Linum Bienne Research Articles

Genetic Relationships of Cultivated Flax and Its Wild Progenitor as Revealed by 454 Pyrosequencing, Sanger Resequencing and Microsatellite Data

Flax (Linum usitatissimum L.), as the earliest oil and fiber crop, is a model plant for genetic inferences of plant domestication processes involving multiple domestication events. However, a puzzle has emerged from several genetic studies, as dehiscent cultivated flax is genetically more related to its progenitor pale flax (L. bienne Mill.), and winter cultivated flax is well mixed with oil or fiber cultivated flax, while capsular dehiscence and winter hardiness are the major characteristics of pale flax. For this, a comparative analysis was conducted with 16 Linum samples representing pale flax and four domestication groups of cultivated flax (oil, fiber, winter, and dehiscent) using 454 pyrosequencing, Sanger resequencing and microsatellite data. It was found that the genomic sampling of genetic variants from the three applied methods yielded similar genetic information on pale flax and four groups of cultivated flax. The revealed genetic relationships did not show significant departures from the previous findings, but instead supported an early, independent domestication of a primitive flax lineage for oil use, followed by a subsequent flax domestication process with multiple domestication events for capsular dehiscence, oil, fiber and winter hardiness. Domestication on capsular dehiscence occurred earlier than domestication on winter hardiness. Domestication on winter hardiness was more complicated than domestication on capsular dehiscence.

Local climate and vernalization sensitivity predict the latitudinal patterns of flowering onset in the crop wild relative Linum bienne Mill.

The timing of flowering onset often correlates with latitude, indicative of climatic gradients. Flowering onset in temperate species commonly requires exposure to cold temperatures, known as vernalization. Hence, population differentiation of flowering onset with latitude might reflect adaptation to the local climatic conditions experienced by populations. Within its Western range, seeds from Linum bienne populations (the wild relative of cultivated L. usitatissimum) were used to describe the latitudinal differentiation of flowering onset to determine its association with the population´s local climate. A vernalization experiment, including different crop cultivars, was used to determine how vernalization accelerates flowering onset, and the vernalization sensitivity response among populations and cultivars. Additionally, genetic differentiation of L. bienne populations along the latitudinal range was scrutinized using microsatellite markers. Flowering onset varied with latitude of origin, with southern populations flowering earlier than their northern counterparts. Vernalization reduced the number of days to flowering onset, but vernalization sensitivity was greater in northern populations compared to southern ones. Conversely, vernalization delayed flowering onset in the crop, exhibiting less variation in sensitivity. In L. bienne, both flowering onset and vernalization sensitivity were better predicted by the population´s local climate than latitude itself. Microsatellite data unveiled genetic differentiation of populations, forming two groups geographically partitioned along latitude. The consistent finding of latitudinal variation across experiments suggests that both flowering onset and vernalization sensitivity in L. bienne populations are under genetic regulation and might depend on climatic cues at the place of origin. The association with climatic gradients along latitude suggests that the climate experienced locally drives population differentiation of the flowering onset and vernalization sensitivity patterns. The genetic population structure suggests that past population history could have influenced the flowering initiation patterns detected, which deserves further work.

Phenotypic analysis of Longya-10 × pale flax hybrid progeny and identification of candidate genes regulating prostrate/erect growth in flax plants.

Flax is a dual-purpose crop that is important for oil and fiber production. The growth habit is one of the crucial targets of selection during flax domestication. Wild hybridization between cultivated flax and wild flax can produce superior germplasms for flax breeding and facilitate the study of the genetic mechanism underlying agronomically important traits. In this study, we used pale flax, Linum grandiflorum, and L. perenne to pollinate Longya-10. Only pale flax interspecific hybrids were obtained, and the trait analysis of the F1 and F2 generations showed that the traits analyzed in this study exhibited disparate genetic characteristics. In the F1 generation, only one trait, i.e., the number of capsules per plant (140) showed significant heterosis, while the characteristics of other traits were closely associated with those of the parents or a decline in hybrid phenotypes. The traits of the F2 generation were widely separated, and the variation coefficient ranged from 9.96% to 146.15%. The quantitative trait locus underlying growth habit was preliminarily found to be situated on chromosome 2 through Bulked-segregant analysis sequencing. Then linkage mapping analysis was performed to fine-map GH2.1 to a 23.5-kb interval containing 4 genes. Among them, L.us.o.m.scaffold22.109 and L.us.o.m.scaffold22.112 contained nonsynonymous SNPs with Δindex=1. Combined with the qRT-PCR results, the two genes might be possible candidate genes for GH2.1. This study will contribute to the development of important germplasms for flax breeding, which would facilitate the elucidation of the genetic mechanisms regulating the growth habit and development of an ideal architecture for the flax plant.

Microsatellite marker development in the crop wild relative Linum bienne using genome skimming.

PremiseNuclear microsatellite markers were developed for Linum bienne, the sister species of the crop L. usitatissimum, to provide molecular genetic tools for the investigation of L. bienne genetic diversity and structure.Methods and ResultsFifty microsatellite loci were identified in L. bienne by means of genome skimming, and 44 loci successfully amplified. Of these, 16 loci evenly spread across the L. usitatissimum reference nuclear genome were used for genotyping six L. bienne populations. Excluding one monomorphic locus, the number of alleles per locus ranged from two to 12. Four out of six populations harbored private alleles. The levels of expected and observed heterozygosity were 0.076 to 0.667 and 0.000 to 1.000, respectively. All 16 loci successfully cross‐amplified in L. usitatissimum.ConclusionsThe 16 microsatellite loci developed here can be used for population genetic studies in L. bienne, and 28 additional loci that successfully amplified are available for further testing.

Genomic Comparison and Population Diversity Analysis Provide Insights into the Domestication and Improvement of Flax.

SummaryFlax has been cultivated for its oil and fiber for thousands of years. However, it remains unclear how the modifications of agronomic traits occurred on the genetic level during flax cultivation. In this study, we conducted genome-wide variation analyses on multiple accessions of oil-use, fiber-use, landraces, and pale flax to identify the genomic variations during flax cultivation. Our findings indicate that, during flax domestication, genes relevant to flowering, dehiscence, oil production, and plant architecture were preferentially selected. Furthermore, regardless of origins, the improvement of the modern oil-use flax preceded that of the fiber-use flax, although the dual selection on oil-use and fiber-use characteristics might have occurred in the early flax domestication. We also found that the expansion of MYB46/MYB83 genes may have contributed to the unique secondary cell wall biosynthesis in flax and the directional selections on MYB46/MYB83 may have shaped the morphological profile of the current oil-use and fiber-use flax.

English

Linseed is the only cultivated species from the genus Linum and selection is the most frequently used method to develop varieties from the crop resulting in the reduction of the genetic diversity. Linum bienne Mill. is genetically more diverse than linseed and produces fertile hybrids with linseed. The author aimed for the development of hybrids with new combinations of genes useful for variety development programme. Morphological characters of parental, F1 and F2 hybrid plants were studied in field and cluster analyses, coefficient of variations (CV) and Nested analysis of variance (NANOVA) were used for the analyses. Cluster analyses from combined quantitative and qualitative characteristics were more powerful in grouping genotype. Selfed F2 hybrids scored the highest CV for all characteristics and seed-weight (20.36%). The degree of boll shattering was different among hybrids. F2 hybrids scored more phenotypic classes from seed coat colour. The differences in seed length and 1000-SW among the groups were significant (P = 0.017 and 0.033, respectively). Except for the differences in seed length, all the mean value differences in quantitative characteristics among sub-groups within the group were significant (P < 0.01). The result showed that the hybrids would be important populations to develop varieties for different traits. There was dragging of unwanted parental characters to hybrids due to a linkage. Assisting the process of crossing with markers associated with a trait would help to minimize the dragging of unwanted characters into hybrids. Key words: Linum bienne, Linum usitatissimum, crop wild relative, segregation, crossing.

SSR and EST-SSR-based population genetic structure of Linum L. (Linaceae) species in Iran

The genus Linum L. (Linaceae) has more than 200 species. These species have agricultural, industrial, and medicinal importance. Domestication and continuous selection reduce genetic diversity of plants and in order to develop new and improved varieties, we should utilize all available genetic diversity in that crop and its wild relatives. The cultivated flax (L. usitatissimum L.) is an important crop plant and is being evaluated as a crop platform for the production of bio-industrial and nutraceutical products. In present study, we studied 230 plants of 23 populations in four Linum species namely, pale flax (L. bienne Mill.), cultivated flax (L. usitatissimum L.), and two wild relatives, L. austriacum L. and L. album L. The objective of this study was to investigate population genetic structure and genetic variability within each species using SSR and EST-SSR markers. The low degree of genetic variability was obtained in these species. AMOVA revealed significant genetic difference among species and populations. The significant correlation was observed between genetic and geographical distances the studied species. Evanno test and K-Means clustering revealed genetic fragmentation in the studied Linum species. Networking and STRUCTURE analysis revealed genetic affinity of the populations. The population assignment test revealed absence of gene flow or the occurrence of very limited gene flow among populations.

The potential of pale flax as a source of useful genetic variation for cultivated flax revealed through molecular diversity and association analyses.

Pale flax (Linum bienne Mill.) is the wild progenitor of cultivated flax (Linum usitatissimum L.) and represents the primary gene pool to broaden its genetic base. Here, a collection of 125 pale flax accessions and the Canadian flax core collection of 407 accessions were genotyped using 112 genome-wide simple sequence repeat markers and phenotyped for nine traits with the aim of conducting population structure, molecular diversity and association mapping analyses. The combined population structure analysis identified two well-supported major groups corresponding to pale and cultivated flax. The L. usitatissimum convar. crepitans accessions most closely resembled its wild progenitor, both having dehiscent capsules. The unbiased Nei’s genetic distance (0.65) confirmed the strong genetic differentiation between cultivated and pale flax. Similar levels of genetic diversity were observed in both species, albeit 430 (48 %) of pale flax alleles were unique, in agreement with their high genetic differentiation. Significant associations were identified for seven and four traits in pale and cultivated flax, respectively. Favorable alleles with potentially positive effect to improve yield through yield components were identified in pale flax. The allelic frequencies of markers associated with domestication-related traits such as capsular dehiscence indicated directional selection with the most common alleles in pale flax being absent or rare in cultivated flax and vice versa. Our results demonstrated that pale flax is a potential source of novel variation to improve multiple traits in cultivated flax and that association mapping is a suitable approach to screening pale flax germplasm to identify favorable quantitative trait locus alleles.Electronic supplementary materialThe online version of this article (doi:10.1007/s11032-014-0165-5) contains supplementary material, which is available to authorized users.

Structural organization and classification of cytochrome P450 genes in flax (Linum usitatissimum L.)

Flax CYPome analysis resulted in the identification of 334 putative cytochrome P450 (CYP450) genes in the cultivated flax genome. Classification of flax CYP450 genes based on the sequence similarity with Arabidopsis orthologs and CYP450 nomenclature, revealed 10 clans representing 44 families and 98 subfamilies. CYP80, CYP83, CYP92, CYP702, CYP705, CYP708, CYP728, CYP729, CYP733 and CYP736 families are absent in the flax genome. The subfamily members exhibited conserved sequences, length of exons and phasing of introns. Similarity search of the genomic resources of wild flax species Linum bienne with CYP450 coding sequences of the cultivated flax, revealed the presence of 127 CYP450 gene orthologs, indicating amplification of novel CYP450 genes in the cultivated flax. Seven families CYP73, 74, 75, 76, 77, 84 and 709, coding for enzymes associated with phenylpropanoid/fatty acid metabolism, showed extensive gene amplification in the flax. About 59% of the flax CYP450 genes were present in the EST libraries.

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.

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.

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 and Relationship of Flax Germplasm as Revealed by AFLP Analysis

Flax (linum usitatissimum) is one of the major fibre and oil yielding crops. AFLP is very useful molecular marker for a number of plant species. To reveal the genetic diversity and find ways to broaden the genetic background of Chinese fibre flax varieties, we analyzed 18 Chinese fibre flax cultivars and other 67 accessions (including linseed, landrace and wild species) from different countries in the world by using AFLP marker. In total, 168 polymorphic alleles were detected with seven pairs of AFLP primer. For each pair of primer, 24 alleles on the average was detected in all accessions. Unweighted pair-group method with arithmetical averages (UPGMA) cluster analysis was used to estimate the genetic distances and to construct a dendrogram by NTSYS 2.1. The results showed that all 85 accessions were classified into four groups at 0.53 of similarity coefficient, which distinguish the varieties of flax (Linum usitatissimum) from three wild flax species. All varieties of flax were classified into five sub-groups (all fibre flax accessions were in one sub-group and the most of linseed accessions divided into other four sub-groups) at 0.67, indicating linseed possessed genetic basis broader than the fibre flax. In Chinese fibre flax varieties, Heiya cultivars had high genetic similarity, and its genetic basis was narrower than that of Shuangya variety. The results also showed that there were close relationship between Linum bienne and Linum usitatissinum, and sustained the hypothesis that fibre flax has evolved from linseed.

Flax and weld: archaeobotanical records from Mutina (Emilia Romagna, Northern Italy), dated to the Imperial Age, first half 1st century a.d.

In the city centre of Modena (Emilia Romagna, Northern Italy) a noteworthy amount of carpological remains attributable to flax and weld came to light dated to the Imperial Age (first half 1st century a.d.). Flax remains, i.e. capsules and extremely small seeds, suggest the presence of either immature seeds and fruits of cultivated flax (Linum usitatissimum L.) or of a wild flax species (Linum bienne Mill.). The quantity of these remains along with the co-presence of seeds of Reseda luteola, an important dye plant, leads to the hypothesis of the cultivation of both flax and weld for textile manufacturing purposes, which are attested as important in the economy of the Roman period.

Variation in phenotypic characters of pale flax (Linum bienne Mill.) from Turkey

The diversity of pale flax (Linum bienne Mill.) as the progenitor of cultivated flax (L. usitatissimum L.) has not been well documented and the domestication syndromes in cultivated flax are poorly understood. An attempt was made to characterize 34 pale flax accessions and six cultivated flax accessions collected during 2007 summer in Turkey. A total of 12 quantitative and 7 qualitative characters covering vegetative and generative plant parts, including phenological traits, were assessed. The occurrence of yellow anthers well known in cultivated flax is reported for the first time in pale flax. Pale flax displayed larger variation in vegetative plant parts and growth habit than the cultivated flax and more heterogeneity within accessions. Within pale flax, a higher degree of variation was observed in many generative parts such as the flower characters than in the capsule and seed characters. Based on the assayed characters, the pale flax from Turkey was grouped into three clusters and these clusters were associated with site elevation and longitude, further confirming local genetic differentiation in pale flax from Turkey. These findings are significant for further studies of flax domestication history and useful for further exploitation of wild flax in genetic improvement of cultivated flax.

Genetic evidence for early flax domestication with capsular dehiscence

Flax (Linum usitatissimum L.) is the earliest oil and fibre crop, but little is known about its domestication process. Attempt was made here to assess genetic relationships of 63 Linum accessions representing seven typical groups of cultivated flax and its wild progenitor, pale flax (Linum bienne Mill.), by using 49 informative expressed sequence tag-derived simple sequence repeat (EST-SSR) primer pairs. The seven groups were pale flax from Turkey, pale flax from other countries, and five groups of cultivated flax (landrace, fibre, oil, winter, and dehiscent). From these 63 samples, 366 polymorphic bands were detected, which likely represented 79 loci. These polymorphic bands had frequencies that ranged from 0.016 to 0.984 and averaged 0.284. Group-specific EST-SSR variation (Fst values) ranged from 0.339 to 0.373 and averaged 0.349 and pairwise group EST-SSR variation ranged from 0.067 to 0.507. A neighbor-joining clustering of these seven groups revealed that dehiscent flax clustered most closely to its wild progenitor, pale flax, followed by oil flax and fibre flax. Winter flax clustered most closely to oil flax and less to pale flax. These clustering patterns were essentially the same when individual samples were analyzed via neighbor-joining. These findings strongly suggest that capsular dehiscence was among the first flax traits modified by human after initial domestication, reflecting the importance that reducing capsular dehiscence likely played in early flax domestication.

Characterization of expressed sequence tag-derived simple sequence repeat markers for 17 Linum species

One major challenge in genetic and evolutionary studies of wild flax species is the lack of informative molecular markers. A set of 100 informative expressed sequence tag-derived simple sequence repeat (EST-SSR) primer pairs developed in cultivated flax ( Linum usitatissimum L.) were characterized on 35 Linum accessions representing 17 Linum species for their transferability to other Linum species. Ninety-nine primer pairs displayed scorable polymorphisms across 35 Linum samples and generated 627 bands likely from 121 loci. About 50% of the detected bands occurred only in three or fewer samples. A total of 393 bands, likely from 116 loci, were detected by 97 primer pairs in Linum bienne Mill. samples, but only up to 60 bands, likely from up to 39 loci, were revealed by 6 to 37 primer pairs in the samples of the other 15 Linum species. The L. bienne samples displayed 23.7% more EST-SSR variation than the L. usitatissimum samples. These characterized EST-SSR markers should be useful for future genetic diversity and evolutionary studies of Linum species, particularly for the progenitor of cultivated flax.

Genetic diversity of cultivated flax (Linum usitatissimum L.) and its wild progenitor pale flax (Linum bienne Mill.) as revealed by ISSR markers

Little is known about the genetic diversity of pale flax (Linum bienne Mill.), the wild progenitor of cultivated flax (L. usitatissimum L.), and ex situ germplasm of pale flax was scarce. Effort was made to collect 34 pale flax accessions and five landrace accessions of cultivated flax in Turkey. The inter simple sequence repeat (ISSR) technique was applied to characterize this set of flax germplasm, along with one Turkish cultivar, one Russian cultivar, five winter and four dehiscent type accessions of cultivated flax. Twenty-four ISSR primer pairs detected a total of 311 DNA fragments, of which 298 bands were polymorphic across 493 flax samples (roughly 10 samples per accession). These polymorphic bands had frequencies ranging from 0.002 to 0.998 and averaging 0.38. Accession-specific ISSR variation (Fst values) ranged from 0.469 to 0.514 and averaged 0.493. There was 49.3% ISSR variation resided among these 50 accessions, 35.9% harbored among landrace, winter, dehiscent types of cultivated flax and pale flax, and 38.2% present among 34 pale flax accessions. Pale flax displayed more ISSR variation than landraces and dehiscent type, but less than winter type, of cultivated flax. Clustering 493 individual plants revealed that these assayed plants were largely grouped according to their plant types and that pale flax was genetically more close to the dehiscent type, followed by the winter type and landrace, of cultivated flax. Pale flax collected within the geographic range of 180 km displayed a significant spatial genetic autocorrelation. Genetic distances among the pale flax accessions were significantly associated with their geographic distances and elevation differences. These findings are significant for understanding flax domestication and its primary gene pool.

Phylogenetic network of Linum species as revealed by non-coding chloroplast DNA sequences

Crop wild relatives are invaluable sources of novel genes for crop improvement and adaptation to changing environments. We assessed phylogenetic relationships among 29 Linum accessions representing 16 species, including cultivated flax and its progenitor pale flax, based on four non-coding regions of chloroplast DNA sequences. We obtained a cpDNA network showing that these 16 Linum species are appropriately connected based on previously defined taxonomic sections; these connections reflect the same evolutionary pathways as determined from earlier morphological and cytological data. These relationships also support an earlier hypothesis that cultivated flax is probably descended from a single domestication of pale flax plants, apparently for oil usage. The detailed species network not only is significant for understanding evolutionary relationships of Linum species, but also useful for classifying exotic gene pools of cultivated flax as a part of the ongoing exploration of new genetic diversity for flax improvement.

Accumulation of Lignans by in vitro Cultures of Three Linum Species

Justicidin B, an arylnaphthalene lignan, has strong cytotoxicity on chronic myeloid and chronic lymphoid leukemia cell lines. The first report of the production of justicidin B in a Linum species concerned in vitro culture of Linum austriacum. Therefore, culture characterization and presence of arylnaphthalene-type lignans in calli and plantlets of Linum tenuifolium from section Linastrum, Linum bienne, and Linum glaucum from section Linum were studied. Seed germination of L. tenuifolium in the light and darkness was significantly higher (p < 0.05) than of L. bienne in the light and L. glaucum in the darkness. L. tenuifolium seedling length in the darkness was significantly higher (p < 0.01) than under light conditions. There were no significant differences in the calli and shoot biomass weight, number and length of shoots in three species over one month, while the shoot diameter of L. bienne was significantly different (p < 0.05) from that of L. glaucum. Justicidin B was detected in L. glaucum callus and plantlet cultures by HPLC/MS/UV-DAD and HPLC coupled with a photodiode array detector. This finding is important from pharmaceutical point of view and shows the chemosystematic relation between L. glaucum and L. austriacum and this method will be a powerful tool for detecting natural products in interested and endangered medicinal plants.