Novel quantitative trait loci from an interspecific Brassica rapa derivative improve pod shatter resistance in Brassica napus.

Pod shatter is a trait of agricultural relevance that ensures plants dehisce seeds in their native environment and has been subjected to domestication and selection for non-shattering types in several broadacre crops. However, pod shattering causes a significant yield reduction in canola (Brassica napus L.) crops. An interspecific breeding line BC95042 derived from a B. rapa/B. napus cross showed improved pod shatter resistance (up to 12-fold than a shatter-prone B. napus variety). To uncover the genetic basis and improve pod shatter resistance in new varieties, we analysed F2 and F2:3 derived populations from the cross between BC95042 and an advanced breeding line, BC95041, and genotyped with 15,498 DArTseq markers. Through genome scan, interval and inclusive composite interval mapping analyses, we identified seven quantitative trait loci (QTLs) associated with pod rupture energy, a measure for pod shatter resistance or pod strength, and they locate on A02, A03, A05, A09 and C01 chromosomes. Both parental lines contributed alleles for pod shatter resistance. We identified five pairs of significant epistatic QTLs for additive x additive, additive dominance and dominance x dominance interactions between A01/C01, A03/A07, A07/C03, A03/C03, and C01/C02 chromosomes for rupture energy. QTL effects on A03/A07 and A01/C01 were in the repulsion phase. Comparative mapping identified several candidate genes (AG, ABI3, ARF3, BP1, CEL6, FIL, FUL, GA2OX2, IND, LATE, LEUNIG, MAGL15, RPL, QRT2, RGA, SPT and TCP10) underlying main QTL and epistatic QTL interactions for pod shatter resistance. Three QTLs detected on A02, A03, and A09 were near the FUL (FRUITFULL) homologues BnaA03g39820D and BnaA09g05500D. Focusing on the FUL, we investigated putative motifs, sequence variants and the evolutionary rate of its homologues in 373 resequenced B. napus accessions of interest. BnaA09g05500D is subjected to purifying selection as it had a low Ka/Ks ratio compared to other FUL homologues in B. napus. This study provides a valuable resource for genetic improvement for yield through an understanding of the genetic mechanism controlling pod shatter resistance in Brassica species.

Quantitative Trait Loci for Genotype and Genotype by Environment Interaction Effects for Seed Yield Plasticity to Terminal Water-Deficit Conditions in Canola (Brassica napus L.).

Canola plants suffer severe crop yield and oil content reductions when exposed to water-deficit conditions, especially during the reproductive stages of plant development. There is a pressing need to develop canola cultivars that can perform better under increased water-deficit conditions with changing weather patterns. In this study, we analysed genetic determinants for the main effects of quantitative trait loci (QTL), (Q), and the interaction effects of QTL and Environment (QE) underlying seed yield and related traits utilising 223 doubled haploid (DH) lines of canola in well-watered and water-deficit conditions under a rainout shelter. Moderate water-deficit at the pre-flowering stage reduced the seed yield to 40.8%. Multi-environmental QTL analysis revealed 23 genomic regions associated with days to flower (DTF), plant height (PH) and seed yield (SY) under well-watered and water-deficit conditions. Three seed yield QTL for main effects were identified on chromosomes A09, C03, and C09, while two were related to QE interactions on A02 and C09. Two QTL regions were co-localised to similar genomic regions for flowering time and seed yield (A09) and the second for plant height and chlorophyll content. The A09 QTL was co-located with a previously mapped QTL for carbon isotope discrimination (Δ13C) that showed a positive relationship with seed yield in the same population. Opposite allelic effects for plasticity in seed yield were identified due to QE interactions in response to water stress on chromosomes A02 and C09. Our results showed that QTL's allelic effects for DTF, PH, and SY and their correlation with Δ13C are stable across environments (field conditions, previous study) and contrasting water regimes (this study). The QTL and DH lines that showed high yield under well-watered and water-deficit conditions could be used to manipulate water-use efficiency for breeding improved canola cultivars.

Genome-Wide Association Analyses Track Genomic Regions for Resistance to Ascochyta rabiei in Australian Chickpea Breeding Germplasm.

Ascochyta blight (AB), caused by a necrotrophic fungus, Ascochyta rabiei (syn. Phoma rabiei) has the potential to destroy the chickpea industry worldwide, due to limited sources of genetic resistance in the cultivated gene pool, high evolutionary potential of the pathogen and challenges with integrated disease management. Therefore, the deployment of stable genetic resistance in new cultivars could provide an effective disease control strategy. To investigate the genetic basis of AB resistance, genotyping-by-sequencing based DArTseq-single nucleotide polymorphism (SNP) marker data along with phenotypic data of 251 advanced breeding lines and chickpea cultivars were used to perform genome-wide association (GWAS) analysis. Host resistance was evaluated seven weeks after sowing using two highly aggressive single spore isolates (F17191-1 and TR9571) of A. rabiei. GWAS analyses based on single-locus and multi-locus mixed models and haplotyping trend regression identified twenty-six genomic regions on Ca1, Ca4, and Ca6 that showed significant association with resistance to AB. Two haplotype blocks (HB) on chromosome Ca1; HB5 (992178-1108145 bp), and HB8 (1886221-1976301 bp) were associated with resistance against both isolates. Nine HB on the chromosome, Ca4, spanning a large genomic region (14.9-56.6 Mbp) were also associated with resistance, confirming the role of this chromosome in providing resistance to AB. Furthermore, trait-marker associations in two F3 derived populations for resistance to TR9571 isolate at the seedling stage under glasshouse conditions were also validated. Eighty-nine significantly associated SNPs were located within candidate genes, including genes encoding for serine/threonine-protein kinase, Myb protein, quinone oxidoreductase, and calmodulin-binding protein all of which are implicated in disease resistance. Taken together, this study identifies valuable sources of genetic resistance, SNP markers and candidate genes underlying genomic regions associated with AB resistance which may enable chickpea breeding programs to make genetic gains via marker-assisted/genomic selection strategies.

A genome-wide association study (GWAS) identifies multiple loci linked with the natural variation for Al3+ resistance in Brassica napus.

Acid soils limit yields of many important crops including canola (Brassica napus ), Australia's third largest crop. Aluminium (Al3+ ) stress is the main cause of this limitation primarily because the toxic Al3+ present inhibits root growth. Breeding programmes do not target acid-soil tolerance in B. napus because genetic variation and convincing quantitative trait loci have not been reported. We conducted a genome-wide association study (GWAS) using the BnASSYST diversity panel of B. napus genotyped with 35 729 high-quality DArTseq markers. We screened 352 B. napus accessions in hydroponics with and without a toxic concentration of AlCl3 (12µM, pH 4.3) for 12days and measured shoot biomass, root biomass, and root length. By accounting for both population structure and kinship matrices, five significant quantitative trait loci for different measures of resistance were identified using incremental Al3+ resistance indices. Within these quantitative trait locus regions of B. napus , 40 Arabidopsis thaliana gene orthologues were identified, including some previously linked with Al3+ resistance. GWAS analysis indicated that multiple genes are responsible for the natural variation in Al3+ resistance in B. napus . The results provide new genetic resources and markers to enhance that Al3+ resistance of B. napus germplasm via genomic and marker-assisted selection.

Multienvironment QTL analysis delineates a major locus associated with homoeologous exchanges for water-use efficiency and seed yield in canola.

Canola varieties exhibit variation in drought avoidance and drought escape traits, reflecting adaptation to water-deficit environments. Our understanding of underlying genes and their interaction across environments in improving crop productivity is limited. A doubled haploid population was analysed to identify quantitative trait loci (QTL) associated with water-use efficiency (WUE) related traits. High WUE in the vegetative phase was associated with low seed yield. Based on the resequenced parental genome data, we developed sequence-capture-based markers and validated their linkage with carbon isotope discrimination (Δ13 C) in an F2 population. RNA sequencing was performed to determine the expression of candidate genes underlying Δ13 C QTL. QTL contributing to main and QTL × environment interaction effects for Δ13 C and yield were identified. One multiple-trait QTL for Δ13 C, days to flower, plant height, and seed yield was identified on chromosome A09. Interestingly, this QTL region overlapped with a homoeologous exchange (HE) event, suggesting its association with the multiple traits. Transcriptome analysis revealed 121 significantly differentially expressed genes underlying Δ13 C QTL on A09 and C09, including in HE regions. Sorting out the negative relationship between vegetative WUE and seed yield is a priority. Genetic and genomic resources and knowledge so developed could improve canola WUE and yield.

Modelling the effects of cold temperature during the reproductive stage on the yield of chickpea (Cicer arietinum L.).

During the reproductive stage, chilling temperatures and frost reduce the yield of chickpea and limit its adaptation. The adverse effects of chilling temperature and frost in terms of the threshold temperatures, impact of cold duration, and genotype-by-environment-by-management interactions are not well quantified. Crop growth models that predict flowering time and yield under diverse climates can identify combinations of cultivars and sowing time to reduce frost risk in target environments. The Agricultural Production Systems Simulator (APSIM-chickpea) model uses daily temperatures to model basic crop growth but does not include penalties for either frost damage or cold temperatures during flowering and podding stages. Regression analysis overcame this limitation of the model for chickpea crops grown at 95 locations in Australia using 70 years of historic data incorporating three cultivars and three sowing times (early, mid, and late). We modified model parameters to include the effect of soil water on thermal time calculations, which significantly improved the prediction of flowering time. Simulated data, and data from field experiments grown in Australia (2013 to 2019), showed robust predictions for flowering time (n = 29; R2 = 0.97), and grain yield (n = 22; R2 = 0.63-0.70). In addition, we identified threshold cold temperatures that significantly affected predicted yield, and combinations of locations, variety, and sowing time where the overlap between peak cold temperatures and peak flowering was minimal. Our results showed that frost and/or cold temperature-induced yield losses are a major limitation in some unexpected Australian locations, e.g., inland, subtropical latitudes in Queensland. Intermediate sowing maximise yield, as it avoids cold temperature, late heat, and drought stresses potentially limiting yield in early and late sowing respectively.

The Rlm13 Gene, a New Player of Brassica napus-Leptosphaeria maculans Interaction Maps on Chromosome C03 in Canola.

Canola exhibits an extensive genetic variation for resistance to blackleg disease, caused by the fungal pathogen Leptosphaeria maculans. Despite the identification of several Avr effectors and R (race-specific) genes, specific interactions between Avr-R genes are not yet fully understood in the Brassica napus-L. maculans pathosystem. In this study, we investigated the genetic basis of resistance in an F2 : 3 population derived from Australian canola varieties CB-Telfer (Rlm4)/ATR-Cobbler (Rlm4) using a single-spore isolate of L. maculans, PHW1223. A genetic linkage map of the CB-Telfer/ATR-Cobbler population was constructed using 7,932 genotyping-by-sequencing-based DArTseq markers and subsequently utilized for linkage and haplotype analyses. Genetic linkage between DArTseq markers and resistance to PHW1223 isolate was also validated using the B. napus 60K Illumina Infinium array. Our results revealed that a major locus for resistance, designated as Rlm13, maps on chromosome C03. To date, no R gene for resistance to blackleg has been reported on the C subgenome in B. napus. Twenty-four candidate R genes were predicted to reside within the quantitative trait locus (QTL) region. We further resequenced both the parental lines of the mapping population (CB-Telfer and ATR-Cobbler, > 80 × coverage) and identified several structural sequence variants in the form of single-nucleotide polymorphisms (SNPs), insertions/deletions (InDels), and presence/absence variations (PAVs) near Rlm13. Comparative mapping revealed that Rlm13 is located within the homoeologous A03/C03 region in ancestral karyotype block "R" of Brassicaceae. Our results provide a "target" for further understanding the Avr-Rlm13 gene interaction as well as a valuable tool for increasing resistance to blackleg in canola germplasm.

Genome-Wide Association Mapping Identifies Novel Loci for Quantitative Resistance to Blackleg Disease in Canola.

Blackleg disease, caused by the fungal pathogen Leptosphaeria maculans, continues to be a major concern for sustainable production of canola (Brassica napus L.) in many parts of the world. The deployment of effective quantitative resistance (QR) is recognized as a durable strategy in providing natural defense to pathogens. Herein, we uncover loci for resistance to blackleg in a genetically diverse panel of canola accessions by exploiting historic recombination events which occurred during domestication and selective breeding by genome-wide association analysis (GWAS). We found extensive variation in resistance to blackleg at the adult plant stage, including for upper canopy infection. Using the linkage disequilibrium and genetic relationship estimates from 12,414 high quality SNPs, GWAS identified 59 statistically significant and "suggestive" SNPs on 17 chromosomes of B. napus genome that underlie variation in resistance to blackleg, evaluated under field and shade-house conditions. Each of the SNP association accounted for up to 25.1% of additive genetic variance in resistance among diverse panel of accessions. To understand the homology of QR genomic regions with Arabidopsis thaliana genome, we searched the synteny between QR regions with 22 ancestral blocks of Brassicaceae. Comparative analyses revealed that 25 SNP associations for QR were localized in nine ancestral blocks, as a result of genomic rearrangements. We further showed that phenological traits such as flowering time, plant height, and maturity confound the genetic variation in resistance. Altogether, these findings provided new insights on the complex genetic control of the blackleg resistance and further expanded our understanding of its genetic architecture.

A copia-like retrotransposon insertion in the upstream region of the SHATTERPROOF1 gene, BnSHP1.A9, is associated with quantitative variation in pod shattering resistance in oilseed rape.

Seed loss resulting from pod shattering is a major constraint in production of oilseed rape (Brassica napus L.). However, the molecular mechanisms underlying pod shatter resistance are not well understood. Here, we show that the pod shatter resistance at quantitative trait locus qSRI.A9.1 is controlled by one of the B. napus SHATTERPROOF1 homologs, BnSHP1.A9, in a doubled haploid population generated from parents designated R1 and R2 as well as in a diverse panel of oilseed rape. The R1 maternal parental line of the doubled haploid population carried the allele for shattering at qSRI.A9.1, while the R2 parental line carried the allele for shattering resistance. Quantitative RT-PCR showed that BnSHP1.A9 was expressed specifically in flower buds, flowers, and developing siliques in R1, while it was not expressed in any tissue of R2. Transgenic plants constitutively expressing either of the BnSHP1.A9 alleles from the R1 and R2 parental lines showed that both alleles are responsible for pod shattering, via a mechanism that promotes lignification of the enb layer. These findings indicated that the allelic differences in the BnSHP1.A9 gene per se are not the causal factor for quantitative variation in shattering resistance at qSRI.A9.1. Instead, a highly methylated copia-like long terminal repeat retrotransposon insertion (4803 bp) in the promotor region of the R2 allele of BnSHP1.A9 repressed the expression of BnSHP1.A9, and thus contributed to pod shatter resistance. Finally, we showed a copia-like retrotransposon-based marker, BnSHP1.A9R2, can be used for marker-assisted breeding targeting the pod shatter resistance trait in oilseed rape.

Genetic and physical mapping of loci for resistance to blackleg disease in canola (Brassica napus L.).

Sustainable canola production is essential to meet growing human demands for vegetable oil, biodiesel, and meal for stock feed markets. Blackleg, caused by the fungal pathogen, Leptosphaeria maculans is a devastating disease that can lead to significant yield loss in many canola production regions worldwide. Breakdown of race-specific resistance to L. maculans in commercial cultivars poses a constant threat to the canola industry. To identify new alleles, especially for quantitative resistance (QR), we analyzed 177 doubled haploid (DH) lines derived from an RP04/Ag-Outback cross. DH lines were evaluated for QR under field conditions in three experiments conducted at Wagga Wagga (2013, 2014) and Lake Green (2015), and under shade house conditions using the 'ascospore shower' test. DH lines were also characterized for qualitative R gene-mediated resistance via cotyledon tests with two differential single spore isolates, IBCN17 and IBCN76, under glasshouse conditions. Based on 18,851 DArTseq markers, a linkage map representing 2,019 unique marker bins was constructed and then utilized for QTL detection. Marker regression analysis identified 22 significant marker associations for resistance, allowing identification of two race-specific resistance R genes, Rlm3 and Rlm4, and 21 marker associations for QR loci. At least three SNP associations for QR were repeatedly detected on chromosomes A03, A07 and C04 across phenotyping environments. Physical mapping of markers linked with these consistent QR loci on the B. napus genome assembly revealed their localization in close proximity of the candidate genes of B. napus BnaA03g26760D (A03), BnaA07g20240D (A07) and BnaC04g02040D (C04). Annotation of these candidate genes revealed their association with protein kinase and jumonji proteins implicated in defense resistance. Both Rlm3 and Rlm4 genes identified in this DH population did not show any association with resistance loci detected under either field and/or shade house conditions (ascospore shower) suggesting that both genes are ineffective in conferring resistance to L. maculans in Australian field conditions. Taken together, our study identified sequence-based molecular markers for dissecting R and QR loci to L. maculans in a canola DH population from the RP04/Ag-Outback cross.

GWAS hints at pleiotropic roles for FLOWERING LOCUS T in flowering time and yield-related traits in canola.

BACKGROUND: Transition to flowering at the right time is critical for local adaptation and to maximize grain yield in crops. Canola is an important oilseed crop with extensive variation in flowering time among varieties. However, our understanding of underlying genes and their role in canola productivity is limited. RESULTS: We report our analyses of a diverse GWAS panel (300-368 accessions) of canola and identify SNPs that are significantly associated with variation in flowering time and response to photoperiod across multiple locations. We show that several of these associations map in the vicinity of FLOWERING LOCUS T (FT) paralogs and its known transcriptional regulators. Complementary QTL and eQTL mapping studies, conducted in an Australian doubled haploid population, also detected consistent genomic regions close to the FT paralogs associated with flowering time and yield-related traits. FT sequences vary between accessions. Expression levels of FT in plants grown in field (or under controlled environment cabinets) correlated with flowering time. We show that markers linked to the FT paralogs display association with variation in multiple traits including flowering time, plant emergence, shoot biomass and grain yield. CONCLUSIONS: Our findings suggest that FT paralogs not only control flowering time but also modulate yield-related productivity traits in canola.

Stable Quantitative Resistance Loci to Blackleg Disease in Canola (Brassica napus L.) Over Continents.

The hemibiotrophic fungus, Leptosphaeria maculans is the most devastating pathogen, causing blackleg disease in canola (Brassica napus L). To study the genomic regions involved in quantitative resistance (QR), 259-276 DH lines from Darmor-bzh/Yudal (DYDH) population were assessed for resistance to blackleg under shade house and field conditions across 3 years. In different experiments, the broad sense heritability varied from 43 to 95%. A total of 27 significant quantitative trait loci (QTL) for QR were detected on 12 chromosomes and explained between 2.14 and 10.13% of the genotypic variance. Of the significant QTL, at least seven were repeatedly detected across different experiments on chromosomes A02, A07, A09, A10, C01, and C09. Resistance alleles were mainly contributed by 'Darmor-bzh' but 'Yudal' also contributed few of them. Our results suggest that plant maturity and plant height may have a pleiotropic effect on QR in our conditions. We confirmed that Rlm9 which is present in 'Darmor-bzh' is not effective to confer resistance in our Australian field conditions. Comparative mapping showed that several R genes coding for nucleotide-binding leucine-rich repeat (LRR) receptors map in close proximity (within 200 Kb) of the significant trait-marker associations on the reference 'Darmor-bzh' genome assembly. More importantly, eight significant QTL regions were detected across diverse growing environments: Australia, France, and United Kingdom. These stable QTL identified herein can be utilized for enhancing QR in elite canola germplasm via marker- assisted or genomic selection strategies.

Molecular Diversity Analysis and Genetic Mapping of Pod Shatter Resistance Loci in Brassica carinata L.

Seed lost due to easy pod dehiscence at maturity (pod shatter) is a major problem in several members of Brassicaceae family. We investigated the level of pod shatter resistance in Ethiopian mustard (Brassica carinata) and identified quantitative trait loci (QTL) for targeted introgression of this trait in Ethiopian mustard and its close relatives of the genus Brassica. A set of 83 accessions of B. carinata, collected from the Australian Grains Genebank, was evaluated for pod shatter resistance based on pod rupture energy (RE). In comparison to B. napus (RE = 2.16 mJ), B. carinata accessions had higher RE values (2.53 to 20.82 mJ). A genetic linkage map of an F2 population from two contrasting B. carinata selections, BC73526 (shatter resistant with high RE) and BC73524 (shatter prone with low RE) comprising 300 individuals, was constructed using a set of 6,464 high quality DArTseq markers and subsequently used for QTL analysis. Genetic analysis of the F2 and F2:3 derived lines revealed five statistically significant QTL (LOD ≥ 3) that are linked with pod shatter resistance on chromosomes B1, B3, B8, and C5. Herein, we report for the first time, identification of genetic loci associated with pod shatter resistance in B. carinata. These characterized accessions would be useful in Brassica breeding programs for introgression of pod shatter resistance alleles in to elite breeding lines. Molecular markers would assist marker-assisted selection for tracing the introgression of resistant alleles. Our results suggest that the value of the germplasm collections can be harnessed through genetic and genomics tools.

Investigation of the Genetic Diversity and Quantitative Trait Loci Accounting for Important Agronomic and Seed Quality Traits in Brassica carinata.

Brassica carinata (BBCC) is an allotetraploid in Brassicas with unique alleles for agronomic traits and has huge potential as source for biodiesel production. To investigate the genome-wide molecular diversity, population structure and linkage disequilibrium (LD) pattern in this species, we genotyped a panel of 81 accessions of B. carinata with genotyping by sequencing approach DArTseq, generating a total of 54,510 polymorphic markers. Two subpopulations were exhibited in the B. carinata accessions. The average distance of LD decay (r2 = 0.1) in B subgenome (0.25 Mb) was shorter than that of C subgenome (0.40 Mb). Genome-wide association analysis (GWAS) identified a total of seven markers significantly associated with five seed quality traits in two experiments. To further identify the quantitative trait loci (QTL) for important agronomic and seed quality traits, we phenotyped a doubled haploid (DH) mapping population derived from the "YW" cross between two parents (Y-BcDH64 and W-BcDH76) representing from the two subpopulations. The YW DH population and its parents were grown in three contrasting environments; spring (Hezheng and Xining, China), semi-winter (Wuhan, China), and spring (Wagga Wagga, Australia) across 5 years for QTL mapping. Genetic bases of phenotypic variation in seed yield and its seven related traits, and six seed quality traits were determined. A total of 282 consensus QTL accounting for these traits were identified including nine major QTL for flowering time, oleic acid, linolenic acid, pod number of main inflorescence, and seed weight. Of these, 109 and 134 QTL were specific to spring and semi-winter environment, respectively, while 39 consensus QTL were identified in both contrasting environments. Two QTL identified for linolenic acid (B3) and erucic acid (C7) were validated in the diverse lines used for GWAS. A total of 25 QTL accounting for flowering time, erucic acid, and oleic acid were aligned to the homologous QTL or candidate gene regions in the C genome of B. napus. These results would not only provide insights for genetic improvement of this species, but will also identify useful genetic variation hidden in the Cc subgenome of B. carinata to improve canola cultivars.

A Major Locus for Manganese Tolerance Maps on Chromosome A09 in a Doubled Haploid Population of Brassica napus L.

Soil acidity poses a major threat to productivity of several crops; mainly due to the prevalence of toxic levels of Al3+ and Mn2+. Crop productivity could be harnessed on acid soils via the development of plant varieties tolerant to phytotoxic levels of these cations. In this study, we investigated the extent of natural variation for Mn2+ tolerance among ten parental lines of the Australian and International canola mapping populations. Response to Mn2+ toxicity was measured on the bases of cotyledon chlorosis, shoot biomass, and leaf area in nutrient solution under control (9 µM of MnCl2⋅4H2O) and Mn treatment (125 µM of MnCl2⋅4H2O). Among parental lines, we selected Darmor-bzh and Yudal that showed significant and contrasting variation in Mn2+ tolerance to understand genetic control and identify the quantitative trait loci (QTL) underlying Mn2+ tolerance. We evaluated parental lines and their doubled haploid (DH) progenies (196 lines) derived from an F1 cross, Darmor-bzh/Yudal for Mn2+ tolerance. Mn2+-tolerant genotypes had significantly higher shoot biomass and leaf area compared to Mn2+-sensitive genotypes. A genetic linkage map based on 7,805 DArTseq markers corresponding to 2,094 unique loci was constructed and further utilized for QTL identification. A major locus, BnMn2+.A09 was further mapped with a SNP marker, Bn-A09-p29012402 (LOD score of 34.6) accounting for most of the variation in Mn2+ tolerance on chromosome A09. This is the first report on the genomic localization of a Mn2+ tolerance locus in B. napus. Additionally, an ortholog of A. thaliana encoding for cation efflux facilitator transporter was located within 3,991 bp from significant SNP marker associated with BnMn2+.A09. A suite of genome sequence based markers (DArTseq and Illumina Infinium SNPs) flanking the BnMn2+.A09 locus would provide an invaluable tool for various molecular breeding applications to improve canola production and profitability on Mn2+ toxic soils.

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.

Multi-environment QTL studies suggest a role for cysteine-rich protein kinase genes in quantitative resistance to blackleg disease in Brassica napus.

BACKGROUND: Resistance to the blackleg disease of Brassica napus (canola/oilseed rape), caused by the hemibiotrophic fungal pathogen Leptosphaeria maculans, is determined by both race-specific resistance (R) genes and quantitative resistance loci (QTL), or adult-plant resistance (APR). While the introgression of R genes into breeding material is relatively simple, QTL are often detected sporadically, making them harder to capture in breeding programs. For the effective deployment of APR in crop varieties, resistance QTL need to have a reliable influence on phenotype in multiple environments and be well defined genetically to enable marker-assisted selection (MAS). RESULTS: Doubled-haploid populations produced from the susceptible B. napus variety Topas and APR varieties AG-Castle and AV-Sapphire were analysed for resistance to blackleg in two locations over 3 and 4 years, respectively. Three stable QTL were detected in each population, with two loci appearing to be common to both APR varieties. Physical delineation of three QTL regions was sufficient to identify candidate defense-related genes, including a cluster of cysteine-rich receptor-like kinases contained within a 49 gene QTL interval on chromosome A01. Individual L. maculans isolates were used to define the physical intervals for the race-specific R genes Rlm3 and Rlm4 and to identify QTL common to both field studies and the cotyledon resistance response. CONCLUSION: Through multi-environment QTL analysis we have identified and delineated four significant and stable QTL suitable for MAS of quantitative blackleg resistance in B. napus, and identified candidate genes which potentially play a role in quantitative defense responses to L. maculans.

A high-throughput SNP array in the amphidiploid species Brassica napus shows diversity in resistance genes.

Single-nucleotide polymorphisms (SNPs)are molecular markers based on nucleotide variation and can be used for genotyping assays across populations and to track genomic inheritance. SNPs offer a comprehensive genotyping alternative to whole-genome sequencing for both agricultural and research purposes including molecular breeding and diagnostics, genome evolution and genetic diversity analyses, genetic mapping, and trait association studies. Here genomic SNPs were discovered between four cultivars of the important amphidiploid oilseed species Brassica napus and used to develop a B. napus Infinium™ array containing 5,306 SNPs randomly dispersed across the genome. Assay success was high, with >94 % of these producing a reproducible, polymorphic genotype in the 1,070 samples screened. Although the assay was designed to B. napus, successful SNP amplification was achieved in the B. napus progenitor species, Brassica rapa and Brassica oleracea, and to a lesser extent in the related species Brassica nigra. Phylogenetic analysis was consistent with the expected relationships between B. napus individuals. This study presents an efficient custom SNP assay development pipeline in the complex polyploid Brassica genome and demonstrates the utility of the array for high-throughput genotyping in a number of related Brassica species. It also demonstrates the utility of this assay in genotyping resistance genes on chromosome A7, which segregate amongst the 1,070 samples.

Genome-wide delineation of natural variation for pod shatter resistance in Brassica napus.

Resistance to pod shattering (shatter resistance) is a target trait for global rapeseed (canola, Brassica napus L.), improvement programs to minimise grain loss in the mature standing crop, and during windrowing and mechanical harvest. We describe the genetic basis of natural variation for shatter resistance in B. napus and show that several quantitative trait loci (QTL) control this trait. To identify loci underlying shatter resistance, we used a novel genotyping-by-sequencing approach DArT-Seq. QTL analysis detected a total of 12 significant QTL on chromosomes A03, A07, A09, C03, C04, C06, and C08; which jointly account for approximately 57% of the genotypic variation in shatter resistance. Through Genome-Wide Association Studies, we show that a large number of loci, including those that are involved in shattering in Arabidopsis, account for variation in shatter resistance in diverse B. napus germplasm. Our results indicate that genetic diversity for shatter resistance genes in B. napus is limited; many of the genes that might control this trait were not included during the natural creation of this species, or were not retained during the domestication and selection process. We speculate that valuable diversity for this trait was lost during the natural creation of B. napus. To improve shatter resistance, breeders will need to target the introduction of useful alleles especially from genotypes of other related species of Brassica, such as those that we have identified.