An R2R3-MYB Transcriptional Factor LuMYB314 Associated with the Loss of Petal Pigmentation in Flax (Linum usitatissimum L.).

The loss of anthocyanin pigments is one of the most common evolutionary transitions in petal color, yet the genetic basis for these changes in flax remains largely unknown. In this study, we used crossing studies, a bulk segregant analysis, genome-wide association studies, a phylogenetic analysis, and transgenic testing to identify genes responsible for the transition from blue to white petals in flax. This study found no correspondence between the petal color and seed color, refuting the conclusion that a locus controlling the seed coat color is associated with the petal color, as reported in previous studies. The locus controlling the petal color was mapped using a BSA-seq analysis based on the F2 population. However, no significantly associated genomic regions were detected. Our genome-wide association study identified a highly significant QTL (BP4.1) on chromosome 4 associated with flax petal color in the natural population. The combination of a local Manhattan plot and an LD heat map identified LuMYB314, an R2R3-MYB transcription factor, as a potential gene responsible for the natural variations in petal color in flax. The overexpression of LuMYB314 in both Arabidopsis thaliana and Nicotiana tabacum resulted in anthocyanin deposition, indicating that LuMYB314 is a credible candidate gene for controlling the petal color in flax. Additionally, our study highlights the limitations of the BSA-seq method in low-linkage genomic regions, while also demonstrating the powerful detection capabilities of GWAS based on high-density genomic variation mapping. This study enhances our genetic insight into petal color variations and has potential breeding value for engineering LuMYB314 to develop colored petals, bast fibers, and seeds for multifunctional use in flax.

The Quantity and Quality of Flax and Hemp Fibers Obtained Using the Osmotic, Water-, and Dew-Retting Processes.

This study presents the quantity and quality of flax (Linum usitatissimum L.) and hemp (Cannabis sativa L.) fibers obtained depending on the fiber extraction method. The extraction methods used in this study were osmotic degumming, dew retting, and water retting. The degummed straw was analyzed for fiber content, while the metrological, chemical, and physical properties were determined for the fibers obtained. It was shown that these properties change based on the method of fiber extraction used. The highest fiber content in the straw was obtained using the osmotic degumming method. These fibers are characterized by a light color, no unpleasant odor, low linear mass, good tenacity, lowest hygroscopicity, and reduced flammability compared to fibers obtained via the dew and water retting of straw.

Identification of new potential molecular actors related to fiber quality in flax through Omics.

One of the biggest challenges for a more widespread utilization of plant fibers is to better understand the different molecular factors underlying the variability in fineness and mechanical properties of both elementary and scutched fibers. Accordingly, we analyzed genome-wide transcription profiling from bast fiber bearing tissues of seven different flax varieties (4 spring, 2 winter fiber varieties and 1 winter linseed) and identified 1041 differentially expressed genes between varieties, of which 97 were related to cell wall metabolism. KEGG analysis highlighted a number of different enriched pathways. Subsequent statistical analysis using Partial Least-Squares Discriminant Analysis showed that 73% of the total variance was explained by the first 3 X-variates corresponding to 56 differentially expressed genes. Calculation of Pearson correlations identified 5 genes showing a strong correlation between expression and morphometric data. Two-dimensional gel proteomic analysis on the two varieties showing the most discriminant and significant differences in morphometrics revealed 1490 protein spots of which 108 showed significant differential abundance. Mass spectrometry analysis successfully identified 46 proteins representing 32 non-redundant proteins. Statistical clusterization based on the expression level of genes corresponding to the 32 proteins showed clear discrimination into three separate clusters, reflecting the variety type (spring-/winter-fiber/oil). Four of the 32 proteins were also highly correlated with morphometric features. Examination of predicted functions for the 9 (5 + 4) identified genes highlighted lipid metabolism and senescence process. Calculation of Pearson correlation coefficients between expression data and retted fiber mechanical measurements (strength and maximum force) identified 3 significantly correlated genes. The genes were predicted to be connected to cell wall dynamics, either directly (Expansin-like protein), or indirectly (NAD(P)-binding Rossmann-fold superfamily protein). Taken together, our results have allowed the identification of molecular actors potentially associated with the determination of both in-planta fiber morphometrics, as well as ex-planta fiber mechanical properties, both of which are key parameters for elementary fiber and scutched fiber quality in flax.

RNA-Seq combined with population-level analysis reveals important candidate genes related to seed size in flax (Linum usitatissimum L.).

Seed size is a key determinant of crop yields. Understanding the regulatory mechanisms of seed size is beneficial for improving flax seed yield. In this study, the development of large flax seeds lagged behind that of small seeds, and 1,751 protein-coding genes were differentially expressed in early seeds, torpedo-stage embryos, and endosperms of CIli2719 and Z11637 using RNA sequencing. Homologous alignment revealed that 129 differentially expressed genes (DEGs) in flax were homologous with 71 known seed size-related genes in Arabidopsis thaliana and rice (Oryza sativa L.). These DEGs controlled seed size through multiple processes and factors, among which phytohormone pathways and transcription factors were the most important. Moreover, 54 DEGs were found to be associated with seed size and weight in a DEG-based association study. Nucleotide diversity (π) analysis of seed size-related candidate DEGs by homologous alignment and association analysis showed that the π values decreased significantly during flax acclimation from oil to fiber flax, suggesting that some seed size-related candidate genes were selected in this acclimation process. These results provide important resources and genetic foundation for further research on seed size regulation and seed improvement in flax.

Raman spectroscopy mapping of changes in the organization and relative quantities of cell wall polymers in bast fiber cell walls of flax plants exposed to gravitropic stress.

Flax is an important fiber crop that is subject to lodging. In order to gain more information about the potential role of the bast fiber cell wall in the return to the vertical position, 6-week-old flax plants were subjected to a long-term (6 week) gravitropic stress by stem tilting in an experimental set-up that excluded autotropism. Stress induced significant morphometric changes (lumen surface, lumen diameter, and cell wall thickness and lumen surface/total fiber surface ratio) in pulling- and opposite-side fibers compared to control fibers. Changes in the relative amounts and spatial distribution of cell wall polymers in flax bast fibers were determined by Raman vibrational spectroscopy. Following spectra acquisition, datasets (control, pulling- and opposite sides) were analyzed by principal component analysis, PC score imaging, and Raman chemical cartography of significant chemical bonds. Our results show that gravitropic stress induces discrete but significant changes in the composition and/or spatial organization of cellulose, hemicelluloses and lignin within the cell walls of both pulling side and opposite side fibers.

Genetic Architecture of Powdery Mildew Resistance Revealed by a Genome-Wide Association Study of a Worldwide Collection of Flax (Linum usitatissimum L.).

Powdery mildew is one of the most important diseases of flax and is particularly prejudicial to its yield and oil or fiber quality. This disease, caused by the obligate biotrophic ascomycete Oïdium lini, is progressing in France. Genetic resistance of varieties is critical for the control of this disease, but very few resistance genes have been identified so far. It is therefore necessary to identify new resistance genes to powdery mildew suitable to the local context of pathogenicity. For this purpose, we studied a worldwide diversity panel composed of 311 flax genotypes both phenotyped for resistance to powdery mildew resistance over 2 years of field trials in France and resequenced. Sequence reads were mapped on the CDC Bethune reference genome revealing 1,693,910 high-quality SNPs, further used for both population structure analysis and genome-wide association studies (GWASs). A number of four major genetic groups were identified, separating oil flax accessions from America or Europe and those from Asia or Middle-East and fiber flax accessions originating from Eastern Europe and those from Western Europe. A number of eight QTLs were detected at the false discovery rate threshold of 5%, located on chromosomes 1, 2, 4, 13, and 14. Taking advantage of the moderate linkage disequilibrium present in the flax panel, and using the available genome annotation, we identified potential candidate genes. Our study shows the existence of new resistance alleles against powdery mildew in our diversity panel, of high interest for flax breeding program.

Genome-Wide Association Study of Salt Tolerance at the Seed Germination Stage in Flax (Linum usitatissimum L.).

Soil salinization seriously affects the growth and distribution of flax. However, there is little information about the salt tolerance of flax. In this study, the salt tolerance of 200 diverse flax accessions during the germination stage was evaluated, and then the Genome-wide Association Study (GWAS) was carried out based on the relative germination rate (RGR), relative shoot length (RSL) and relative root length (RRL), whereby quantitative trait loci (QTLs) related to salt tolerance were identified. The results showed that oil flax had a better salt tolerance than fiber flax. A total of 902 single nucleotide polymorphisms (SNPs) were identified on 15 chromosomes. These SNPs were integrated into 64 QTLs, explaining 14.48 to 29.38% (R2) of the phenotypic variation. In addition, 268 candidate genes were screened by combining previous transcriptome data and homologous gene annotation. Among them, Lus10033213 is a single-point SNP repeat mapping gene, which encodes a Glutathione S-transferase (GST). This study is the first to use GWAS to excavate genes related to salt tolerance during the germination stage of flax. The results of this study provide important information for studying the genetic mechanism of salt tolerance of flax, and also provide the possibility to improve the salt tolerance of flax.

Resequencing 200 Flax Cultivated Accessions Identifies Candidate Genes Related to Seed Size and Weight and Reveals Signatures of Artificial Selection.

Seed size and weight are key traits determining crop yield, which often undergo strongly artificial selection during crop domestication. Although seed sizes differ significantly between oil flax and fiber flax, the genetic basis of morphological differences and artificial selection characteristics in seed size remains largely unclear. Here we re-sequenced 200 flax cultivated accessions to generate a genome variation map based on chromosome assembly reference genomes. We provide evidence that oil flax group is the ancestor of cultivated flax, and the oil-fiber dual purpose group (OF) is the evolutionary intermediate transition state between oil and fiber flax. Genome-wide association studies (GWAS) were combined with LD Heatmap to identify candidate regions related to seed size and weight, then candidate genes were screened based on detailed functional annotations and estimation of nucleotide polymorphism effects. Using this strategy, we obtained 13 candidate genes related to seed size and weight. Selective sweeps analysis indicates human-involved selection of small seeds during the oil to fiber flax transition. Our study shows the existence of elite alleles for seed size and weight in flax germplasm and provides molecular insights into approaches for further improvement.

Genome-Wide Association Study Identifying Candidate Genes Influencing Important Agronomic Traits of Flax (Linum usitatissimum L.) Using SLAF-seq.

Flax (Linum usitatissimum L.) is an important cash crop, and its agronomic traits directly affect yield and quality. Molecular studies on flax remain inadequate because relatively few flax genes have been associated with agronomic traits or have been identified as having potential applications. To identify markers and candidate genes that can potentially be used for genetic improvement of crucial agronomic traits, we examined 224 specimens of core flax germplasm; specifically, phenotypic data for key traits, including plant height, technical length, number of branches, number of fruits, and 1000-grain weight were investigated under three environmental conditions before specific-locus amplified fragment sequencing (SLAF-seq) was employed to perform a genome-wide association study (GWAS) for these five agronomic traits. Subsequently, the results were used to screen single nucleotide polymorphism (SNP) loci and candidate genes that exhibited a significant correlation with the important agronomic traits. Our analyses identified a total of 42 SNP loci that showed significant correlations with the five important agronomic flax traits. Next, candidate genes were screened in the 10 kb zone of each of the 42 SNP loci. These SNP loci were then analyzed by a more stringent screening via co-identification using both a general linear model (GLM) and a mixed linear model (MLM) as well as co-occurrences in at least two of the three environments, whereby 15 final candidate genes were obtained. Based on these results, we determined that UGT and PL are candidate genes for plant height, GRAS and XTH are candidate genes for the number of branches, Contig1437 and LU0019C12 are candidate genes for the number of fruits, and PHO1 is a candidate gene for the 1000-seed weight. We propose that the identified SNP loci and corresponding candidate genes might serve as a biological basis for improving crucial agronomic flax traits.