Genome-wide association study reveals the genetic architecture of flowering time in rapeseed (Brassica napus L.)

Flowering time adaptation is a major breeding goal in the allopolyploid species Brassica napus. To investigate the genetic architecture of flowering time, a genome-wide association study (GWAS) of flowering time was conducted with a diversity panel comprising 523 B. napus cultivars and inbred lines grown in eight different environments. Genotyping was performed with a Brassica 60K Illumina Infinium SNP array. A total of 41 single-nucleotide polymorphisms (SNPs) distributed on 14 chromosomes were found to be associated with flowering time, and 12 SNPs located in the confidence intervals of quantitative trait loci (QTL) identified in previous researches based on linkage analyses. Twenty-five candidate genes were orthologous to Arabidopsis thaliana flowering genes. To further our understanding of the genetic factors influencing flowering time in different environments, GWAS was performed on two derived traits, environment sensitivity and temperature sensitivity. The most significant SNPs were found near Bn-scaff_16362_1-p380982, just 13 kb away from BnaC09g41990D, which is orthologous to A. thaliana CONSTANS (CO), an important gene in the photoperiod flowering pathway. These results provide new insights into the genetic control of flowering time in B. napus and indicate that GWAS is an effective method by which to reveal natural variations of complex traits in B. napus.     

Effect of ellagic acid on growth and physiology of canola (Brassica napus L.) under saline conditions

Salinity stress is limiting growth and productivity of plants in many areas of the world. Plants adopted different strategies to minimize the effect of salt stress. A pot experiment was conducted to investigate the morphological and physiological changes produced in Canola (Brassica napus) by exogenous application of ellagic acid (EA) under saline conditions. EA is an antioxidant, expected to reduce the effect of salinity stress. The seeds of two canola cultivars, Rainbow and Oscar, were soaked for 6?h with different concentrations of EA (0, 55 and 110?µg/ml). The soaked seeds were sown in small pots. Salt stress was imposed on the plants by applying NaCl solutions of different concentrations (0, 60 and 120?mM) and the duration of stress was for four weeks. Salinity stress reduced seed germination and disturbed the morphological and physiological attributes of B. napus. Application of EA as seed soaking reduced the effect of salinity and enhanced the growth of plants. Overall, we could confirm a significant role of EA by inducing salinity tolerance in B. napus.     

Triacontanol modulates photosynthesis and osmoprotectants in canola (Brassica napus L.) under saline stress

Abstract

An experiment was conducted to assess the effect of pre-sowing seed treatment with triacontanol (TRIA) in canola (Brassica napus L.) cultivar (RBN-3060) under saline stress. Canola seeds were soaked in three levels of TRIA (0, 0.5, and 1 mg L^?1) for 12 hours. Three levels of salt stress (0, 100, and 150 mM NaCl) in full strength Hoagland's nutrient solution were applied to 56-days-old plants. Salt stress caused a significant reduction in growth, gas exchange, photochemical quenching (qP), and shoot and root K⁺ contents, while increased leaf glycine betaine, free proline, and shoot Na⁺ contents. Pre-sowing seed treatment with TRIA increased shoot fresh weight, number of seeds per plant, photosynthetic rate, transpiration rate, ratio of chlorophyll a/b, qP, electron transport rate, shoot and root K⁺ contents, and free proline and glycine betaine contents of canola plants at various TRIA levels under nonsaline or saline conditions.     

Phytohormone production and colonization of canola (Brassica napus L.) roots by Pseudomonas fluorescens 6-8 under gnotobiotic conditions

Pseudomonas fluorescens 6-8, a rhizosphere isolate previously shown to enhance root elongation of canola ( Brassica napus L.), was characterized for its ability to produce indole-3-acetic acid and cytokinins in pure culture and in the rhizosphere of canola under gnotobiotic conditions in comparison with the cytokinin-producing strain P. fluorescens G20-18 and its mutant CNT2. Strain 6-8 produced isopentenyl adenosine, zeatin riboside, and dihydroxyzeatin riboside at levels similar to those of G20-18, but only very low concentrations of indole-3-acetic acid. In a gnotobiotic assay canola inoculated with 6-8 and G20-18 had higher concentrations of isopentenyl adenosine and zeatin riboside in the rhizosphere and greater root length than the noninoculated control. The ability of strain 6-8 to colonize canola roots was assessed following transformation with the green fluorescent protein and inoculation onto canola seed in a gnotobiotic assay. Higher populations of strain 6-8 were observed on the proximal region of the root closest to the seed than on the mid and distal portions 9?days after seed inoculation. The ability of P. fluorescens 6-8 to produce cytokinins, colonize the roots of canola seedlings, and enhance root elongation may contribute to its ability to survive in the rhizosphere and may benefit seedling growth.     

Confirmation of QTL controlling seed yield in spring canola (Brassica napus L.) hybrids

Allelic effects observed in QTL discovery experiments must be confirmed to be useful in subsequent breeding efforts. Two QTL affecting seed yield of spring hybrid canola (Brassica napus L.) were previously identified in two populations of inbred backcross lines (IBLs) containing germplasm introgressed from a winter cultivar. The effects of favorable alleles at these QTL were retested by crossing two selected IBLs (M5 and M31) to three spring canola lines having different genetic backgrounds. Doubled haploid (DH) lines derived from each F₁ were genotyped with RFLP markers flanking the QTL and grouped into the four possible QTL genotypes. For the first field experiment, DH lines derived by crossing the M5 line to one spring line were crossed to two female testers and evaluated as individual testcross progenies in one environment. QTL genotypes had large variances and were not significantly different. A second field experiment was conducted using the DH lines from the first experiment and two other sets of DH lines derived from the M31 line crossed to two different spring canola lines. Individual lines within each QTL genotype of each set were bulked and crossed to the same testers used in Experiment 1. Bulked hybrid seeds of each QTL genotype were planted in a split-split plot randomized block design and 12 replicates. QTL genotypes had smaller variances in this experiment, and the effects of one QTL were confirmed in some genetic backgrounds. These results suggest that bulking of QTL genotypes and use of an appropriate experimental design with many replicates are needed to detect small differences between QTL genotypes.     

Chelate-assisted phytoextraction using canola (Brassica napus L.) in outdoors pot and lysimeter experiments

Phytoextraction is an emerging technology for non-destructive remediation of heavy metal-polluted soils. This study was conducted to test chelate-assisted phytoextraction of Cu, Pb and Zn using EDTA and canola (Brassica napus L. cv. Petranova) on a moderately polluted industrial soil (loamy sand) in the sub-continental climate of Eastern Austria. The effects of the rate (up to 2.1 g kg^–1 soil) and mode (single versus split) of EDTA application on the biomass, water contents and metal concentrations in shoots and roots were investigated along with changes of metal lability in soil and leaching from the root zone in parallel outdoors pot and lysimeter experiments. Labile (1 M NH₄NO₃-extractable) metal concentrations in soil increased considerably upon application of EDTA, indicating enhanced phytoavailability. However, this was also associated with enormously increased metal concentrations in the leachates collected below the root zone. Enhanced metal labilities and leachate concentrations persisted for more than 1 year after harvest. Metal lability was more enhanced by EDTA in rhizosphere relative to bulk soil, indicating interactions of EDTA with root activities. Shoot biomass and water contents of canola were virtually unaffected by EDTA, revealing that canola can tolerate excessive metal concentrations in soil pore water. Metal concentrations in shoots were increased considerably, but were insufficient to obtain reasonable extraction rates. Split applications were generally more effective than the same amounts of EDTA added at once. Metal concentrations in roots decreased after each application of EDTA, possibly indicating metal removal from roots by free protonated EDTA, but increased again within several days. As the application of chelate-assisted phytoextraction is limited by the risk of groundwater pollution, further work should focus on natural, continuous phytoextraction technologies.     

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.     

Diversity of root-associated bacteria associated with field-grown canola (Brassica napus L.) and wheat (Triticum aestivum L.)

Little is known about the composition and diversity of the bacterial community associated with plant roots. The purpose of this study was to investigate the diversity of bacteria associated with the roots of canola plants grown at three field locations in Saskatchewan, Canada. Over 300 rhizoplane and 220 endophytic bacteria were randomly selected from agar-solidified trypticase soy broth, and identified using fatty acid methyl ester (FAME) profiles. Based on FAME profiles, 18 bacterial genera were identified with a similarity index >0.3, but 73% of the identified isolates belonged to four genera: Bacillus (29%), Flavobacterium (12%), Micrococcus (20%) and Rathayibacter (12%). The endophytic community had a lower Shannon-Weaver diversity index (1.35) compared to the rhizoplane (2.15), and a higher proportion of Bacillus, Flavobacterium, Micrococcus and Rathayibacter genera compared to rhizoplane populations. Genera identified in the endophytic isolates were also found in the rhizoplane isolates. Furthermore, principal component analysis indicated three clusters of bacteria regardless of their site of origin, i.e., rhizoplane or endophytic. In addition, the rhizoplane communities of canola and wheat grown at the same site differed significantly. These results indicate that diverse groups of bacteria are associated with field-grown plants and that endophytes are a subset of the rhizoplane community.     

Extent and structure of linkage disequilibrium in canola quality winter rapeseed (Brassica napus L.)

Linkage disequilibrium was investigated in canola quality winter rapeseed to analyze (1) the prospects for whole-genome association analyses and (2) the impact of the recent breeding history of rapeseed on linkage disequilibrium. A total of 845 mapped AFLP markers with allele frequencies ≥0.1 were used for the analysis of linkage disequilibrium in a population of 85 canola quality winter rapeseed genotypes. A low overall level of linkage disequilibrium was found with a mean r ² of only 0.027 over all 356,590 possible marker pairs. At a significance threshold of P = 2.8 × 10⁻⁷, which was derived by a Bonferroni correction from a global α-level of 0.1, only 0.78% of the marker pairs were in significant linkage disequilibrium. Among physically linked marker pairs, the level of linkage disequilibrium was about five times higher with more than 10% of marker pairs in significant linkage disequilibrium. Linkage disequilibrium decayed rapidly with distance between linked markers with high levels of linkage disequilibrium extending only for about 2 cM. Owing to the rapid decay of linkage disequilibrium with distance association analyses in canola quality rapeseed will have a significantly higher resolution than QTL analyses in segregating populations by interval mapping, but much larger number of markers will be necessary to cover the whole genome. A major impact of the recent breeding history of rapeseed on linkage disequilibrium could not be observed.     

Conservation of S-locus for self-incompatibility in Brassica napus (L.) and Brassica oleracea (L.)

 Self-incompatibility (SI) in Brassica is a sporophytic system, genetically determined by alleles at the S-locus, which prevents self-fertilization and encourages outbreeding. This system occurs naturally in diploid Brassica species but is introduced into amphidiploid Brassica species by interspecific breeding, so that in both cases there is a potential for yield increase due to heterosis and the combination of desirable characteristics from both parental lines. Using a polymerase chain reaction (PCR) based analysis specific for the alleles of the SLG (S-locus glycoprotein gene) located on the S-locus, we genetically mapped the S-locus of B. oleracea for SI using a F₂ population from a cross between a rapid-cycling B. oleracea line (CrGC-85) and a cabbage line (86-16-5). The linkage map contained both RFLP (restriction fragment length polymorphism) and RAPD (random amplified polymorphic DNA) markers. Similarly, the S-loci were mapped in B. napus using two different crosses (91-SN-5263×87-DHS-002; 90-DHW-1855-4×87-DHS-002) where the common male parent was self-compatible, while the S-alleles introgressed in the two different SI female parents had not been characterized. The linkage group with the S-locus in B. oleracea showed remarkable homology to the corresponding linkage group in B. napus except that in the latter there was an additional locus present, which might have been introgressed from B. rapa. The S-allele in the rapid-cycling Brassica was identified as the S₂₉ allele, the S-allele of the cabbage was the S ₅ allele. These same alleles were present in our two B. napus SI lines, but there was evidence that it might not be the active or major SI allele that caused self-incompatibility in these two B. napus crosses. Received: 7 June 1996/Accepted: 6 September 1996     

Water use dynamics of dryland canola (Brassica napus L.) grown on contrasting soils under elevated CO2

Plant and Soil - Increasing atmospheric carbon dioxide concentration ([CO2]) stimulates the leaf-level (intrinsic) water use efficiency (iWUE), which may mitigate the adverse effects of drought by...     

Identification and characterization of CBL and CIPK gene families in canola (Brassica napus L.)

Background

Canola (Brassica napus L.) is one of the most important oil-producing crops in China and worldwide. The yield and quality of canola is frequently threatened by environmental stresses including drought, cold and high salinity. Calcium is a ubiquitous intracellular secondary messenger in plants. Calcineurin B-like proteins (CBLs) are Ca²⁺ sensors and regulate a group of Ser/Thr protein kinases called CBL-interacting protein kinases (CIPKs). Although the CBL-CIPK network has been demonstrated to play crucial roles in plant development and responses to various environmental stresses in Arabidopsis, little is known about their function in canola.

Results

In the present study, we identified seven CBL and 23 CIPK genes from canola by database mining and cloning of cDNA sequences of six CBLs and 17 CIPKs. Phylogenetic analysis of CBL and CIPK gene families across a variety of species suggested genome duplication and diversification. The subcellular localization of three BnaCBLs and two BnaCIPKs were determined using green fluorescence protein (GFP) as the reporter. We also demonstrated interactions between six BnaCBLs and 17 BnaCIPKs using yeast two-hybrid assay, and a subset of interactions were further confirmed by bimolecular fluorescence complementation (BiFC). Furthermore, the expression levels of six selected BnaCBL and 12 BnaCIPK genes in response to salt, drought, cold, heat, ABA, methyl viologen (MV) and low potassium were examined by quantitative RT-PCR and these CBL or CIPK genes were found to respond to multiple stimuli, suggesting that the canola CBL-CIPK network may be a point of convergence for several different signaling pathways. We also performed a comparison of interaction patterns and expression profiles of CBL and CIPK in Arabidospsis, canola and rice, to examine the differences between orthologs, highlighting the importance of studying CBL-CIPK in canola as a prerequisite for improvement of this crop.

Conclusions

Our findings indicate that CBL and CIPK family members may form a dynamic complex to respond to different abiotic or hormone signaling. Our comparative analyses of the CBL-CIPK network between canola, Arabidopsis and rice highlight functional differences and the necessity to study CBL-CIPK gene functions in canola. Our data constitute a valuable resource for CBL and CPK genomics.     

Genome-wide association study for electrolyte leakage in rapeseed/canola (<Emphasis Type="Italic">Brassica napus</Emphasis> L.)

Freezing temperature/frosts can cause significant damage to plants by rupturing plant cells. Rapeseed/canola (Brassica napus L.) is susceptible to freezing temperature at early seedling stage. The degree of cell rupture or seedling damage can be evaluated through the measurement of electrolyte leakage. Here, we measured the electrolyte leakage of a diversity panel of B. napus germplasm accessions under simulated freezing conditions. Preliminary data for electrolyte leakage measurement indicated that cold acclimation of two-week-old seedlings for 7 days at 4 °C followed by freezing treatment at ??12 °C for 2 h provided a reasonable diversity in response. With this protocol for electrolyte leakage, a genome-wide association study was conducted on 157 winter, semi-winter, and spring types of B. napus accessions that originated from 17 countries. A total of 37,454 single-nucleotide polymorphism (SNP) markers based upon genotyping-by-sequencing were used for the analysis. Ten QTL were identified as associated with electrolyte leakage of canola seedlings, which together explained 43% phenotypic variation. Five of the QTL were located on A-genome. We identified at least 33 orthologs of the functional candidate genes. Although no well-characterized cold regulatory genes were identified, there were some indications that genes involved in membrane structure, developmental processes, and extracellular transport may be involved in altering the electrolyte leakage following the short-term hard freeze and rapid defrosting suffered by the plants in our protocol.     

Hybridisation between Brassica napus L. and Raphanus raphanistrum L. under agronomic field conditions

The frequency of hybridisation between Brassica napus L. and Raphanus raphanistrum L. under agronomic conditions was assessed in field experiments, where R. raphanistrum were randomly planted at two different densities into large plots of B. napus. An acetolacate synthase (ALS)-inhibiting herbicide-resistant trait was used to detect potential hybrid individuals. No hybrids were detected amongst 25,000 seedlings grown from seed collected from R. raphanistrum plants. Two hybrids were obtained from more than 52-million B. napus seedlings. Both hybrids were characterised as amphidiploids (AACCRrRr, 2n = 56) and were fertile. The frequency of hybridisation into B. napus in this experiment using male-fertile B. napus was 4 × 10^–8. Received: 31 August 2000 / Accepted: 23 January 2001     

Relationship between genetic distance and heterosis for yield and morphological traits in winter canola (Brassica napus L.)

Genetic distance among canola cultivars was estimated through multivariate analysis. Thirty cultivars from various sources were analyzed and clustered based upon five morphological characteristics and yield components-crown diameter, number of branches plant^-1, number of pods plant^-1, number of seeds pod^-1 and yield plant^-1 -and placed in three distinct clusters. Two cultivars from each cluster were selected as parents and 15 partial-diallel inter- and intra-cluster crosses were made between the six selected parents and evaluated at two locations in Michigan in 1990/1991. The association between genetic distance and mid-parent heterosis was investigated. The correlation between genetic distance and heterosis was positive and highly significant for seed yield, number of pods plant^-1, and number of seeds pod^-1. Clustering, based on yield and yield-component traits, demonstrated that inter-cluster heterosis was greater than intra-cluster heterosis in the majority of cases.     

Study on salt tolerance with YHem1 transgenic canola (Brassica napus)

5‐Aminolevulinic acid (5‐ALA) has been suggested for improving plant salt tolerance via exogenous application. In this study, we used a transgenic canola (Brassica napus), which contained a constituted gene YHem1 and biosynthesized more 5‐ALA, to study salt stress responses. In a long‐term pot experiment, the transgenic plants produced higher yield under 200 mmol L^?1 NaCl treatment than the wild type (WT). In a short‐term experiment, the YHem1 transformation accelerated endogenous 5‐ALA metabolism, leading to more chlorophyll accumulation, higher diurnal photosynthetic rates and upregulated expression of the gene encoding Rubisco small subunit. Furthermore, the activities of antioxidant enzymes, including superoxide dismutase, guaiacol peroxidase, catalase and ascorbate peroxidase, were significantly higher in the transgenic plants than the WT, while the levels of O₂·^? and malondialdehyde were lower than the latter. Additionally, the Na⁺ content was higher in the transgenic leaves than that in the WT under salinity, but K⁺ and Cl^? were significantly lower. The levels of N, P, Cu, and S in the transgenic plants were also significantly lower than those in the WT, but the Fe content was significantly improved. As the leaf Fe content was decreased by salinity, it was suggested that the stronger salt tolerance of the transgenic plants was related to the higher Fe acquisition. Lastly, YHem1 transformation improved the leaf proline content, but salinity decreased rather than increased it. The content of free amino acids and soluble sugars was similarly decreased as salinity increased, but it was higher in the transgenic plants than that in the WT.     

Gene transfer between canola (Brassica napus L. and B. campestris L.) and related weed species

Brassica species are particularly receptive to gene transformation techniques. There now exists canola genotypes with transgenic herbicide resistance for glyphosate, imidazolinone, sulfonylurea and glufosinate herbicides. The main concern of introducing such herbicide resistance into commercial agriculture is the introgression of the engineered gene to related weed species. The potential of gene transfer between canola (Brassica napus and B. campestris) and related weed species was determined by hand pollination under controlled greenhouse conditions. Canola was used as both male and female parent in crosses to the related weed species collected in the Inland Northwest region of the United States. Weed species used included: field mustard (B. rapa), wild mustard (S. arvensis) and black mustard (B. nigra). Biological and cytological aspects necessary for successful hybrid seed production were investigated including: pollen germination on the stigma; pollen tube growth down the style; attraction of pollen tubes to the ovule; ovule fertilisation; embryo and endosperm developmental stages. Pollen germination was observed in all 25 hybrid combinations. Pollen tubes were found in the ovary of over 80% of combinations. About 30% of the hybrid combinations developed to the heart stage of embryo development or further. In an additional study involving transgenic glufosinate herbicide resistant B. napus and field mustard it was found that hybrids occurred with relatively high frequency, hybrids exhibited glufosinate herbicide resistance and a small proportion of hybrids produced self fertile seeds. These fertile plants were found to backcross to either canola or weed parent.     

Genome-wide association study for candidate genes controlling seed yield and its components in rapeseed (Brassica napus subsp. napus)

Genetic improvement of seed yield per plant (SY) is one of the major objectives in Brassica napus breeding programme. SY, being a complex quantitative trait is directly and indirectly influenced by yield-component traits such as siliqua length (SL), number of seeds per siliqua (NSS), and thousand seed weight (TSW). Therefore, concurrent improvement in SL, NSS and TSW can lead to higher SY in B. napus. This study was conducted to identify significant SNPs and putative candidate genes governing SY and its component traits (SL, NSS, TSW). All these traits were evaluated in a diverse set of 200 genotypes representing diversity from wide geographical locations. Of these, a set of 125 genotypes were chosen based on pedigree diversity and multi-location trait variation for genotyping by sequencing (GBS). Best linear unbiased predictors (BLUPs) of all the traits were used for genome-wide association study (GWAS) with 85,126 SNPs obtained from GBS. A total of 16, 18, 27 and 18 SNPs were found to be significantly associated for SL, NSS, TSW and SY respectively. Based on linkage disequilibrium decay analysis, 150 kb genomic region flanking the SNP was used for the identification of underlying candidate genes for each test trait. Important candidate genes involved in phytohormone signaling (WAT1, OSR1, ARR8, CKX1, REM7, REM9, BG1) and seed storage proteins (Cruciferin) were found to have significant influence on seed weight and yield. Genes involved in sexual reproduction and fertilization (PERK7, PERK13, PRK3, GATA15, NFD6) were found to determine the number of seeds per siliqua. Several genes found in this study namely ATS3A, CKX1, SPL2, SPL6, SPL9, WAT1 showed pleiotropic effect with yield component traits. Significant SNPs and putative candidate genes identified for SL, NSS, TSW and SY could be used in marker-assisted breeding for improvement of crop yield in B. napus. Genotypes identified with high SL, NSS, TSW and SY could serve as donors in crop improvement programs in B. napus.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12298-021-01060-9.