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.

Protease Inhibitors Purified from the Canola Meal Extracts of Two Genetically Diverse Genotypes Exhibit Antidiabetic and Antihypertension Properties.

Valorization of vegetable oil waste residues is gaining importance due to their high protein and polyphenol contents. Protease inhibitors (PIs), proteins from these abundantly available waste residues, have recently gained importance in treating chronic diseases. This research aimed to use canola meal of genetically diverse Brassica napus genotypes, BLN-3347 and Rivette, to identify PIs with diverse functionalities in therapeutic and pharmacological applications. The canola meal PI purification steps involved: native PAGE and trypsin inhibition activity, followed by ammonium sulfate fractionation, anion exchange, gel filtration, and reverse-phase chromatography. The purified PI preparations were characterized using SDS-PAGE, isoelectric focusing (IEF), and N terminal sequencing. SDS-PAGE analysis of PI preparations under native reducing and nonreducing conditions revealed three polymorphic PIs in each genotype. The corresponding IEF of the genotype BLN-3347, exhibited three acidic isoforms with isoelectric points (pI) of 4.6, 4.0, and 3.9, while Rivette possessed three isoforms, exhibiting two basic forms of pI 8.65 and 9.9, and one acidic of pI 6.55. Purified PI preparations from both the genotypes displayed dipeptidyl peptidase-IV (DPP-IV) and angiotensin-converting enzyme (ACE) inhibition activities; the BLN-3347 PI preparation exhibited a strong inhibitory effect with lower IC50 values (DPP-IV 37.42 µg/mL; ACE 129 µg/mL) than that from Rivette (DPP-IV 67.97 µg/mL; ACE 376.2 µg/mL). In addition to potential human therapy, these highly polymorphic PIs, which can inhibit damaging serine proteases secreted by canola plant pathogens, have the potential to be used by canola plant breeders to seek qualitative trait locus (QTLs) linked to genes conferring resistance to canola diseases.

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.

Phenolic Compounds with Antioxidant Properties from Canola Meal Extracts Inhibit Adipogenesis.

The extraction of phenolic compounds from canola meal produces functional health products and renders the canola meal a more digestible animal feed. The extracted phenolics may have novel bioactivity worth investigation. In this study, several solvents were evaluated for their ability to extract phenolic compounds from canola meal: water (WE) and various 80% organic solvent/water mixtures of methanol (ME), acetone (AE), ethanol (EE), butanol (BE), chloroform (CE) and hexane (HE). The in vitro antioxidant and anti-obesity properties of various extracts were investigated. Anti-obesity properties were studied using adipogenic differentiation inhibition of a murine mesenchymal stem cell line (C3H10T1/2) and a pancreatic lipase inhibition assay. AE, ME, and BE showed significant (p < 0.05) adipogenesis and pancreatic lipase inhibitory activities and may have more pharmacological properties. AE down-regulated the gene expression of the major adipogenic transcription factor, peroxisome proliferator-activated receptor gamma (PPARγ), correlating to phenolic content in a dose-dependent manner. The chemical characterization of AE revealed the presence of sinapic acid, ferulic acid, and kaempferol derivatives as main bioactive phenols.

A rapid method for the simultaneous quantification of the major tocopherols, carotenoids, free and esterified sterols in canola (Brassica napus) oil using normal phase liquid chromatography.

A normal phase high performance liquid chromatography (HPLC) method was developed to simultaneously quantify several prominent bioactive compounds in canola oil vis. α-tocopherol, γ-tocopherol, δ-tocopherol, ß-carotene, lutein, ß-sitosterol, campesterol and brassicasterol. The use of sequential diode array detection (DAD) and tandem mass spectrometry (MS/MS) allowed direct injection of oils, diluted in hexane without derivatisation or saponification, greatly reducing sample preparation time, and permitting the quantification of both free sterols and intact sterol esters. Further advantages over existing methods included increased analytical selectivity, and a chromatographic run time substantially less than other reported normal phase methods. The HPLC-DAD-MS/MS method was applied to freshly extracted canola oil samples as well as commercially available canola, palm fruit, sunflower and olive oils.

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.

Identification of QTLs associated with resistance to Phomopsis pod blight (Diaporthe toxica) in Lupinus albus.

Phomopsis blight in Lupinus albus is caused by a fungal pathogen, Diaporthe toxica. It can invade all plant parts, leading to plant material becoming toxic to grazing animals, and potentially resulting in lupinosis. Identifying sources of resistance and breeding for resistance remains the best strategy for controlling Phomopsis and reducing lupinosis risks. However, loci associated with resistance to Phomopsis blight have not yet been identified. In this study, quantitative trait locus (QTL) analysis identified genomic regions associated with resistance to Phomopsis pod blight (PPB) using a linkage map of L. albus constructed previously from an F8 recombinant inbred line population derived from a cross between Kiev-Mutant (susceptible to PPB) and P27174 (resistant to PPB). Phenotyping was undertaken using a detached pod assay. In total, we identified eight QTLs for resistance to PPB on linkage group (LG) 3, LG6, LG10, LG12, LG17 and LG27 from different phenotyping environments. However, at least one QTL, QTL-5 on LG10 was consistently detected in both phenotyping environments and accounted for up to 28.2% of the total phenotypic variance. The results of this study showed that the QTL-2 on LG3 interacts epistatically with QTL-5 and QTL-6, which map on LG10 and LG12, respectively.

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.

Metabolomics differentiation of canola genotypes: toward an understanding of canola allelochemicals.

Allelopathy is one crop attribute that could be incorporated in an integrated weed management system as a supplement to synthetic herbicides. However, the underlying principles of crop allelopathy and secondary metabolite production are still poorly understood including in canola. In this study, an allelopathic bioassay and a metabolomic analysis were conducted to compare three non-allelopathic and three allelopathic canola genotypes. Results from the laboratory bioassay showed that there were significant differences among canola genotypes in their ability to inhibit root and shoot growth of the receiver annual ryegrass; impacts ranged from 14% (cv. Atr-409) to 76% (cv. Pak85388-502) and 0% (cv. Atr-409) to 45% (cv. Pak85388-502) inhibition respectively. The root length of canola also differed significantly between genotypes, there being a non-significant negative interaction (r = -0.71; y = 0.303x + 21.33) between the root length of donor canola and of receiver annual ryegrass. Variation in chemical composition was detected between organs (root extracts, shoot extracts) and root exudates and also between canola genotypes. Root extracts contained more secondary metabolites than shoot extracts while fewer compounds were recorded in the root exudates. Individual compound assessments identified a total of 14 secondary metabolites which were identified from the six tested genotypes. However, only Pak85388-502 and Av-opal exuded sinapyl alcohol, p-hydroxybenzoic acid and 3,5,6,7,8-pentahydroxy flavones in agar growth medium, suggesting that the synergistic effect of these compounds playing a role for canola allelopathy against annual ryegrass in vitro.

Construction of integrated linkage map of a recombinant inbred line population of white lupin (Lupinus albus L.).

We report the development of a Diversity Arrays Technology (DArT) marker panel and its utilisation in the development of an integrated genetic linkage map of white lupin (Lupinus albus L.) using an F8 recombinant inbred line population derived from Kiev Mutant/P27174. One hundred and thirty-six DArT markers were merged into the first genetic linkage map composed of 220 amplified fragment length polymorphisms (AFLPs) and 105 genic markers. The integrated map consists of 38 linkage groups of 441 markers and spans a total length of 2,169 cM, with an average interval size of 4.6 cM. The DArT markers exhibited good genome coverage and were associated with previously identified genic and AFLP markers linked with quantitative trait loci for anthracnose resistance, flowering time and alkaloid content. The improved genetic linkage map of white lupin will aid in the identification of markers for traits of interest and future syntenic studies.

Genetic and physical mapping of flowering time loci in canola (Brassica napus L.).

We identified quantitative trait loci (QTL) underlying variation for flowering time in a doubled haploid (DH) population of vernalisation-responsive canola (Brassica napus L.) cultivars Skipton and Ag-Spectrum and aligned them with physical map positions of predicted flowering genes from the Brassica rapa genome. Significant genetic variation in flowering time and response to vernalisation were observed among the DH lines from Skipton/Ag-Spectrum. A molecular linkage map was generated comprising 674 simple sequence repeat, sequence-related amplified polymorphism, sequence characterised amplified region, Diversity Array Technology, and candidate gene based markers loci. QTL analysis indicated that flowering time is a complex trait and is controlled by at least 20 loci, localised on ten different chromosomes. These loci each accounted for between 2.4 and 28.6% of the total genotypic variation for first flowering and response to vernalisation. However, identification of consistent QTL was found to be dependant upon growing environments. We compared the locations of QTL with the physical positions of predicted flowering time genes located on the sequenced genome of B. rapa. Some QTL associated with flowering time on A02, A03, A07, and C06 may represent homologues of known flowering time genes in Arabidopsis; VERNALISATION INSENSITIVE 3, APETALA1, CAULIFLOWER, FLOWERING LOCUS C, FLOWERING LOCUS T, CURLY LEAF, SHORT VEGETATIVE PHASE, GA3 OXIDASE, and LEAFY. Identification of the chromosomal location and effect of the genes influencing flowering time may hasten the development of canola varieties having an optimal time for flowering in target environments such as for low rainfall areas, via marker-assisted selection.

Molecular mapping of qualitative and quantitative loci for resistance to Leptosphaeria maculans causing blackleg disease in canola (Brassica napus L.).

Blackleg, caused by Leptosphaeria maculans, is one of the most important diseases of oilseed and vegetable crucifiers worldwide. The present study describes (1) the construction of a genetic linkage map, comprising 255 markers, based upon simple sequence repeats (SSR), sequence-related amplified polymorphism, sequence tagged sites, and EST-SSRs and (2) the localization of qualitative (race-specific) and quantitative (race non-specific) trait loci controlling blackleg resistance in a doubled-haploid population derived from the Australian canola (Brassica napus L.) cultivars Skipton and Ag-Spectrum using the whole-genome average interval mapping approach. Marker regression analyses revealed that at least 14 genomic regions with LOD ≥ 2.0 were associated with qualitative and quantitative blackleg resistance, explaining 4.6-88.9 % of genotypic variation. A major qualitative locus, designated RlmSkipton (Rlm4), was mapped on chromosome A7, within 0.8 cM of the SSR marker Xbrms075. Alignment of the molecular markers underlying this QTL region with the genome sequence data of B. rapa L. suggests that RlmSkipton is located approximately 80 kb from the Xbrms075 locus. Molecular marker-RlmSkipton linkage was further validated in an F(2) population from Skipton/Ag-Spectrum. Our results show that SSR markers linked to consistent genomic regions are suitable for enrichment of favourable alleles for blackleg resistance in canola breeding programs.