Effects of vanilla odor on hypoxia-related periodic breathing in premature newborns: A pilot study.

BACKGROUND: Periodic breathing (PB)-related intermittent hypoxia can have long-lasting deleterious consequences in preterm infants. Olfactory stimulation using vanilla odor is beneficial for apnea of prematurity in the first postnatal days/weeks. We aimed to determine for the first time whether vanilla odor can also decrease PB-related intermittent hypoxia. METHOD: This pilot study was a balanced crossover clinical trial including 27 premature infants born between 30 and 33+6 weeks of gestation. We performed 12-h recordings on two nights separated by a 24-h period. All infants were randomly exposed to vanilla odor on the first or second study night. The primary outcome was the desaturation index, defined as the number per hour of pulse oximetry (SpO2) values <90 % for at least 5 s, together with a drop of ≥5 % from the preceding value. Univariate mixed linear models were used for the statistical analysis. RESULTS: Overall, exposure to vanilla odor did not significantly decrease the desaturation index (52 ± 22 events/h [mean ± SD] on the intervention night vs. 57 ± 26, p = 0.2); furthermore, it did not significantly alter any secondary outcome. In a preliminary post hoc subgroup analysis, however, the effect of vanilla odor was statistically significant in infants with a desaturation index of ≥70/h (from 86 ± 12 to 65 ± 23, p = 0.04). CONCLUSION: In this pilot study, vanilla odor overall did not decrease PB-related intermittent hypoxia in infants born at 30-33+6 weeks of gestation, which is when they are close to term. Preliminary results suggesting a beneficial effect in infants with the highest desaturation index, however, justify further studies in the presence of PB-related intermittent hypoxia as well as in infants born more prematurely.

Bradyrhizobium ontarionense sp. nov., a novel bacterial symbiont isolated from Aeschynomene indica (Indian jointvetch), harbours photosynthesis, nitrogen fixation and nitrous oxide (N2O) reductase genes.

A novel bacterial symbiont, strain A19T, was previously isolated from a root-nodule of Aeschynomene indica and assigned to a new lineage in the photosynthetic clade of the genus Bradyrhizobium. Here data are presented for the detailed genomic and taxonomic analyses of novel strain A19T. Emphasis is placed on the analysis of genes of practical or ecological significance (photosynthesis, nitrous oxide reductase and nitrogen fixation genes). Phylogenomic analysis of whole genome sequences as well as 50 single-copy core gene sequences placed A19T in a highly supported lineage distinct from described Bradyrhizobium species with B. oligotrophicum as the closest relative. The digital DNA-DNA hybridization and average nucleotide identity values for A19T in pair-wise comparisons with close relatives were far lower than the respective threshold values of 70% and ~ 96% for definition of species boundaries. The complete genome of A19T consists of a single 8.44 Mbp chromosome and contains a photosynthesis gene cluster, nitrogen-fixation genes and genes encoding a complete denitrifying enzyme system including nitrous oxide reductase implicated in the reduction of N2O, a potent greenhouse gas, to inert dinitrogen. Nodulation and type III secretion system genes, needed for nodulation by most rhizobia, were not detected. Data for multiple phenotypic tests complemented the sequence-based analyses. Strain A19T elicits nitrogen-fixing nodules on stems and roots of A. indica plants but not on soybeans or Macroptilium atropurpureum. Based on the data presented, a new species named Bradyrhizobium ontarionense sp. nov. is proposed with strain A19T (= LMG 32638T = HAMBI 3761T) as the type strain.

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.

Genome-wide association studies using multi-models and multi-SNP datasets provide new insights into pasmo resistance in flax.

Introduction: Flax (Linum usitatissimum L.) is an economically important crop due to its oil and fiber. However, it is prone to various diseases, including pasmo caused by the fungus Septoria linicola. Methods: In this study, we conducted field evaluations of 445 flax accessions over a five-year period (2012-2016) to assess their resistance to pasmo A total of 246,035 single nucleotide polymorphisms (SNPs) were used for genetic analysis. Four statistical models, including the single-locus model GEMMA and the multi-locus models FarmCPU, mrMLM, and 3VmrMLM, were assessed to identify quantitative trait nucleotides (QTNs) associated with pasmo resistance. Results: We identified 372 significant QTNs or 132 tag QTNs associated with pasmo resistance from five pasmo resistance datasets (PAS2012-PAS2016 and the 5-year average, namely PASmean) and three genotypic datasets (the all SNPs/ALL, the gene-based SNPs/GB and the RGA-based SNPs/RGAB). The tag QTNs had R2 values of 0.66-16.98% from the ALL SNP dataset, 0.68-20.54%from the GB SNP dataset, and 0.52-22.42% from the RGAB SNP dataset. Of these tag QTNs, 93 were novel. Additionally, 37 resistance gene analogs (RGAs)co-localizing with 39 tag QTNs were considered as potential candidates for controlling pasmo resistance in flax and 50 QTN-by-environment interactions(QEIs) were identified to account for genes by environmental interactions. Nine RGAs were predicted as candidate genes for ten QEIs. Discussion: Our results suggest that pasmo resistance in flax is polygenic and potentially influenced by environmental factors. The identified QTNs provide potential targets for improving pasmo resistance in flax breeding programs. This study sheds light on the genetic basis of pasmo resistance and highlights the importance of considering both genetic and environmental factors in breeding programs for flax.

Genome scans capture key adaptation and historical hybridization signatures in tetraploid wheat.

Tetraploid wheats (Triticum turgidum L.), including durum wheat (T. turgidum ssp. durum (Desf.) Husn.), are important crops with high nutritional and cultural values. However, their production is constrained by sensitivity to environmental conditions. In search of adaptive genetic signatures tracing historical selection and hybridization events, we performed genome scans on two datasets: (1) Durum Global Diversity Panel comprising a total of 442 tetraploid wheat and wild progenitor accessions including durum landraces (n = 286), domesticated emmer (T. turgidum ssp. dicoccum (Schrank) Thell.; n = 103) and wild emmer (T. turgidum ssp. dicoccoides (Korn. ex Asch. & Graebn.) Thell.; n = 53) wheats genotyped using the 90K single nucleotide polymorphism (SNP) array, and (2) a second dataset comprising a total 121 accessions of nine T. turgidum subspecies including wild emmer genotyped with >100 M SNPs from whole-genome resequencing. The genome scan on the first dataset detected six outlier loci on chromosomes 1A, 1B, 3A (n = 2), 6A, and 7A. These loci harbored important genes for adaptation to abiotic stresses, phenological responses, such as seed dormancy, circadian clock, flowering time, and key yield-related traits, including pleiotropic genes, such as HAT1, KUODA1, CBL1, and ZFN1. The scan on the second dataset captured a highly differentiated region on chromosome 2B that shows significant differentiation between two groups: one group consists of Georgian (T. turgidum ssp. paleocolchicum A. Love & D. Love) and Persian (T. turgidum ssp. carthlicum (Nevski) A. Love & D. Love) wheat accessions, while the other group comprises all the remaining tetraploids including wild emmer. This is consistent with a previously reported introgression in this genomic region from T. timopheevii Zhuk. which naturally cohabit in the Georgian and neighboring areas. This region harbored several adaptive genes, including the thermomorphogenesis gene PIF4, which confers temperature-resilient disease resistance and regulates other biological processes. Genome scans can be used to fast-track germplasm housed in gene banks and in situ; which helps to identify environmentally resilient accessions for breeding and/or to prioritize them for conservation.

Lifting of the 1,000 wheat exome project SNPs from Triticum aestivum cv. Chinese Spring assembly RefSeq v1.0 to RefSeq v2.1.

OBJECTIVE: The 1,000 wheat exome project captured the single nucleotide variants in the coding regions of a diverse set of 890 wheat accessions to analyse the contribution of introgression to adaptation of wheat. However, this highly useful single nucleotide polymorphism (SNP) dataset is based on RefSeq v1.0 of the International Wheat Genome Sequencing Consortium (IWGSC) assembly of the bread wheat genome of Chinese Spring. This reference sequence has recently been updated using optical maps and long-read sequencing to produce the improved RefSeq v2.1. Our objective was to develop a reliable high-density SNP dataset positioned onto RefSeq v2.1 because it is the current standard reference sequence used by wheat researchers. RESULTS: The 3,039,822 SNPs originally positioned on RefSeq v1.0 were projected to v2.1 using Liftoff with four different flanking regions, and 2,946,536 SNPs were consistently lifted to the same location irrespective of the flanking region lengths. Of these, 2,799,166 were located on the '+' ve strand. The distribution of the SNPs across the 21 chromosomes on RefSeq v2.1 was similar to that of RefSeq v1.0. Among the SNPs that were based on unanchored scaffolds in RefSeq v1.0, 11,938 were projected to one of the 21 pseudomolecules in the upgraded assembly. This SNP dataset constitutes a much-needed standardized resource for the wheat research community.

Ensifer canadensis sp. nov. strain T173T isolated from Melilotus albus (sweet clover) in Canada possesses recombinant plasmid pT173b harbouring symbiosis and type IV secretion system genes apparently acquired from Ensifer medicae.

A bacterial strain, designated T173T, was previously isolated from a root-nodule of a Melilotus albus plant growing in Canada and identified as a novel Ensifer lineage that shared a clade with the non-symbiotic species, Ensifer adhaerens. Strain T173T was also previously found to harbour a symbiosis plasmid and to elicit root-nodules on Medicago and Melilotus species but not fix nitrogen. Here we present data for the genomic and taxonomic description of strain T173T. Phylogenetic analyses including the analysis of whole genome sequences and multiple locus sequence analysis (MLSA) of 53 concatenated ribosome protein subunit (rps) gene sequences confirmed placement of strain T173T in a highly supported lineage distinct from named Ensifer species with E. morelensis Lc04T as the closest relative. The highest digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) values of genome sequences of strain T173T compared with closest relatives (35.7 and 87.9%, respectively) are well below the respective threshold values of 70% and 95-96% for bacterial species circumscription. The genome of strain T173T has a size of 8,094,229 bp with a DNA G + C content of 61.0 mol%. Six replicons were detected: a chromosome (4,051,102 bp) and five plasmids harbouring plasmid replication and segregation (repABC) genes. These plasmids were also found to possess five apparent conjugation systems based on analysis of TraA (relaxase), TrbE/VirB4 (part of the Type IV secretion system (T4SS)) and TraG/VirD4 (coupling protein). Ribosomal RNA operons encoding 16S, 23S, and 5S rRNAs that are usually restricted to bacterial chromosomes were detected on plasmids pT173d and pT173e (946,878 and 1,913,930 bp, respectively) as well as on the chromosome of strain T173T. Moreover, plasmid pT173b (204,278 bp) was found to harbour T4SS and symbiosis genes, including nodulation (nod, noe, nol) and nitrogen fixation (nif, fix) genes that were apparently acquired from E. medicae by horizontal transfer. Data for morphological, physiological and symbiotic characteristics complement the sequence-based characterization of strain T173T. The data presented support the description of a new species for which the name Ensifer canadensis sp. nov. is proposed with strain T173T (= LMG 32374T = HAMBI 3766T) as the species type strain.

Historical Selection, Adaptation Signatures, and Ambiguity of Introgressions in Wheat.

Wheat was one of the crops domesticated in the Fertile Crescent region approximately 10,000 years ago. Despite undergoing recent polyploidization, hull-to-free-thresh transition events, and domestication bottlenecks, wheat is now grown in over 130 countries and accounts for a quarter of the world's cereal production. The main reason for its widespread success is its broad genetic diversity that allows it to thrive in different environments. To trace historical selection and hybridization signatures, genome scans were performed on two datasets: approximately 113K SNPs from 921 predominantly bread wheat accessions and approximately 110K SNPs from about 400 wheat accessions representing all ploidy levels. To identify environmental factors associated with the loci, a genome-environment association (GEA) was also performed. The genome scans on both datasets identified a highly differentiated region on chromosome 4A where accessions in the first dataset were dichotomized into a group (n = 691), comprising nearly all cultivars, wild emmer, and most landraces, and a second group (n = 230), dominated by landraces and spelt accessions. The grouping of cultivars is likely linked to their potential ancestor, bread wheat cv. Norin-10. The 4A region harbored important genes involved in adaptations to environmental conditions. The GEA detected loci associated with latitude and temperature. The genetic signatures detected in this study provide insight into the historical selection and hybridization events in the wheat genome that shaped its current genetic structure and facilitated its success in a wide spectrum of environmental conditions. The genome scans and GEA approaches applied in this study can help in screening the germplasm housed in gene banks for breeding, and for conservation purposes.

Benchmarking Enrichment Efforts in the US & Canada Across Species and Enrichment Categories.

Enrichment is important for animal welfare and data quality. Provision of enrichment opportunities varies between species and enrichment category. However, data benchmarking these differences does not exist. Our objective was to characterize enrichment provision and associated factors across species in the US and Canada. Personnel who work with research animals (n = 1098) in the US and Canada voluntarily responded to online promotions and completed a survey about enrichment used for the species they worked with most, their control of and wish for more enrichment, stress or pain in the animals they worked the most with, and demographics. All participants (except those working with rats) received the same questionnaire regardless of species to allow objectivity, as the effects of many enrichment items on some species have not yet been determined. The questionnaire asked about enrichments that were beneficial to at least one species. The provision of enrichment was allocated into 2 outcome variables: diversity and frequency per enrichment category. Results showed a significant interaction between enrichment category and species. Generally, physical, nutritional, and sensory enrichments were provided less often than social enrichment. In addition, nonhuman primates received more diverse and more frequent enrichment than did other species (twice as much as rats and mice). Enrichment was provided less frequently by personnel who wished they could do more than the status quo. Both enrichment frequency and diversity were higher in respondents from Canada, those who had more control over provision, and those who had been in the field longer. While our results cannot be used to determine the quality of enrichment provided to various species, they do provide information on current enrichment practices in the US and Canada and identify differences in implementation by species and enrichment category. The data also indicate provision of enrichment is influenced by factors such as country and individual control over enrichment. This information can also be used to identify areas for greater enrichment efforts for some species (for example, rats and mice) and categories, with the ultimate goal of improving animal welfare.

Global transcriptome analysis of allopolyploidization reveals large-scale repression of the D-subgenome in synthetic hexaploid wheat.

Synthetic hexaploid wheat (SHW) lines are created as pre-breeding germplasm to diversify the D subgenome of hexaploid wheat and capitalize upon the untapped genetic diversity of the Aegilops tauschii gene pool. However, the phenotypes observed in the Ae. tauschii parents are not always recovered in the SHW lines, possibly due to inter-subgenome interactions. To elucidate this post-polyploidization genome reprogramming phenomenon, we performed RNA-seq of four SHW lines and their corresponding tetraploid and diploid parents, across ten tissues and three biological replicates. Homoeologue expression bias (HEB) analysis using more than 18,000 triads suggests massive suppression of homoeoalleles of the D subgenome in SHWs. Comparative transcriptome analysis of the whole-genome gene set further corroborated this finding. Alternative splicing analysis of the high-confidence genes indicates an additional layer of complexity where all five splice events are identified, and retained intron is predominant. Homoeologue expression upon resynthesis of hexaploid wheat has implications to the usage and handling of this germplasm in breeding as it relates to capturing the effects of epistatic interaction across subgenomes upon polyploidization. Special considerations must be given to this germplasm in pre-breeding activities to consider the extent of the inter-subgenome interactions on gene expression and their impact on traits for crop improvement.

Genetic architecture of rust resistance in a wheat (Triticum turgidum) diversity panel.

Introduction: Wheat rust diseases are widespread and affect all wheat growing areas around the globe. Breeding strategies focus on incorporating genetic disease resistance. However, pathogens can quickly evolve and overcome the resistance genes deployed in commercial cultivars, creating a constant need for identifying new sources of resistance. Methods: We have assembled a diverse tetraploid wheat panel comprised of 447 accessions of three Triticum turgidum subspecies and performed a genome-wide association study (GWAS) for resistance to wheat stem, stripe, and leaf rusts. The panel was genotyped with the 90K Wheat iSelect single nucleotide polymorphism (SNP) array and subsequent filtering resulted in a set of 6,410 non-redundant SNP markers with known physical positions. Results: Population structure and phylogenetic analyses revealed that the diversity panel could be divided into three subpopulations based on phylogenetic/geographic relatedness. Marker-trait associations (MTAs) were detected for two stem rust, two stripe rust and one leaf rust resistance loci. Of them, three MTAs coincide with the known rust resistance genes Sr13, Yr15 and Yr67, while the other two may harbor undescribed resistance genes. Discussion: The tetraploid wheat diversity panel, developed and characterized herein, captures wide geographic origins, genetic diversity, and evolutionary history since domestication making it a useful community resource for mapping of other agronomically important traits and for conducting evolutionary studies.

Quantitative Trait Locus Mapping of Marsh Spot Disease Resistance in Cranberry Common Bean (Phaseolus vulgaris L.).

Common bean (Phaseolus vulgaris L.) is a food crop that is an important source of dietary proteins and carbohydrates. Marsh spot is a physiological disorder that diminishes seed quality in beans. Prior research suggested that this disease is likely caused by manganese (Mn) deficiency during seed development and that marsh spot resistance is controlled by at least four genes. In this study, genetic mapping was performed to identify quantitative trait loci (QTL) and the potential candidate genes associated with marsh spot resistance. All 138 recombinant inbred lines (RILs) from a bi-parental population were evaluated for marsh spot resistance during five years from 2015 to 2019 in sandy and heavy clay soils in Morden, Manitoba, Canada. The RILs were sequenced using a genotyping by sequencing approach. A total of 52,676 single nucleotide polymorphisms (SNPs) were identified and filtered to generate a high-quality set of 2066 SNPs for QTL mapping. A genetic map based on 1273 SNP markers distributed on 11 chromosomes and covering 1599 cm was constructed. A total of 12 stable and 4 environment-specific QTL were identified using additive effect models, and an additional two epistatic QTL interacting with two of the 16 QTL were identified using an epistasis model. Genome-wide scans of the candidate genes identified 13 metal transport-related candidate genes co-locating within six QTL regions. In particular, two QTL (QTL.3.1 and QTL.3.2) with the highest R2 values (21.8% and 24.5%, respectively) harbored several metal transport genes Phvul.003G086300, Phvul.003G092500, Phvul.003G104900, Phvul.003G099700, and Phvul.003G108900 in a large genomic region of 16.8-27.5 Mb on chromosome 3. These results advance the current understanding of the genetic mechanisms of marsh spot resistance in cranberry common bean and provide new genomic resources for use in genomics-assisted breeding and for candidate gene isolation and functional characterization.

Insights into the Genetic Architecture and Genomic Prediction of Powdery Mildew Resistance in Flax (Linum usitatissimum L.).

Powdery mildew (PM), caused by the fungus Oidium lini in flax, can cause defoliation and reduce seed yield and quality. To date, one major dominant gene (Pm1) and three quantitative trait loci (QTL) on chromosomes 1, 7 and 9 have been reported for PM resistance. To fully dissect the genetic architecture of PM resistance and identify QTL, a diverse flax core collection of 372 accessions augmented with an additional 75 breeding lines were sequenced, and PM resistance was evaluated in the field for eight years (2010-2017) in Morden, Manitoba, Canada. Genome-wide association studies (GWAS) were performed using two single-locus and seven multi-locus statistical models with 247,160 single nucleotide polymorphisms (SNPs) and the phenotypes of the 447 individuals for each year separately as well as the means over years. A total of 349 quantitative trait nucleotides (QTNs) were identified, of which 44 large-effect QTNs (R2 = 10-30%) were highly stable over years. The total number of favourable alleles per accession was significantly correlated with PM resistance (r = 0.74), and genomic selection (GS) models using all identified QTNs generated significantly higher predictive ability (r = 0.93) than those constructed using the 247,160 genome-wide random SNP (r = 0.69), validating the overall reliability of the QTNs and showing the additivity of PM resistance in flax. The QTNs were clustered on the distal ends of all 15 chromosomes, especially on chromosome 5 (0.4-5.6 Mb and 9.4-16.9 Mb) and 13 (4.7-5.2 Mb). To identify candidate genes, a dataset of 3230 SNPs located in resistance gene analogues (RGAs) was used as input for GWAS, from which an additional 39 RGA-specific QTNs were identified. Overall, 269 QTN loci harboured 445 RGAs within the 200 Kb regions spanning the QTNs, including 45 QTNs located within the RGAs. These RGAs supported by significant QTN/SNP allele effects were mostly nucleotide binding site and leucine-rich repeat receptors (NLRs) belonging to either coiled-coil (CC) NLR (CNL) or toll interleukin-1 (TIR) NLR (TNL), receptor-like kinase (RLK), receptor-like protein kinase (RLP), transmembrane-coiled-coil (TM-CC), WRKY, and mildew locus O (MLO) genes. These results constitute an important genomic tool for resistance breeding and gene cloning for PM in flax.

Variations in exons 11 and 12 of the multi-pest resistance wheat gene Lr34 are independently additive for leaf rust resistance.

Introduction: Characterization of germplasm collections for the wheat leaf rust gene Lr34 previously defined five haplotypes in spring wheat. All resistant lines had a 3-bp TTC deletion (null) in exon 11, resulting in the absence of a phenylalanine residue in the ABC transporter, as well as a single nucleotide C (Tyrosine in Lr34+) to T (Histidine in Lr34-) transition in exon 12. A rare haplotype present in Odesskaja 13 and Koktunkulskaja 332, both of intermediate rust resistance, had the 3-bp deletion typical of Lr34+ in exon 11 but the T nucleotide of Lr34- in exon 12. Methods: To quantify the role of each mutation in leaf rust resistance, Odesskaja 13 and Koktunkulskaja 332 were crossed to Thatcher and its near-isogenic line Thatcher-Lr34 (RL6058). Single seed descent populations were generated and evaluated for rust resistance in six different rust nurseries. Results: The Odesskaja 13 progeny with the TTC/T haplotype were susceptible with an average severity rating of 62.3%, the null/T haplotype progeny averaged 39.7% and the null/C haplotype was highly resistant, averaging 13.3% severity. The numbers for the Koktunkulskaja 332 crosses were similar with 63.5%, 43.5% and 23.7% severity ratings, respectively. Differences between all classes in all crosses were statistically significant, indicating that both mutations are independently additive for leaf rust resistance. The three-dimensional structural models of LR34 were used to analyze the locations and putative interference of both amino acids with the transport channel. Koktunkulskaja 332 also segregated for marker csLV46 which is linked to Lr46. Rust severity in lines with Lr34+ and csLV46+ had significantly lower rust severity ratings than those without, indicating the additivity of the two loci. Discussion: This has implications for the deployment of Lr34 in wheat cultivars and for the basic understanding of this important wheat multi-pest durable resistance gene.

Bradyrhizobium septentrionale sp. nov. (sv. septentrionale) and Bradyrhizobium quebecense sp. nov. (sv. septentrionale) associated with legumes native to Canada possess rearranged symbiosis genes and numerous insertion sequences.

Six bacterial strains isolated from root nodules of soybean plants that had been inoculated with root-zone soil of legumes native to Canada were previously characterized and 1) placed in two novel lineages within the genus Bradyrhizobium and 2) assigned to symbiovar septentrionale. Here we verified the taxonomic status of these strains using genomic and phenotypic analyses. Phylogenetic analyses of five protein encoding partial gene sequences as well as 52 full length ribosome protein subunit gene sequences confirmed placement of the novel strains in two highly supported lineages distinct from named Bradyrhizobium species. The highest average nucleotide identity values of strains representing these two lineages relative to type strains of closest relatives were 90.7 and 92.3% which is well below the threshold value for bacterial species circumscription. The genomes of representative strains 1S1T, 162S2 and 66S1MBT have sizes of 10598256, 10733150 and 9032145 bp with DNA G+C contents of 63.5, 63.4 and 63.8 mol%, respectively. These strains possess between one and three plasmids based on copy number of plasmid replication and segregation (repABC) genes. Novel strains also possess numerous insertion sequences, and, relative to reference strain Bradyrhizobium diazoefficiens USDA110T, exhibit inversion and fragmentation of nodulation (nod) and nitrogen-fixation (nif) gene clusters. Phylogenetic analyses of nodC and nifH gene sequences confirmed placement of novel strains in a distinct lineage corresponding to symbiovar septentrionale. Data for morphological, physiological and symbiotic characteristics complement the sequence-based results. The data presented here support the description of two new species for which the names Bradyrhizobium septentrionale sp. nov. (sv. septentrionale) and Bradyrhizobium quebecense sp. nov. (sv. septentrionale) are proposed, with 1S1T (=LMG 29930T=HAMBI 3676T) and 66S1MBT (=LMG 31547T=HAMBI 3720T) as type strains, respectively.

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 signatures in flax pinpoint to adaptive evolution along its ecological gradient.

BACKGROUND: Flax is one of the eight founder crops of agriculture. It is believed to have been domesticated as a long-day plant that has since spread to survive in a wide range of eco-geographic regions extending from the warm Indian subcontinent to the low latitude east African highlands and to the cool and high-latitude Eurasia. Understanding the genetic basis underlying its adaptation and selection events throughout its dispersion is essential to develop cultivars adapted to local environmental conditions. METHODS: Here we detected genetic signatures of local adaptation and selection events of flax based on 385 accessions from all major flax growing regions of the world using genome scan methods and three genomic datasets: (1) a genome-wide dataset of more than 275K single nucleotide polymorphisms (SNPs), (2) a filtered dataset of 23K SNPs with minor allele frequency >10% and, (3) a 34K exon-derived SNP dataset. RESULTS: Principal component (PC) and fixation index (FS⁢T)-based genome scans yielded consistent outlier SNP loci on chromosomes 1, 8, 9 and 12. Additional loci on chromosomes 3, 7, 8, 10, 11, 13 and 14 were detected using both the PC and FS⁢T methods in two of the three datasets. A genome-environment association (GEA) analysis using the 23K dataset and the first PC of cropping season temperature, day-length and latitude identified significant SNPs on chromosomes 3, 7, 9 and 13. CONCLUSIONS: Most of the loci detected by the three methods harbored relevant genes for local adaptation, including some that play roles in day-length, light and other biotic and abiotic stresses responses. Such genetic signatures may help to select pre-breeding materials potentially adapted to specific growing niches prior to field performance trials. Given the current low genotyping cost and freely available environmental data, the genome scans along with GEA can readily provide opportunity to sort out materials suitable to various environmental conditions from large set of germplasm in gene banks and/or in situ, thereby assisting the breeding and genetic conservation efforts.

Identification of New Leaf Rust Resistance Loci in Wheat and Wild Relatives by Array-Based SNP Genotyping and Association Genetics.

Leaf rust caused by Puccinia triticina is the most widespread rust disease of wheat. As pathogen populations are constantly evolving, identification of novel sources of resistance is necessary to maintain disease resistance and stay ahead of this plant-pathogen evolutionary arms race. The wild genepool of wheat is a rich source of genetic diversity, accounting for 44% of the Lr genes identified. Here we performed a genome-wide association study (GWAS) on a diverse germplasm of 385 accessions, including 27 different Triticum and Aegilops species. Genetic characterization using the wheat 90 K array and subsequent filtering identified a set of 20,501 single nucleotide polymorphic (SNP) markers. Of those, 9,570 were validated using exome capture and mapped onto the Chinese Spring reference sequence v1.0. Phylogenetic analyses illustrated four major clades, clearly separating the wild species from the T. aestivum and T. turgidum species. GWAS was conducted using eight statistical models for infection types against six leaf rust isolates and leaf rust severity rated in field trials for 3-4 years at 2-3 locations in Canada. Functional annotation of genes containing significant quantitative trait nucleotides (QTNs) identified 96 disease-related loci associated with leaf rust resistance. A total of 21 QTNs were in haplotype blocks or within flanking markers of at least 16 known Lr genes. The remaining significant QTNs were considered loci that putatively harbor new Lr resistance genes. Isolation of these candidate genes will contribute to the elucidation of their role in leaf rust resistance and promote their usefulness in marker-assisted selection and introgression.