, independent domestication of a primitive flax lineage, in which the loss of seed dispersal through capsular indehiscence was not established, but increased oil content was likely occurred. A subsequent flax domestication process occurred that probably involved multiple domestications and includes lineages that contain oil, fiber, and winter varieties. In agreement with previous studies, oil rather than fiber varieties occupy basal phylogenetic positions. The data support multiple paths of flax domestication for oil-associated traits before selection of the other domestication-associated traits of seed dispersal loss and fiber production. The sad2 locus is less revealing about the origin of winter tolerance. In this case, a single domestication-associated locus is informative about the history of domesticated forms with the associated trait while partially informative on forms less associated with the trait.     

Genetic diversity of cultivated flax (Linum usitatissimum L.) germplasm assessed by retrotransposon-based markers

Retrotransposon segments were characterized and inter-retrotransposon amplified polymorphism (IRAP) markers developed for cultivated flax (Linum usitatissimum L.) and the Linum genus. Over 75 distinct long terminal repeat retrotransposon segments were cloned, the first set for Linum, and specific primers designed for them. IRAP was then used to evaluate genetic diversity among 708 accessions of cultivated flax comprising 143 landraces, 387 varieties, and 178 breeding lines. These included both traditional and modern, oil (86), fiber (351), and combined-use (271) accessions, originating from 36 countries, and 10 wild Linum species. The set of 10 most polymorphic primers yielded 141 reproducible informative data points per accession, with 52% polymorphism and a 0.34 Shannon diversity index. The maximal genetic diversity was detected among wild Linum species (100% IRAP polymorphism and 0.57 Jaccard similarity), while diversity within cultivated germplasm decreased from landraces (58%, 0.63) to breeding lines (48%, 0.85) and cultivars (50%, 0.81). Application of Bayesian methods for clustering resulted in the robust identification of 20 clusters of accessions, which were unstratified according to origin or user type. This indicates an overlap in genetic diversity despite disruptive selection for fiber versus oil types. Nevertheless, eight clusters contained high proportions (70?C100%) of commercial cultivars, whereas two clusters were rich (60%) in landraces. These findings provide a basis for better flax germplasm management, core collection establishment, and exploration of diversity in breeding, as well as for exploration of the role of retrotransposons in flax genome dynamics.     

Osmotic stress alters the balance between organic and inorganic solutes in flax (Linum usitatissimum)

Flax (Linum usitatissimum) is grown for its oil and its fiber. This crop, cultivated in temperate regions, has seen a renewed interest due to the presence of abundant molecules of interest for many applications. Little information is available about the behavior of flax during osmotic stress; yet this is considered a major stress that causes significant yield losses in most crops. To control the presence of this stress better, flax behavior was investigated following the application of osmotic stress and the response was examined by applying increasing concentrations of PEG 8000. This resulted in the reorganization of 32 metabolites and 6 mineral ions in the leaves. The analysis of these two types of solute highlighted the contrasting behavior between a higher metabolite content (particularly fructose, glucose and proline) and a decrease in mineral ions (especially nitrate and potassium) following PEG treatment. However, this reorganization did not lead to a greater accumulation of solutes, with the total amount remaining unchanged in leaves during osmotic stress.     

Population-based resequencing revealed an ancestral winter group of cultivated flax: implication for flax domestication processes

Cultivated flax (Linum usitatissimum L.) is the earliest oil and fiber crop and its early domestication history may involve multiple events of domestication for oil, fiber, capsular indehiscence, and winter hardiness. Genetic studies have demonstrated that winter cultivated flax is closely related to oil and fiber cultivated flax and shows little relatedness to its progenitor, pale flax (L. bienne Mill.), but winter hardiness is one major characteristic of pale flax. Here, we assessed the genetic relationships of 48 Linum samples representing pale flax and four trait-specific groups of cultivated flax (dehiscent, fiber, oil, and winter) through population-based resequencing at 24 genomic regions, and revealed a winter group of cultivated flax that displayed close relatedness to the pale flax samples. Overall, the cultivated flax showed a 27% reduction of nucleotide diversity when compared with the pale flax. Recombination frequently occurred at these sampled genomic regions, but the signal of selection and bottleneck was relatively weak. These findings provide some insight into the impact and processes of flax domestication and are significant for expanding our knowledge about early flax domestication, particularly for winter hardiness.     

Genome-wide analysis of genomic imprinting in the endosperm and allelic variation in flax

Genomic imprinting is an epigenetic phenomenon that causes biased expression of maternally and paternally inherited alleles. In flowering plants, genomic imprinting predominantly occurs in the triploid endosperm and plays a vital role in seed development. In this study, we identified 248 candidate imprinted genes including 114 maternally expressed imprinted genes (MEGs) and 134 paternally expressed imprinted genes (PEGs) in flax (Linum usitatissimum L.) endosperm using deep RNA sequencing. These imprinted genes were neither clustered in specific chromosomal regions nor well conserved among flax and other plant species. MEGs tended to be expressed specifically in the endosperm, whereas the expression of PEGs was not tissue-specific. Imprinted single nucleotide polymorphisms differentiated 200 flax cultivars into the oil flax, oil-fiber dual purpose flax and fiber flax subgroups, suggesting that genomic imprinting contributed to intraspecific variation in flax. The nucleotide diversity of imprinted genes in the oil flax subgroup was significantly higher than that in the fiber flax subgroup, indicating that some imprinted genes underwent positive selection during flax domestication from oil flax to fiber flax. Moreover, imprinted genes that underwent positive selection were related to flax functions. Thirteen imprinted genes related to flax seed size and weight were identified using a candidate gene-based association study. Therefore, our study provides information for further exploration of the function and genomic variation of imprinted genes in the flax population.     

Differential Uptake of Tritiated Thymidine into Hetero- and Euchromatin in Melanoplus and Secale

Grasshoppers of the species Melanoplus differentialis were injected with tritium-labelled thymidine. At intervals thereafter autoradiographic stripping film was applied over Feulgen squashes and sections. In this species during early prophase of meiosis the sex chromosome forms a heterochromatic block large enough to be resolved in tritium autoradiographs. A study of the squash preparations reveals that the sex chromosome is synthesizing DNA at a different period of time from the euchromatic autosomes. Since there is a developmental sequence of spermatocyte cysts along the testicular tubes it is possible from the sections to show that the heterochromatin synthesizes DNA later than does the euchromatin. To find out whether the results obtained in Melanoplus were characteristic of heterochromatin in general, young seedlings of rye were grown in a tritiated thymidine solution and Feulgen squashes were made as for Melanoplus. In rye leaf nuclei there is a large block of heterochromatin constituted by the proximal regions of the chromosomes and a euchromatic one formed by the median and distal regions of the same chromosomes. Here also the heterochromatin synthesizes DNA at a different period of time from the euchromatin. It is concluded that in rye the asynchrony of synthesis occurs within each chromosome. Counts of silver grains over the two types of chromatin in nuclei of Melanoplus and Secale disclosed that the number of grains per unit area was two to three times higher over the heterochromatin. To check the DNA content, Feulgen photometric measurements were made of Melanoplus nuclei at the same stage. The Feulgen and grain counts agree in showing that the heterochromatin contains two to three times more DNA per unit area than the euchromatin.     

不同覆盖方式对旱地胡麻土壤硝态氮和籽粒产量的影响

为了探讨黄土高原旱作雨养条件下覆盖种植胡麻田土壤硝态氮动态特征和增产效果,在2015和2016年胡麻生长季,以常规种植模式为对照(CK),研究了3种覆盖方式(全膜微垄覆土穴播、全膜覆土穴播和秸秆覆盖条播)对胡麻籽粒产量和关键生育时期农田硝态氮分布的影响.结果表明: 与CK相比,覆盖处理的胡麻籽粒产量分别增加56.1%(2015)和22.7%(2016),且均以全膜覆土穴播处理的籽粒产量最高.覆膜提高了胡麻根际土壤水分含量,在整个生育期的影响程度为先增加后降低;随着胡麻生育进程的推进,土壤硝态氮含量逐渐降低.两年试验中,覆盖处理下胡麻现蕾期0～40 cm土层硝态氮含量分别比CK增加3.1%～18.6%(2015)和5.1%～16.4%(2016),其中,全膜覆土穴播的增幅最大.2015年,胡麻盛花期和成熟期0～100 cm土层土壤硝态氮积累量较CK分别增加10.2%～22.2%和8.6%～21.4%,特别是在降雨较多的成熟期,0～40 cm土层土壤硝态氮含量较40～100 cm土层显著提高3.3%～4.9%,说明覆盖处理可以减缓硝态氮向下层的迁移.2016年,生育后期的高温干旱对胡麻生长造成了极大的影响,胡麻成熟期覆盖处理0～100 cm土层硝态氮积累量较CK显著增加6.6%～18.0%.相关分析表明,胡麻关键生育时期不同土层的土壤硝态氮含量与籽粒产量之间有显著正相关关系.综合考虑,全膜覆土穴播模式是适宜干旱、半干旱区推广应用的胡麻高产高效栽培方式.     

Importance of galacturonic Acid in controlling the retting of flax

The empirical test was compared with the determination of pH, total acidity, and galacturonic acid (GA) as methods for establishing the termination time of the retting process in flax. The GA dosage was the best method for determining the end point of this process by distinctly dividing the phases of retting and over-retting in the bacterial loosening of flax fiber. This end point is critical in the production of standard fibers for the weaving industry.     

Homofusion of Golgi secretory vesicles in flax phloem fibers during formation of the gelatinous secondary cell wall

The gelatinous type of secondary cell wall is present in tension wood and in phloem fibers of many plants. It is characterized by the absence of xylan and lignin, a high cellulose content and axially orientated microfibrils in the huge S2 layer. In flax phloem fiber, the major non-cellulosic component of such cell walls is tissue-specific galactan, which is tightly bound to cellulose. Ultrastructural analysis of flax fiber revealed that initiation of gelatinous secondary cell wall formation was accompanied by the accumulation of specific Golgi vesicles, which had a characteristic bicolor (dark-light) appearance and were easily distinguishable from vesicles made in different tissues and during the other stages of fiber development. Many of the bicolor vesicles appeared to fuse with each other, forming large vacuoles. The largest observed was 4 mum in diameter. Bicolor vesicles and vacuoles fused with the plasma membrane and spread their content in a characteristic "syringe-like" manner, covering a significant area of periplasm and forming "dark" stripes on the inner wall surface. Both Golgi derivatives and cell wall layers were labeled by LM5 antibody, indicating the presence of tissue- and stage-specific (1-->4)-beta-galactan. We suggest that this specific type of galactan secretion, which allows coverage of a large area of periplasm, is designed to increase the chance of the galactan meeting the cellulose microfibrils while they are still in the process of construction. The membrane fusion machinery of flax fiber must possess special components, which may be crucial for the formation of the gelatinous type cell wall.     

Pattern of the Cyanide-Potential in Developing Fruits : Implications for Plants Accumulating Cyanogenic Monoglucosides (Phaseolus lunatus) or Cyanogenic Diglucosides in Their Seeds (Linum usitatissimum, Prunus amygdalus)

The absolute cyanide content of developing fruits was determined in Costa Rican wild lima beans (Phaseolus lunatus), oil flax (Linum usitatissimum), and bitter almonds (Prunus amygdalus). The cyanide potential (HCN-p) of the lima bean and the almond fruit began to increase shortly after anthesis and then stopped before fruit maturity. In contrast, the flax inflorescence had a higher HCN-p in absolute terms than the mature flax fruit. At all times of its development the bean fruit contained the monoglucosides linamarin and lotaustralin. The almond and the flax fruits contained, at anthesis, the monoglucosides prunasin, and linamarin and lotaustralin, respectively, while, at maturity, only the corresponding diglucosides amygdalin, and linustatin and neolinustatin, respectively, were present.     

Identification of QTLs Associated with Oil Content in a High-Oil Brassica napus Cultivar and Construction of a High-Density Consensus Map for QTLs Comparison in B. napus

Increasing seed oil content is one of the most important goals in breeding of rapeseed (B. napus L.). To dissect the genetic basis of oil content in B. napus, a large and new double haploid (DH) population containing 348 lines was obtained from a cross between ‘KenC-8’ and ‘N53-2’, two varieties with >10% difference in seed oil content, and this population was named the KN DH population. A genetic linkage map consisting of 403 markers was constructed, which covered a total length of 1783.9 cM with an average marker interval of 4.4 cM. The KN DH population was phenotyped in eight natural environments and subjected to quantitative trait loci (QTL) analysis for oil content. A total of 63 identified QTLs explaining 2.64–17.88% of the phenotypic variation were identified, and these QTLs were further integrated into 24 consensus QTLs located on 11 chromosomes using meta-analysis. A high-density consensus map with 1335 marker loci was constructed by combining the KN DH map with seven other published maps based on the common markers. Of the 24 consensus QTLs in the KN DH population, 14 were new QTLs including five new QTLs in A genome and nine in C genome. The analysis revealed that a larger population with significant differences in oil content gave a higher power detecting new QTLs for oil content, and the construction of the consensus map provided a new clue for comparing the QTLs detected in different populations. These findings enriched our knowledge of QTLs for oil content and should be a potential in marker-assisted breeding of B. napus.     

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.     

水氮耦合对旱地胡麻产量形成与花后氮素积累转运的影响

为明确旱地胡麻在有限灌水条件下的最佳水氮耦合管理模式,采用完全随机裂区试验设计,以灌水(I₀: 0 m³·hm^-2; I₁₂₀₀: 1200 m³·hm^-2; I₁₈₀₀: 1800 m³·hm^-2)为主区,施氮量(N₀: 0 kg·hm^-2; N₆₀₀: 60 kg·hm^-2; N₁₂₀: 120 kg·hm^-2)为副区,测定胡麻不同生育阶段氮素积累量、花后氮素转运特征、产量和氮肥利用率。结果表明: 不同水氮处理对旱地胡麻不同生育时期各器官氮素吸收、积累及产量的耦合效应不同。不灌水条件下,施氮有利于胡麻花期和成熟期茎秆对氮素的吸收,不同灌水水平下N₁₂₀均抑制了茎秆对氮的吸收;I₁₂₀₀水平下,花期叶片氮含量随施氮量的增加先升高后下降,N₆₀较N₀和N₁₂₀高11.0%和28.9%;I₁₈₀₀水平下,施氮提高了成熟期胡麻叶片中氮含量,N₆₀和N₁₂₀较N₀高39.7%和26.9%。水氮对胡麻阶段氮素积累量影响的耦合效应主要表现在现蕾期以后,同一灌水水平下,N₆₀促进了胡麻现蕾期以后各阶段氮素积累量,而N₁₂₀具有抑制作用。施氮分别提高了I₁₂₀₀和I₁₈₀₀水平下叶片和茎秆氮素转运率和贡献率。灌水1800 m³·hm^-2、施氮60 kg·hm^-2显著增加了胡麻单株有效蒴果数和籽粒产量(6.6%~22.8%),是试验区比较适宜的水氮耦合管理模式。