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.

Fully resolved assembly of Fusarium proliferatum DSM106835 genome.

In the United Arab Emirates, sudden decline syndrome (SDS) is a destructive disease of date palm caused by the soil-borne fungal pathogen Fusarium proliferatum (Fp) DSM106835. Here, a high-resolution genome assembly of Fp DSM106835 was generated using PacBio HiFi sequencing with Omni-C data to provide a high-quality chromatin-organised reference genome with 418 scaffolds, totalling 58,468,907 bp in length and an N50 value of 4,383,091 bp from which 15,580 genes and 16,321 transcripts were predicted. The assembly achieved a complete BUSCO score of 99.2% for 758 orthologous genes. Compared to seven other Fp strains, Fp DSM106835 exhibited the highest continuity with a cumulative size of 44.26 Mbp for the first ten scaffolds/contigs, surpassing the assemblies of all examined Fp strains. Our findings of the high-quality genome of Fp DSM106835 provide an important resource to investigate its genetics, biology and evolutionary history. This study also contributes to fulfill the gaps in fungal knowledge, particularly the genes/metabolites associated with pathogenicity during the plant-pathogen interaction responsible for SDS.

Plant Genomics-Advancing Our Understanding of Plants.

Plant genomics has made significant progress in recent years, enabling researchers to identify genes and genomic regions responsible for plant growth, development, and stress response [...].

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.

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.

Fine-mapping of a putative glutathione S-transferase (GST) gene responsible for yellow seed colour in flax (Linum usitatissimum).

OBJECTIVE: The brown seed coat colour of flax (Linum ustiatissimum) results from proanthocyanidin synthesis and accumulation. Glutathione S-transferases (GSTs), such as the TT19 protein in Arabidopsis, have been implicated in the transport of anthocyanidins during the synthesis of the brown proanthocyanidins. This study fine mapped the g allele responsible for yellow seed colour in S95407 and identified it as a putative mutated GST. RESULTS: We developed a Recombinant Inbred Line population with 320 lines descended from a cross between CDC Bethune (brown seed coat) and S95407 (yellow seed) and used molecular markers to fine map the G gene on Chromosome 6 (Chr 6). We used Next Generation Sequencing (NGS) to identify a putative GST was identified in this region and Sanger sequenced the gene from CDC Bethune, S95407 and other yellow seeded genotypes. The putative GST from S95407 had 13 SNPs encoding, including four non-synonymous amino acid changes, compared to the CDC Bethune reference sequence and the other genotypes. The GST encoded by Lus10019895 is a lambda-GST in contrast to the Arabidopsis TT19 which is a phi-GST.

Aegilops tauschii genome assembly Aet v5.0 features greater sequence contiguity and improved annotation.

Aegilops tauschii is the donor of the D subgenome of hexaploid wheat and an important genetic resource. The reference-quality genome sequence Aet v4.0 for Ae. tauschii acc. AL8/78 was therefore an important milestone for wheat biology and breeding. Further advances in sequencing acc. AL8/78 and release of the Aet v5.0 sequence assembly are reported here. Two new optical maps were constructed and used in the revision of pseudomolecules. Gaps were closed with Pacific Biosciences long-read contigs, decreasing the gap number by 38,899. Transposable elements and protein-coding genes were reannotated. The number of annotated high-confidence genes was reduced from 39,635 in Aet v4.0 to 32,885 in Aet v5.0. A total of 2245 biologically important genes, including those affecting plant phenology, grain quality, and tolerance of abiotic stresses in wheat, was manually annotated and disease-resistance genes were annotated by a dedicated pipeline. Disease-resistance genes encoding nucleotide-binding site domains, receptor-like protein kinases, and receptor-like proteins were preferentially located in distal chromosome regions, whereas those encoding transmembrane coiled-coil proteins were dispersed more evenly along the chromosomes. Discovery, annotation, and expression analyses of microRNA (miRNA) precursors, mature miRNAs, and phasiRNAs are reported, including miRNA target genes. Other small RNAs, such as hc-siRNAs and tRFs, were characterized. These advances enhance the utility of the Ae. tauschii genome sequence for wheat genetics, biotechnology, and breeding.

Genome Assembly of the Canadian two-row Malting Barley cultivar AAC Synergy.

Barley (Hordeum vulgare L.) is one of the most important global crops. The six-row barley cultivar Morex reference genome has been used by the barley research community worldwide. However, this reference genome can have limitations when used for genomic and genetic diversity analysis studies, gene discovery, and marker development when working in two-row germplasm that is more common to Canadian barley. Here we assembled, for the first time, the genome sequence of a Canadian two-row malting barley, cultivar AAC Synergy. We applied deep Illumina paired-end reads, long mate-pair reads, PacBio sequences, 10X chromium linked read libraries, and chromosome conformation capture sequencing (Hi-C) to generate a contiguous assembly. The genome assembled from super-scaffolds had a size of 4.85 Gb, N50 of 2.32 Mb, and an estimated 93.9% of complete genes from a plant database (BUSCO, benchmarking universal single-copy orthologous genes). After removal of small scaffolds (< 300 Kb), the assembly was arranged into pseudomolecules of 4.14 Gb in size with seven chromosomes plus unanchored scaffolds. The completeness and annotation of the assembly were assessed by comparing it with the updated version of six-row Morex and recently released two-row Golden Promise genome assemblies.

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.

Genome-wide identification of ATP binding cassette (ABC) transporter and heavy metal associated (HMA) gene families in flax (Linum usitatissimum L.).

BACKGROUND: The recent release of the reference genome sequence assembly of flax, a self-pollinated crop with 15 chromosome pairs, into chromosome-scale pseudomolecules enables the characterization of gene families. The ABC transporter and HMA gene families are important in the control of cadmium (Cd) accumulation in crops. To date, the genome-wide analysis of these two gene families has been successfully conducted in some plant species, but no systematic evolutionary analysis is available for the flax genome. RESULTS: Here we describe the ABC transporter and HMA gene families in flax to provide a comprehensive overview of its evolution and some support towards the functional annotation of its members. The 198 ABC transporter and 12 HMA genes identified in the flax genome were classified into eight ABC transporter and four HMA subfamilies based on their phylogenetic analysis and domains' composition. Nine of these genes, i.e., LuABCC9, LuABCC10, LuABCG58, LuABCG59, LuABCG71, LuABCG72, LuABCG73, LuHMA3, and LuHMA4, were orthologous with the Cd associated genes in Arabidopsis, rice and maize. Ten motifs were identified from all ABC transporter and HMA genes. Also, several motifs were conserved among genes of similar length, but each subfamily each had their own motif structures. Both the ABC transporter and HMA gene families were highly conserved among subfamilies of flax and with those of Arabidopsis. While four types of gene duplication were observed at different frequencies, whole-genome or segmental duplications were the most frequent with 162 genes, followed by 29 dispersed, 14 tandem and 4 proximal duplications, suggesting that segmental duplications contributed the most to the expansion of both gene families in flax. The rates of non-synonymous to synonymous (Ka/Ks) mutations of paired duplicated genes were for the most part lower than one, indicative of a predominant purifying selection. Only five pairs of genes clearly exhibited positive selection with a Ka/Ks ratio greater than one. Gene ontology analyses suggested that most flax ABC transporter and HMA genes had a role in ATP binding, transport, catalytic activity, ATPase activity, and metal ion binding. The RNA-Seq analysis of eight different organs demonstrated diversified expression profiling patterns of the genes and revealed their functional or sub-functional conservation and neo-functionalization. CONCLUSION: Characterization of the ABC transporter and HMA gene families will help in the functional analysis of candidate genes in flax and other crop species.

Evaluation of variant calling tools for large plant genome re-sequencing.

BACKGROUND: Discovering single nucleotide polymorphisms (SNPs) from agriculture crop genome sequences has been a widely used strategy for developing genetic markers for several applications including marker-assisted breeding, population diversity studies for eco-geographical adaption, genotyping crop germplasm collections, and others. Accurately detecting SNPs from large polyploid crop genomes such as wheat is crucial and challenging. A few variant calling methods have been previously developed but they show a low concordance between their variant calls. A gold standard of variant sets generated from one human individual sample was established for variant calling tool evaluations, however hitherto no gold standard of crop variant set is available for wheat use. The intent of this study was to evaluate seven SNP variant calling tools (FreeBayes, GATK, Platypus, Samtools/mpileup, SNVer, VarScan, VarDict) with the two most popular mapping tools (BWA-mem and Bowtie2) on wheat whole exome capture (WEC) re-sequencing data from allohexaploid wheat. RESULTS: We found the BWA-mem mapping tool had both a higher mapping rate and a higher accuracy rate than Bowtie2. With the same mapping quality (MQ) cutoff, BWA-mem detected more variant bases in mapping reads than Bowtie2. The reads preprocessed with quality trimming or duplicate removal did not significantly affect the final mapping performance in terms of mapped reads. Based on the concordance and receiver operating characteristic (ROC), the Samtools/mpileup variant calling tool with BWA-mem mapping of raw sequence reads outperformed other tests followed by FreeBayes and GATK in terms of specificity and sensitivity. VarDict and VarScan were the poorest performing variant calling tools with the wheat WEC sequence data. CONCLUSION: The BWA-mem and Samtools/mpileup pipeline, with no need to preprocess the raw read data before mapping onto the reference genome, was ascertained the optimum for SNP calling for the complex wheat genome re-sequencing. These results also provide useful guidelines for reliable variant identification from deep sequencing of other large polyploid crop genomes.

A phenylpropanoid diglyceride associates with the leaf rust resistance Lr34res gene in wheat.

The gene Lr34res is one of the most long-lasting sources of quantitative fungal resistance in wheat. It is shown to be effective against leaf, stem, and stripe rusts, as well as powdery mildew and spot blotch. Recent biochemical characterizations of the encoded ABC transporter have outlined a number of allocrites, including phospholipids and abscisic acid, consistent with the established general promiscuity of ABC transporters, but ultimately leaving its mechanism of rust resistance unclear. Working with flag leaves of Triticum aestivum L. variety 'Thatcher' (Tc) and a near-isogenic line of 'Thatcher' into which the Lr34res allele was introgressed (Tc+Lr34res; RL6058), a comparative semi-targeted metabolomics analysis of flavonoid-rich extracts revealed virtually identical profiles with the exception of one metabolite accumulating in Tc+Lr34res, which was not present at comparable levels in Tc. Structural characterization of the purified metabolite revealed a phenylpropanoid diglyceride structure, 1-O-p-coumaroyl-3-O-feruloylglycerol (CFG). Additional profiling of CFG across a collection of near-isogenic lines and representative Lr34 haplotypes highlighted a broad association between the presence of Lr34res and elevated accumulations of CFG. Depletion of CFG upon infection, juxtaposed to its relatively lower anti-fungal activity, suggests CFG may serve as a storage form of the more potent anti-microbial hydroxycinnamic acids that are accessed during defense responses. Altogether these findings suggest a role for the encoded LR34res ABC transporter in modifying the accumulation of CFG, leading to increased accumulation of anti-fungal metabolites, essentially priming the wheat plant for defense.

Mapping Quantitative Trait Loci onto Chromosome-Scale Pseudomolecules in Flax.

Quantitative trait loci (QTL) are genomic regions associated with phenotype variation of quantitative traits. To date, a total of 313 QTL for 31 quantitative traits have been reported in 14 studies on flax. Of these, 200 QTL from 12 studies were identified based on genetic maps, the scaffold sequences, or the pre-released chromosome-scale pseudomolecules. Molecular markers for QTL identification differed across studies but the most used ones were simple sequence repeats (SSRs) or single nucleotide polymorphisms (SNPs). To uniquely map the SSR and SNP markers from different references onto the recently released chromosome-scale pseudomolecules, methods with several scripts and database files were developed to locate PCR- and SNP-based markers onto the same reference, co-locate QTL, and scan genome-wide candidate genes. Using these methods, 195 out of 200 QTL were successfully sorted onto the 15 flax chromosomes and grouped into 133 co-located QTL clusters; the candidate genes that co-located with these QTL clusters were also predicted. The methods and tools presented in this article facilitate marker re-mapping to a new reference, genome-wide QTL analysis, candidate gene scanning, and breeding applications in flax and other crops.

Genomic Prediction Accuracy of Seven Breeding Selection Traits Improved by QTL Identification in Flax.

Molecular markers are one of the major factors affecting genomic prediction accuracy and the cost of genomic selection (GS). Previous studies have indicated that the use of quantitative trait loci (QTL) as markers in GS significantly increases prediction accuracy compared with genome-wide random single nucleotide polymorphism (SNP) markers. To optimize the selection of QTL markers in GS, a set of 260 lines from bi-parental populations with 17,277 genome-wide SNPs were used to evaluate the prediction accuracy for seed yield (YLD), days to maturity (DTM), iodine value (IOD), protein (PRO), oil (OIL), linoleic acid (LIO), and linolenic acid (LIN) contents. These seven traits were phenotyped over four years at two locations. Identification of quantitative trait nucleotides (QTNs) for the seven traits was performed using three types of statistical models for genome-wide association study: two SNP-based single-locus (SS), seven SNP-based multi-locus (SM), and one haplotype-block-based multi-locus (BM) models. The identified QTNs were then grouped into QTL based on haplotype blocks. For all seven traits, 133, 355, and 1,208 unique QTL were identified by SS, SM, and BM, respectively. A total of 1420 unique QTL were obtained by SS+SM+BM, ranging from 254 (OIL, LIO) to 361 (YLD) for individual traits, whereas a total of 427 unique QTL were achieved by SS+SM, ranging from 56 (YLD) to 128 (LIO). SS models alone did not identify sufficient QTL for GS. The highest prediction accuracies were obtained using single-trait QTL identified by SS+SM+BM for OIL (0.929 ± 0.016), PRO (0.893 ± 0.023), YLD (0.892 ± 0.030), and DTM (0.730 ± 0.062), and by SS+SM for LIN (0.837 ± 0.053), LIO (0.835 ± 0.049), and IOD (0.835 ± 0.041). In terms of the number of QTL markers and prediction accuracy, SS+SM outperformed other models or combinations thereof. The use of all SNPs or QTL of all seven traits significantly reduced the prediction accuracy of traits. The results further validated that QTL outperformed high-density genome-wide random markers, and demonstrated that the combined use of single and multi-locus models can effectively identify a comprehensive set of QTL that improve prediction accuracy, but further studies on detection and removal of redundant or false-positive QTL to maximize prediction accuracy and minimize the number of QTL markers in GS are warranted.

Genome-wide introgression from a bread wheat × Lophopyrum elongatum amphiploid into wheat.

KEY MESSAGE: We introgressed wheatgrass germplasm from the octoploid amphiploid Triticum aestivum× Lophopyrum elongatum into wheat by manipulating the wheat Ph1 gene and discovered and characterized 130 introgression lines harboring single or, in various combinations, complete and recombined L. elongatum chromosomes. Diploid wheatgrass Lophopyrum elongatum (genomes EE) possesses valuable traits for wheat genetics and breeding. We evaluated several strategies for introgression of this germplasm into wheat. To detect it, we developed and validated multiplexed sets of Sequenom MassARRAY single nucleotide polymorphism (SNP) markers, which differentiated disomic and monosomic L. elongatum chromosomes from wheat chromosomes. We identified 130 introgression lines (ILs), which harbored 108 complete and 89 recombined L. elongatum chromosomes. Of the latter, 59 chromosomes were recombined by one or more crossovers and 30 were involved in centromeric (Robertsonian) translocations or were telocentric. To identify wheat chromosomes substituted for or recombined with L. elongatum chromosomes, we genotyped the ILs with the wheat 90-K Infinium SNP array. We found that most of the wheat 90-K probes correctly detected their targets in the L. elongatum genome and showed that some wheat SNPs are ancient and had originated prior to the divergence of the wheat and L. elongatum lineages. Of the 130 ILs, 52% were homozygous for Ph1 deletion and thus are staged to be recombined further. We failed to detect in the L. elongatum genome the 4/5 reciprocal translocation that has been reported in Thinopyrum bessarabicum and several other Triticeae genomes.

Dominant inhibition of awn development by a putative zinc-finger transcriptional repressor expressed at the B1 locus in wheat.

Awns, bristle-like structures extending from grass lemmas, provide protection against predators, contribute to photosynthesis and aid in grain dispersal. In wheat, selection of awns with minimal extension, termed awnletted, has occurred during domestication by way of loci that dominantly inhibit awn development, such as Tipped1 (B1), Tipped2 (B2), and Hooded (Hd). Here we identify and characterize the B1 gene. B1 was identified using bulked segregant RNA-sequencing of an F2 durum wheat population and through deletion mapping of awned bread wheat mutants. Functional characterization was accomplished by gene overexpression while haplotype analyses assessed B1 polymorphisms and genetic variation. Located on chromosome 5A, B1 is a C2H2 zinc finger encoding gene with ethylene-responsive element binding factor-associated amphiphilic repression (EAR) motifs. Constitutive overexpression of B1 in awned wheat produced an awnletted phenotype with pleiotropic effects on plant height and fertility. Transcriptome analysis of B1 overexpression plants suggests a role as transcriptional repressor, putatively targeting pathways involved in cell proliferation. Haplotype analysis revealed a conserved B1 coding region with proximal polymorphisms and supported the contention that B1 is mainly responsible for awnletted wheats globally. B1, predominantly responsible for awn inhibition in wheat, encodes a C2H2 zinc finger protein with EAR motifs which putatively functions as a transcriptional repressor.

The Complex Genetic Architecture of Early Root and Shoot Traits in Flax Revealed by Genome-Wide Association Analyses.

Roots are fundamental organs for water and nutrient uptake as well as for signal transduction in response to biotic and abiotic stresses. Flax has a shallow tap root system that relies mostly on top soil nutrient and moisture resources. The crop can easily be outcompeted by weeds or other crops in intercropping systems, especially in moisture deficit conditions. However, there is a wide range of variation among genotypes in terms of performance under scarce resources such as moisture limitation. Here we phenotyped 15 root, two shoot traits and shoot to root dry weight ratio on 115 flax accessions grown in a hydroponic pouch system and performed a genome-wide association study (GWAS) based on seven different models to identify quantitative trait loci underlying these traits. Significant variation among genotypes was observed for the two shoot and 12 of the 14 root traits. Shoot dry weight was correlated with root network volume, length, surface area, and root dry weight (r > 0.5, P < 0.001) but not significantly correlated with root depth (r = 0.033, P > 0.05). The seven GWAS models detected a total of 228 quantitative trait nucleotides (QTNs) for 16 traits. Most loci, defined by an interval of 100 kb up and downstream of the QTNs, harbored genes known to play role(s) in root and shoot development, suggesting them as candidates. Examples of candidate genes linked to root network QTNs included genes encoding GRAS transcription factors, mitogen-activated protein kinases, and auxin related lateral organ boundary proteins while QTN loci for shoot dry weight harbored genes involved in photomorphogenesis and plant immunity. These results provide insights into the genetic bases of early shoot and root development traits in flax that could be capitalized upon to improve its root architecture, particularly in view of better withstanding water limiting conditions during the cropping season.