Global Integrated Genomic and Transcriptomic Analyses of MYB Transcription Factor Superfamily in C3 Model Plant Oryza sativa (L.) Unravel Potential Candidates Involved in Abiotic Stress Signaling.

Plant transcription factors (TFs) are significant players in transcriptional regulations, signal transduction, and constitute an integral part of signaling networks. MYB TFs are major TF superfamilies that play pivotal roles in regulation of transcriptional reprogramming, physiological processes, and abiotic stress (AbS) responses. To explore the understanding of MYB TFs, genome and transcriptome-wide identification was performed in the C3 model plant, Oryza sativa (OsMYB). This study retrieved 114 OsMYB TFs that were computationally analyzed for their expression profiling, gene organization, cis-acting elements, and physicochemical properties. Based on the microarray datasets, six OsMYB genes which were sorted out and identified by a differential expression pattern were noted in various tissues. Systematic expression profiling of OsMYB TFs showed their meta-differential expression of different AbS treatments, spatio-temporal gene expression of various tissues and their growth in the field, and gene expression profiling in responses to phytohormones. In addition, the circular ideogram of OsMYB genes in related C4 grass plants conferred the gene synteny. Protein-protein interactions of these genes revealed the molecular crosstalk of OsMYB TFs. Transcriptional analysis (qPCR) of six OsMYB players in response to drought and salinity stress suggested the involvement in individual and combined AbS responses. To decipher how these OsMYB play functional roles in AbS dynamics, further research is a prerequisite.

GrainGenes: Tools and Content to Assist Breeders Improving Oat Quality.

GrainGenes is the USDA-ARS database and Web resource for wheat, barley, oat, rye, and their relatives. As a community Web hub and database for small grains, GrainGenes strives to provide resources for researchers, students, and plant breeders to improve traits such as quality, yield, and disease resistance. Quantitative trait loci (QTL), genes, and genetic maps for quality attributes in GrainGenes represent the historical approach to mapping genes for groat percentage, test weight, protein, fat, and ß-glucan content in oat (Avena spp.). Genetic maps are viewable in CMap, the comparative mapping tool that enables researchers to take advantage of highly populated consensus maps to increase the marker density around their genes-of-interest. GrainGenes hosts over 50 genome browsers and is launching an effort for community curation, including the manually curated tracks with beta-glucan QTL and significant markers found via GWAS and cloned cellulose synthase-like AsClF6 alleles.

Comparative chromosome painting in Chacoan peccary, Catagonus wagneri.

Chacoan peccary (Catagonus wagneri, 2n=20) is the most endangered of three extant species of Tayassuidae. Its karyotype has been studied only by differential chromosome staining methods so far. To establish a comparative cytogenetic map of the peccary, we used cross-species hybridization with porcine (Sus scrofa, 2n=38) painting probes. Painting revealed 30 evolutionary conserved autosomal segments between pig and peccary. The q-arm of the submetacentric chromosome X is homologous to the porcine X chromosome, while the p-arm is composed of heterochromatin. Nucleolar organizer regions were detected on chromosomes 8 and 9 which are homologous to pig chromosomes 8 and 4/18, respectively. Fusions of chromosomes homologous to pig chromosomes 4/7 and 4/18 and fission of chromosome 7 are synapomorphic characters shared by Catagonus wagneri and Tayassu pecari but not by Pecari tajacu. Our results confirmed a high rate of karyotype evolution in Tayassuidae and a closer relationship of Catagonus wagneri with Tayassu pecari than with Pecari tajacu.

The Puzzling Fate of a Lupin Chromosome Revealed by Reciprocal Oligo-FISH and BAC-FISH Mapping.

Old World lupins constitute an interesting model for evolutionary research due to diversity in genome size and chromosome number, indicating evolutionary genome reorganization. It has been hypothesized that the polyploidization event which occurred in the common ancestor of the Fabaceae family was followed by a lineage-specific whole genome triplication (WGT) in the lupin clade, driving chromosome rearrangements. In this study, chromosome-specific markers were used as probes for heterologous fluorescence in situ hybridization (FISH) to identify and characterize structural chromosome changes among the smooth-seeded (Lupinus angustifolius L., Lupinus cryptanthus Shuttlew., Lupinus micranthus Guss.) and rough-seeded (Lupinus cosentinii Guss. and Lupinus pilosus Murr.) lupin species. Comparative cytogenetic mapping was done using FISH with oligonucleotide probes and previously published chromosome-specific bacterial artificial chromosome (BAC) clones. Oligonucleotide probes were designed to cover both arms of chromosome Lang06 of the L. angustifolius reference genome separately. The chromosome was chosen for the in-depth study due to observed structural variability among wild lupin species revealed by BAC-FISH and supplemented by in silico mapping of recently released lupin genome assemblies. The results highlighted changes in synteny within the Lang06 region between the lupin species, including putative translocations, inversions, and/or non-allelic homologous recombination, which would have accompanied the evolution and speciation.

Genome-Wide Novel Genic Microsatellite Marker Resource Development and Validation for Genetic Diversity and Population Structure Analysis of Banana.

Trait tagging through molecular markers is an important molecular breeding tool for crop improvement. SSR markers encoded by functionally relevant parts of a genome are well suited for this task because they may be directly related to traits. However, a limited number of these markers are known for Musa spp. Here, we report 35136 novel functionally relevant SSR markers (FRSMs). Among these, 17,561, 15,373 and 16,286 FRSMs were mapped in-silico to the genomes of Musa acuminata, M. balbisiana and M. schizocarpa, respectively. A set of 273 markers was validated using eight accessions of Musa spp., from which 259 markers (95%) produced a PCR product of the expected size and 203 (74%) were polymorphic. In-silico comparative mapping of FRSMs onto Musa and related species indicated sequence-based orthology and synteny relationships among the chromosomes of Musa and other plant species. Fifteen FRSMs were used to estimate the phylogenetic relationships among 50 banana accessions, and the results revealed that all banana accessions group into two major clusters according to their genomic background. Here, we report the first large-scale development and characterization of functionally relevant Musa SSR markers. We demonstrate their utility for germplasm characterization, genetic diversity studies, and comparative mapping in Musa spp. and other monocot species. The sequences for these novel markers are freely available via a searchable web interface called Musa Marker Database.

Preferential Mapping of Sex-Biased Differentially-Expressed Genes of Larvae to the Sex-Determining Region of Flathead Grey Mullet (Mugil cephalus).

Flathead gray mullet (Mugil cephalus) is a cosmopolitan mugilid species popular in fishery and aquaculture with an economic preference for all-female population. However, it displays neither sexual dimorphisms nor heteromorphic sex chromosomes. We have previously presented a microsatellite-based linkage map for this species locating a single sex determination region (SDR) on linkage group 9 (LG9) with evidence for XX/XY sex determination (SD) mechanism. In this work, we refine the critical SDR on LG9, and propose positional- and functional- candidate genes for SD. To elucidate the genetic mechanism of SD, we assembled and compared male and female genomic sequences of 19 syntenic genes within the putative SDR on mullet's LG9, based on orthology to tilapia's LG8 (tLG8) physical map. A total of 25 sequence-based markers in 12 genes were developed. For all markers, we observed association with sex in at least one of the two analyzed M. cephalus full-sib families, but not in the wild-type population. Recombination events were inferred within families thus setting the SDR boundaries to a region orthologous to ∼0.9 Mbp with 27 genes on tLG8. As the sexual phenotype is evident only in adults, larvae were assigned into two putative sex-groups according to their paternal haplotypes, following a model of XY/XX SD-system. A total of 107 sex-biased differentially expressed genes in larvae were observed, of which 51 were mapped to tLG8 (48% enrichment), as compared to 5% in random control. Furthermore, 23 of the 107 genes displayed sex-specific expression; and 22 of these genes were positioned to tLG8, indicating 96% enrichment. Of the 27 SDR genes, BCCIP, DHX32A, DOCK1, and FSHR (GTH-RI) are suggested as positional and functional gene candidates for SD.

Integration of mandarin (Citrus reticulata) cytogenetic map with its genome sequence.

Citrus is an extremely important genus in terms of world fruit production. Despite its economic importance and the small genome sizes of its species (2n = 18, 1C = 430 ± 68 Mbp), entire genomic assemblies have only recently become available for some of its representatives. Together with the previous CMA/DAPI banding and fluorescence in situ hybridization (FISH) in the group, these data are important for understanding the complex relationships between its species and for assisting breeding programs. To anchor genomic data with the cytogenetic map of mandarin (Citrus reticulata), the parental species of several economically important hybrids such as sweet orange and clementine, 18 BAC (bacterial artificial chromosome) clones were used. Eleven clementine BACs were positioned by BAC-FISH, doubling the number of chromosome markers so far available for BAC-FISH in citrus. Additionally, six previously mapped BACs were end-sequenced, allowing, together with one BAC previously sequenced, their assignment to scaffolds and the subsequent integration of chromosomes and the genome assembly. This study therefore established correlations between mandarin scaffolds and chromosomes, allowing further structural genomic and comparative study with the sweet orange genome, as well as insights into the chromosomal evolution of the group.

Genomic dissection and expression analysis of stress-responsive genes in C4 panicoid models, Setaria italica and Setaria viridis.

The study reports the identification and expression profiling of five major classes of C4 pathway-specific genes, namely, carbonic anhydrase (CaH), phosphoenolpyruvate carboxylase (PEPC), pyruvate orthophosphate dikinase (PPDK), NADP-dependent malate dehydrogenase (MDH) and NADP-dependent malic enzyme (NADP-ME), in the model species, Setaria italica and Setaria viridis. A total of 42 and 41 genes were identified in S. italica and S. viridis, respectively. Further analysis revealed that segmental and tandem duplications have contributed to the expansion of these gene families. RNA-Seq derived expression profiles of the gene family members showed their differential expression pattern in tissues and dehydration stress. Comparative genome mapping and Ks dating provided insights into their duplication and divergence in the course of evolution. Expression profiling of candidate genes in contrasting S. italica cultivars subjected to abiotic stresses and hormone treatments showed distinct stress-specific upregulation of SiαCaH1, SißCaH5, SiPEPC2, SiPPDK2, SiMDH8, and SiNADP-ME5 in the tolerant cultivar. Overexpression of SiNADP-ME5 in heterologous yeast system enabled the transgenic cells to survive and grow in dehydration stress conditions, which highlights the putative role of SiNADP-ME5 in conferring tolerance to dehydration stress. Altogether, the study highlights key genes that could be potential candidates for elucidating their functional roles in abiotic stress response.

Global integrated omics expression analyses of abiotic stress signaling HSF transcription factor genes in Oryza sativa L.: An in silico approach.

Among the significant transcription factors (TFs), HSF proteins play pivotal roles in the regulation of hormonal signal transduction and different abiotic stress (AbS) responses. Hence considering its importance, global omics expression analysis of HSF candidates was performed in rice (OsHSF). The current study identified 25 HSF family members and physically plotted them against the rice genome. These proteins were systematically analyzed for their physicochemical features, organization and expression signatures. Further, heatmap of both spatio-temporal and global plant hormones revealed the developmental tissues and hormone specific expression profiling of these genes respectively. Comparative genome mapping between OsHSF players in interrelated C4 grass species revealed the chromosome level synteny. Signalome analysis revealed the protein - protein interactions of OsHSF. Expression profiling of key players in response to stresses exhibited the new involvement in combined AbS (CAbS) responses. Our results are significantly valuable to decipher their functional analysis of CAbS tolerant in rice.

QTL Fine Mapping for Sex Determination Region in Bighead Carp (Hypophthalmichthys nobilis) and Comparison with Silver Carp (Hypophthalmichthys molitrix).

Bighead carp (Hypophthalmichthys nobilis) and silver carp (Hypophthalmichthys molitrix) are genetically close aquaculture fish in the Cyprinidae, which have been confirmed to hold XX/XY sex determination. However, genomic locations of potential sex-related loci in these two fishes are still unknown. In this study, a high-resolution genetic linkage map was constructed by using 2976 SNP and 924 microsatellite markers in a F1 full-sib family of bighead carp, the length of which spanned 2022.34 cM with an average inter-marker distance of 0.52 cM. Comparative genomics revealed a high level of genomic synteny between bighead carp and zebrafish as well as grass carp. QTL fine mapping for sex trait was performed based on this linkage map of bighead carp and an unpublished linkage map of silver carp. A map distance of 3.863 cM (69.787-73.650 cM) on LG19 of bighead carp and 4.705 cM (79.096-83.801 cM) on LG21 of silver carp was significantly associated with sex phenotypes, and these two LGs are homologous between two fish species. Fourteen markers harboring in these regions were in strong linkage disequilibrium with the sex phenotype variance explained (PVE) varying from 89 to 100%. Two common markers were mapped on the QTL regions of bighead carp and silver carp, suggesting that these two carp species may have similar genetic bases for sex determination. Eleven potentially sex-related genes were identified within or near the sex QTL markers in two species. This study provided insights into elucidating mechanisms and evolution of sex determination in cyprinid fishes.

Revealing Functional Crosstalk between Distinct Bioprocesses through Reciprocal Functional Tests of Genetically Interacting Genes.

To systematically explore the genes mediating functional crosstalk between metazoan biological processes, we apply comparative genetic interaction (GI) mapping in Saccharomyces cerevisiae and Caenorhabditis elegans to generate an inter-bioprocess network consisting of 178 C. elegans GIs. The GI network spans six annotated biological processes including aging, intracellular transport, microtubule-based processes, cytokinesis, lipid metabolic processes, and anatomical structure development. By proposing a strategy called "reciprocal functional test" for interacting gene pairs, we discover a group of genes that mediate crosstalk between distinct biological processes. In particular, we identify the ribosomal S6 Kinase/RSKS-1, previously characterized as an mTOR (mechanistic target of rapamycin) effector, as a regulator of DAF-2 endosomal recycling transport, which traces a functional correlation between endocytic recycling and aging processes. Together, our results provide an alternative and effective strategy for identifying genes and pathways that mediate crosstalk between bioprocesses with little prior knowledge.

Two Loci, RiAF3 and RiAF4, Contribute to the Annual-Fruiting Trait in Rubus.

Most Rubus species have a biennial cycle of flowering and fruiting with an intervening period of winter dormancy, in common with many perennial fruit crops. Annual-fruiting (AF) varieties of raspberry (Rubus idaeus and Rubus occidentalis L.) and blackberry (Rubus subgenus Rubus) are able to flower and fruit in one growing season, without the intervening dormant period normally required in biennial-fruiting (BF) varieties. We used a red raspberry (R. idaeus) population segregating for AF obtained from a cross between NC493 and 'Chilliwack' to identify genetic factors controlling AF. Genotyping by sequencing (GBS) was used to generate saturated linkage maps in both parents. Trait mapping in this population indicated that AF is controlled by two newly identified loci (RiAF3 and RiAF4) located on Rubus linkage groups (LGs) 3 and 4. The location of these loci was analyzed using single-nucleotide polymorphism (SNP) markers on independent red raspberry and blackberry populations segregating for the AF trait. This confirmed that AF in Rubus is regulated by loci on LG 3 and 4, in addition to a previously reported locus on LG 7. Comparative RNAseq analysis at the time of floral bud differentiation in an AF and a BF variety revealed candidate genes potentially regulating the trait.

Gene Expression Profiling and Fine Mapping Identifies a Gibberellin 2-Oxidase Gene Co-segregating With the Dominant Dwarfing Gene Ddw1 in Rye (Secale cereale L.).

The gibberellin (GA)-sensitive dwarfing gene Ddw1 provides an opportunity to genetically reduce plant height in rye. Genetic analysis in a population of recombinant inbred lines confirmed a monogenetic dominant inheritance of Ddw1. Significant phenotypic differences in PH between homo- and heterozygotic genotypes indicate an incomplete dominance of Ddw1. De novo transcriptome sequencing of Ddw1 mutant as well as tall genotypes resulted in 113,547 contigs with an average length of 318 bp covering 36.18 Mbp rye DNA. A hierarchical cluster analysis based on individual groups of rye homologs of functionally characterized rice genes controlling morphological or physiological traits including plant height, flowering time, and source activity identified the gene expression profile of stems at the begin of heading to most comprehensively mirror effects of Ddw1. Genome-wide expression profiling identified 186 transcripts differentially expressed between semi-dwarf and tall genotypes in stems. In total, 29 novel markers have been established and mapped to a 27.2 cM segment in the distal part of the long arm of chromosome 5R. Ddw1 could be mapped within a 0.4 cM interval co-segregating with a marker representing the C20-GA2-oxidase gene ScGA2ox12, that is up-regulated in stems of Ddw1 genotypes. The increased expression of ScGA2ox12 observed in semi-dwarf rye as well as structural alterations in transcript sequences associated with the ScGA2ox12 gene implicate, that Ddw1 is a dominant gain-of-function mutant. Integration of the target interval in the wheat reference genome sequence indicated perfect micro-colinearity between the Ddw1 locus and a 831 kb segment on chromosome 5A, which resides inside of a 11.21 Mb interval carrying the GA-sensitive dwarfing gene Rht12 in wheat. The potential of Ddw1 as a breeder's option to improve lodging tolerance in rye is discussed.

Mapping Genes Is Good for You.

I abandoned my original career choice of high school teaching to pursue dentistry and soon abandoned that path for genetics. The latter decision was due to a challenge by a professor that led to me reading Nobel speeches by pioneer geneticists before I had formal exposure to the subject. Even then, I was 15 years into my career before my interest in rodent genomes gave way to mapping cattle genes. Events behind these twists and turns in my career path comprise the first part of this review. The remainder is a review of the development of the field of bovine genomics from my personal perspective. I have had the pleasure of working with outstanding graduate students, postdocs, and colleagues to contribute my small part to a discipline that has evolved from a few individuals mapping an orphan genome to a discipline underlying a revolution in animal breeding.

Fine Physical Bin Mapping of the Powdery Mildew Resistance Gene Pm21 Based on Chromosomal Structural Variations in Wheat.

Pm21, derived from wheat wild relative Dasypyrum villosum, is one of the most effective powdery mildew resistance genes and has been widely applied in wheat breeding in China. Mapping and cloning Pm21 are of importance for understanding its resistance mechanism. In the present study, physical mapping was performed using different genetic stocks involving in structural variations of chromosome 6VS carrying Pm21. The data showed that 6VS could be divided into eight distinguishable chromosomal bins, and Pm21 was mapped to the bin FLb4-b5/b6 closely flanked by the markers 6VS-08.6 and 6VS-10.2. Comparative genomic mapping indicated that the orthologous regions of FLb4-b5/b6 carrying Pm21 were narrowed to a 117.7 kb genomic region harboring 19 genes in Brachypodium and a 37.7 kb region harboring 5 genes in rice, respectively. The result was consistent with that given by recent genetic mapping in diploid D. villosum. In conclusion, this study demonstrated that physical mapping based on chromosomal structural variations is an efficient method for locating alien genes in wheat background.

Genetic, Physical and Comparative Mapping of the Powdery Mildew Resistance Gene Pm21 Originating from Dasypyrum villosum.

Pm21, originating from wheat wild relative Dasypyrum villosum, confers immunity to all known races of Blumeria graminis f. sp. tritici (Bgt) and has been widely utilized in wheat breeding. However, little is known on the genetic basis of the Pm21 locus. In the present study, four seedling-susceptible D. villosum lines (DvSus-1 ∼ DvSus-4) were identified from different natural populations. Based on the collinearity among genomes of Brachypodium distachyon, Oryza, and Triticeae, a set of 25 gene-derived markers were developed declaring the polymorphisms between DvRes-1 carrying Pm21 and DvSus-1. Fine genetic mapping of Pm21 was conducted by using an extremely large F2 segregation population derived from the cross DvSus-1/DvRes-1. Then Pm21 was narrowed to a 0.01-cM genetic interval defined by the markers 6VS-08.4b and 6VS-10b. Three DNA markers, including a resistance gene analog marker, were confirmed to co-segregate with Pm21. Moreover, based on the susceptible deletion line Y18-S6 induced by ethyl methanesulfonate treatment conducted on Yangmai 18, Pm21 was physically mapped into a similar interval. Comparative analysis revealed that the orthologous regions of the interval carrying Pm21 were narrowed to a 112.5 kb genomic region harboring 18 genes in Brachypodium, and a 23.2 kb region harboring two genes in rice, respectively. This study provides a high-density integrated map of the Pm21 locus, which will contribute to map-based cloning of Pm21.

Global Transcriptome Analysis of Combined Abiotic Stress Signaling Genes Unravels Key Players in Oryza sativa L.: An In silico Approach.

Combined abiotic stress (CAbS) affects the field grown plants simultaneously. The multigenic and quantitative nature of uncontrollable abiotic stresses complicates the process of understanding the stress response by plants. Considering this, we analyzed the CAbS response of C3 model plant, Oryza sativa by meta-analysis. The datasets of commonly expressed genes by drought, salinity, submergence, metal, natural expression, biotic, and abiotic stresses were data mined through publically accessible transcriptomic abiotic stress (AbS) responsive datasets. Of which 1,175, 12,821, and 42,877 genes were commonly expressed in meta differential, individual differential, and unchanged expressions respectively. Highly regulated 100 differentially expressed AbS genes were derived through integrative meta-analysis of expression data (INMEX). Of this 30 genes were identified from AbS gene families through expression atlas that were computationally analyzed for their physicochemical properties. All AbS genes were physically mapped against O. sativa genome. Comparative mapping of these genes demonstrated the orthologous relationship with related C4 panicoid genome. In silico expression analysis of these genes showed differential expression patterns in different developmental tissues. Protein-protein interaction of these genes, represented the complexity of AbS. Computational expression profiling of candidate genes in response to multiple stresses suggested the putative involvement of OS05G0350900, OS02G0612700, OS05G0104200, OS03G0596200, OS12G0225900, OS07G0152000, OS08G0119500, OS06G0594700, and Os01g0393100 in CAbS. These potential candidate genes need to be studied further to decipher their functional roles in AbS dynamics.

Integrating genomic resources of flatfish (Pleuronectiformes) to boost aquaculture production.

Flatfish have a high market acceptance thus representing a profitable aquaculture production. The main farmed species is the turbot (Scophthalmus maximus) followed by Japanese flounder (Paralichthys olivaceous) and tongue sole (Cynoglossus semilaevis), but other species like Atlantic halibut (Hippoglossus hippoglossus), Senegalese sole (Solea senegalensis) and common sole (Solea solea) also register an important production and are very promising for farming. Important genomic resources are available for most of these species including whole genome sequencing projects, genetic maps and transcriptomes. In this work, we integrate all available genomic information of these species within a common framework, taking as reference the whole assembled genomes of turbot and tongue sole (>210× coverage). New insights related to the genetic basis of productive traits and new data useful to understand the evolutionary origin and diversification of this group were obtained. Despite a general 1:1 chromosome syntenic relationship between species, the comparison of turbot and tongue sole genomes showed huge intrachromosomic reorganizations. The integration of available mapping information supported specific chromosome fusions along flatfish evolution and facilitated the comparison between species of previously reported genetic associations for productive traits. When comparing transcriptomic resources of the six species, a common set of ~2500 othologues and ~150 common miRNAs were identified, and specific sets of putative missing genes were detected in flatfish transcriptomes, likely reflecting their evolutionary diversification.

New insights into sex chromosome evolution in anole lizards (Reptilia, Dactyloidae).

Anoles are a clade of iguanian lizards that underwent an extensive radiation between 125 and 65 million years ago. Their karyotypes show wide variation in diploid number spanning from 26 (Anolis evermanni) to 44 (A. insolitus). This chromosomal variation involves their sex chromosomes, ranging from simple systems (XX/XY), with heterochromosomes represented by either micro- or macrochromosomes, to multiple systems (X1X1X2X2/X1X2Y). Here, for the first time, the homology relationships of sex chromosomes have been investigated in nine anole lizards at the whole chromosome level. Cross-species chromosome painting using sex chromosome paints from A. carolinensis, Ctenonotus pogus and Norops sagrei and gene mapping of X-linked genes demonstrated that the anole ancestral sex chromosome system constituted by microchromosomes is retained in all the species with the ancestral karyotype (2n = 36, 12 macro- and 24 microchromosomes). On the contrary, species with a derived karyotype, namely those belonging to genera Ctenonotus and Norops, show a series of rearrangements (fusions/fissions) involving autosomes/microchromosomes that led to the formation of their current sex chromosome systems. These results demonstrate that different autosomes were involved in translocations with sex chromosomes in closely related lineages of anole lizards and that several sequential microautosome/sex chromosome fusions lead to a remarkable increase in size of Norops sagrei sex chromosomes.

Genome-wide Association Study Identifies New Loci for Resistance to Leptosphaeria maculans in Canola.

Key message "We identified both quantitative and quantitative resistance loci to Leptosphaeria maculans, a fungal pathogen, causing blackleg disease in canola. Several genome-wide significant associations were detected at known and new loci for blackleg resistance. We further validated statistically significant associations in four genetic mapping populations, demonstrating that GWAS marker loci are indeed associated with resistance to L. maculans. One of the novel loci identified for the first time, Rlm12, conveys adult plant resistance in canola." Blackleg, caused by Leptosphaeria maculans, is a significant disease which affects the sustainable production of canola (Brassica napus). This study reports a genome-wide association study based on 18,804 polymorphic SNPs to identify loci associated with qualitative and quantitative resistance to L. maculans. Genomic regions delimited with 694 significant SNP markers, that are associated with resistance evaluated using 12 single spore isolates and pathotypes from four canola stubble were identified. Several significant associations were detected at known disease resistance loci including in the vicinity of recently cloned Rlm2/LepR3 genes, and at new loci on chromosomes A01/C01, A02/C02, A03/C03, A05/C05, A06, A08, and A09. In addition, we validated statistically significant associations on A01, A07, and A10 in four genetic mapping populations, demonstrating that GWAS marker loci are indeed associated with resistance to L. maculans. One of the novel loci identified for the first time, Rlm12, conveys adult plant resistance and mapped within 13.2 kb from Arabidopsis R gene of TIR-NBS class. We showed that resistance loci are located in the vicinity of R genes of Arabidopsis thaliana and Brassica napus on the sequenced genome of B. napus cv. Darmor-bzh. Significantly associated SNP markers provide a valuable tool to enrich germplasm for favorable alleles in order to improve the level of resistance to L. maculans in canola.