The Genetic Dissection of Nitrogen Use-Related Traits in Flax (Linum usitatissimum L.) at the Seedling Stage through the Integration of Multi-Locus GWAS, RNA-seq and Genomic Selection.

Nitrogen (N), the most important macro-nutrient for plant growth and development, is a key factor that determines crop yield. Yet its excessive applications pollute the environment and are expensive. Hence, studying nitrogen use efficiency (NUE) in crops is fundamental for sustainable agriculture. Here, an association panel consisting of 123 flax accessions was evaluated for 21 NUE-related traits at the seedling stage under optimum N (N+) and N deficiency (N-) treatments to dissect the genetic architecture of NUE-related traits using a multi-omics approach integrating genome-wide association studies (GWAS), transcriptome analysis and genomic selection (GS). Root traits exhibited significant and positive correlations with NUE under N- conditions (r = 0.33 to 0.43, p < 0.05). A total of 359 QTLs were identified, accounting for 0.11% to 23.1% of the phenotypic variation in NUE-related traits. Transcriptomic analysis identified 1034 differentially expressed genes (DEGs) under contrasting N conditions. DEGs involved in N metabolism, root development, amino acid transport and catabolism and others, were found near the QTLs. GS models to predict NUE stress tolerance index (NUE_STI) trait were tested using a random genome-wide SNP dataset and a GWAS-derived QTLs dataset. The latter produced superior prediction accuracy (r = 0.62 to 0.79) compared to the genome-wide SNP marker dataset (r = 0.11) for NUE_STI. Our results provide insights into the QTL architecture of NUE-related traits, identify candidate genes for further studies, and propose genomic breeding tools to achieve superior NUE in flax under low N input.

Genetic dissection of flowering time in flax (Linum usitatissimum L.) through single- and multi-locus genome-wide association studies.

In a rapidly changing climate, flowering time (FL) adaptation is important to maximize seed yield in flax (Linum usitatissimum L.). However, our understanding of the genetic mechanism underlying FL in this multipurpose crop remains limited. With the aim of dissecting the genetic architecture of FL in flax, a genome-wide association study (GWAS) was performed on 200 accessions of the flax core collection evaluated in four environments. Two single-locus and six multi-locus models were applied using 70,935 curated single nucleotide polymorphism (SNP) markers. A total of 40 quantitative trait nucleotides (QTNs) associated with 27 quantitative trait loci (QTL) were identified in at least two environments. The number of QTL with positive-effect alleles in accessions was significantly correlated with FL (r = 0.77 to 0.82), indicating principally additive gene actions. Nine QTL were significant in at least three of the four environments accounting for 3.06-14.71% of FL variation. These stable QTL spanned regions that harbored 27 Arabidopsis thaliana and Oryza sativa FL-related orthologous genes including FLOWERING LOCUS T (Lus10013532), FLOWERING LOCUS D (Lus10028817), transcriptional regulator SUPERMAN (Lus10021215), and gibberellin 2-beta-dioxygenase 2 (Lus10037816). In silico gene expression analysis of the 27 FL candidate gene orthologous suggested that they might play roles in the transition from vegetative to reproductive phase, flower development and fertilization. Our results provide new insights into the QTL architecture of flowering time in flax, identify potential candidate genes for further studies, and demonstrate the effectiveness of combining different GWAS models for the genetic dissection of complex traits.

Loci harboring genes with important role in drought and related abiotic stress responses in flax revealed by multiple GWAS models.

KEY MESSAGE: QTNs associated with drought tolerance traits and indices were identified in a flax mini-core collection through multiple GWAS models and phenotyping at multiple locations under irrigated and non-irrigated field conditions. Drought is a critical phenomenon challenging today's agricultural sector. Crop varieties adapted to moisture deficit are becoming vital. Flax can be greatly affected by limiting moisture conditions, especially during the early development and reproductive stages. Here, a mini-core collection comprising genotypes from more than 20 major growing countries was evaluated for 11 drought-related traits in irrigated and non-irrigated fields for 3 years. Heritability of the traits ranged from 44.7 to 86%. Six of the 11 traits showed significant phenotypic difference between irrigated and non-irrigated conditions. A genome-wide association study (GWAS) was performed for these six traits and their corresponding stress indices based on 106 genotypes and 12,316 single nucleotide polymorphisms (SNPs) using six multi-locus and one single-locus models. The SNPs were then assigned to 8050 linkage disequilibrium (LD) blocks to which a restricted two-stage multi-locus multi-allele GWAS was applied. A total of 144 quantitative trait nucleotides (QTNs) and 13 LD blocks were associated with at least one trait or stress index. Of these, 16 explained more than 15% of the genetic variance. Most large-effect QTN loci harbored gene(s) previously predicted to play role(s) in the associated traits. Genes mediating responses to abiotic stresses resided at loci associated with stress indices. Flax genes Lus10009480 and Lus10030150 that are predicted to encode WAX INDUCER1 and STRESS-ASSOCIATED PROTEIN (SAP), respectively, are among the important candidates detected. Accessions with multiple favorable alleles outperformed others for grain yield, thousand seed weight and fiber/biomass in non-irrigated conditions, suggesting their potential usefulness in breeding and genomic selection.

Genome-Wide Association Analysis of Mucilage and Hull Content in Flax (Linum usitatissimum L.) Seeds.

New flaxseed cultivars differing in seed mucilage content (MC) with low hull content (HC) represent an attractive option to simultaneously target the food and feed markets. Here, a genome-wide association study (GWAS) was conducted for MC and HC in 200 diverse flaxseed accessions genotyped with 1.7 million single nucleotide polymorphism (SNP) markers. The data obtained for MC and HC indicated a broad phenotypic variation and high (~70%) and a moderate (~49%) narrow sense heritability, respectively. MC and HC did not differ statistically between fiber and oil morphotypes, but yellow-seeded accessions had 2.7% less HC than brown-seeded ones. The genome-wide linkage disequilibrium (LD) decayed to r² = 0.1 at a physical distance of ~100 kb. Seven and four quantitative trait loci (QTL) were identified for MC and HC, respectively. Promising candidate genes identified include Linum usitatissimum orthologs of the Arabidopsis thaliana genes TRANSPARENT TESTA 8, SUBTILISIN-LIKE SERINE PROTEASE, GALACTUROSYL TRANSFERASE-LIKE 5, MUCILAGE-MODIFIED 4, AGAMOUS-LIKE MADS-BOX PROTEIN AGL62, GLYCOSYL HYDROLASE FAMILY 17, and UDP-GLUCOSE FLAVONOL 3-O-GLUCOSYLTRANSFERASE. These genes have been shown to play a role in mucilage synthesis and release, seed coat development and anthocyanin biosynthesis in A. thaliana. The favorable alleles will be useful in flaxseed breeding towards the goal of achieving the ideal MC and HC composition for food and feed by genomic-based breeding.

Association mapping of seed quality traits using the Canadian flax (Linum usitatissimum L.) core collection.

KEY MESSAGE: The identification of stable QTL for seed quality traits by association mapping of a diverse panel of linseed accessions establishes the foundation for assisted breeding and future fine mapping in linseed. Linseed oil is valued for its food and non-food applications. Modifying its oil content and fatty acid (FA) profiles to meet market needs in a timely manner requires clear understanding of their quantitative trait loci (QTL) architectures, which have received little attention to date. Association mapping is an efficient approach to identify QTL in germplasm collections. In this study, we explored the quantitative nature of seed quality traits including oil content (OIL), palmitic acid, stearic acid, oleic acid, linoleic acid (LIO) linolenic acid (LIN) and iodine value in a flax core collection of 390 accessions assayed with 460 microsatellite markers. The core collection was grown in a modified augmented design at two locations over 3 years and phenotypic data for all seven traits were obtained from all six environments. Significant phenotypic diversity and moderate to high heritability for each trait (0.73-0.99) were observed. Most of the candidate QTL were stable as revealed by multivariate analyses. Nine candidate QTL were identified, varying from one for OIL to three for LIO and LIN. Candidate QTL for LIO and LIN co-localized with QTL previously identified in bi-parental populations and some mapped nearby genes known to be involved in the FA biosynthesis pathway. Fifty-eight percent of the QTL alleles were absent (private) in the Canadian cultivars suggesting that the core collection possesses QTL alleles potentially useful to improve seed quality traits. The candidate QTL identified herein will establish the foundation for future marker-assisted breeding in linseed.

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.

Genomic regions underlying agronomic traits in linseed (Linum usitatissimum L.) as revealed by association mapping.

The extreme climate of the Canadian Prairies poses a major challenge to improve yield. Although it is possible to breed for yield per se, focusing on yield-related traits could be advantageous because of their simpler genetic architecture. The Canadian flax core collection of 390 accessions was genotyped with 464 simple sequence repeat markers, and phenotypic data for nine agronomic traits including yield, bolls per area, 1,000 seed weight, seeds per boll, start of flowering, end of flowering, plant height, plant branching, and lodging collected from up to eight environments was used for association mapping. Based on a mixed model (principal component analysis (PCA) + kinship matrix (K)), 12 significant marker-trait associations for six agronomic traits were identified. Most of the associations were stable across environments as revealed by multivariate analyses. Statistical simulation for five markers associated with 1000 seed weight indicated that the favorable alleles have additive effects. None of the modern cultivars carried the five favorable alleles and the maximum number of four observed in any accessions was mostly in breeding lines. Our results confirmed the complex genetic architecture of yield-related traits and the inherent difficulties associated with their identification while illustrating the potential for improvement through marker-assisted selection.

Genetic characterization of a core collection of flax (Linum usitatissimum L.) suitable for association mapping studies and evidence of divergent selection between fiber and linseed types.

BACKGROUND: Flax is valued for its fiber, seed oil and nutraceuticals. Recently, the fiber industry has invested in the development of products made from linseed stems, making it a dual purpose crop. Simultaneous targeting of genomic regions controlling stem fiber and seed quality traits could enable the development of dual purpose cultivars. However, the genetic diversity, population structure and linkage disequilibrium (LD) patterns necessary for association mapping (AM) have not yet been assessed in flax because genomic resources have only recently been developed. We characterized 407 globally distributed flax accessions using 448 microsatellite markers. The data was analyzed to assess the suitability of this core collection for AM. Genomic scans to identify candidate genes selected during the divergent breeding process of fiber flax and linseed were conducted using the whole genome shotgun sequence of flax. RESULTS: Combined genetic structure analysis assigned all accessions to two major groups with six sub-groups. Population differentiation was weak between the major groups (F(ST) = 0.094) and for most of the pairwise comparisons among sub-groups. The molecular coancestry analysis indicated weak relatedness (mean = 0.287) for most individual pairs. Abundant genetic diversity was observed in the total panel (5.32 alleles per locus), and some sub-groups showed a high proportion of private alleles. The average genome-wide LD (r²) was 0.036, with a relatively fast decay of 1.5 cM. Genomic scans between fiber flax and linseed identified candidate genes involved in cell-wall biogenesis/modification, xylem identity and fatty acid biosynthesis congruent with genes previously identified in flax and other plant species. CONCLUSIONS: Based on the abundant genetic diversity, weak population structure and relatedness and relatively fast LD decay, we concluded that this core collection is suitable for AM studies targeting multiple agronomic and quality traits aiming at the improvement of flax as a true dual purpose crop. Our genomic scans provide the first insights into candidate regions affected by divergent selection in flax. In combination with AM, genomic scans have the ability to increase the power to detect loci influencing complex traits.

Outlier Loci and Selection Signatures of Simple Sequence Repeats (SSRs) in Flax (Linum usitatissimum L.).

Genomic microsatellites (gSSRs) and expressed sequence tag-derived SSRs (EST-SSRs) have gained wide application for elucidating genetic diversity and population structure in plants. Both marker systems are assumed to be selectively neutral when making demographic inferences, but this assumption is rarely tested. In this study, three neutrality tests were assessed for identifying outlier loci among 150 SSRs (85 gSSRs and 65 EST-SSRs) that likely influence estimates of population structure in three differentiated flax sub-populations (FST = 0.19). Moreover, the utility of gSSRs, EST-SSRs, and the combined sets of SSRs was also evaluated in assessing genetic diversity and population structure in flax. Six outlier loci were identified by at least two neutrality tests showing footprints of balancing selection. After removing the outlier loci, the STRUCTURE analysis and the dendrogram topology of EST-SSRs improved. Conversely, gSSRs and combined SSRs results did not change significantly, possibly as a consequence of the higher number of neutral loci assessed. Taken together, the genetic structure analyses established the superiority of gSSRs to determine the genetic relationships among flax accessions, although the combined SSRs produced the best results. Genetic diversity parameters did not differ statistically (P > 0.05) between gSSRs and EST-SSRs, an observation partially explained by the similar number of repeat motifs. Our study provides new insights into the ability of gSSRs and EST-SSRs to measure genetic diversity and structure in flax and confirms the importance of testing for the occurrence of outlier loci to properly assess natural and breeding populations, particularly in studies considering only few loci.

Characterization of novel genic SSR markers in Linum usitatissimum (L. ) and their transferability across eleven Linum species

Little is known about the evolutionary relationships among Linum species, basically because of the lack of transferable molecular markers. Currently, expressed sequence tags available in public databases provide an opportunity for the rapid and inexpensive development of simple sequence repeat (SSR) markers in wild flax species. In this regard, fifty expressed sequence tag-derived microsatellite markers (EST-SSRs) were evaluated for polymorphism and transferability in 50 Linum usitatissimum cultivars/accessions and 11 Linum species. Among them 23 EST-SSRs were polymorphic in L. usitatissimum, while 2-4 alleles were detected (average 2.26 per locus). The polymorphism information content value ranged from 0.08 to 0.55 (average 0.38). Forty one genic markers (95.3 percent) produced strong amplicons in at least two of the 11 Linum species. The percentage of cross amplification ranged from 34.1 percent to 92.7 percent in L. tauricum and L. bienne, respectively. Moreover, the rate of transferability was associated positively with the botanical section. Our results suggest that the high degree of EST-SSRs transferability to Linum species can be a useful enhancement of the current database of SSR markers for future genetic and evolutionary studies.