Rozprawy doktorskie na temat „Brassica – Breeding”

[Truncated abstract] In order to expand the gene pool of canola-quality rapeseed (Brassica napus) reciprocal interspecific crosses were made between B. napus cv. Mystic and near canola-quality B. juncea breeding line JN29. F1 progeny from these crosses were used to make backcrosses to both parents in all possible combinations and directions, and were selfed to form F2-derived lines. The highest frequencies of viable F2 and BC1 progeny were obtained when B. napus was the maternal parent of the interspecific hybrid. BC1 and F2 progeny (and subsequent generations) were grown under field conditions to identify agronomic improvements over the parents. Transgressive segregation was observed in F2 and BC1 and in subsequent generations for agronomic traits (seed yield under high or low rainfall conditions, plant biomass, harvest index, height, branching and days to anthesis) and seed quality traits (oil, protein, glucosinolates, oleic acid). The majority of progeny conformed to B. napus morphology, and a minority segregated to B. juncea morphology in subsequent generations. Some of the B. juncea morphotypes had lower glucosinolates and higher oleic acid than the parent JN29, with no detectable erucic acid, and thereby conformed to canola quality. Methods were developed for tracing B-genome in interspecific progeny. A repetitive DNA sequence pBNBH35 from B. nigra (genome BB, 2n = 16) was used to identify B-genome chromosomes and introgressions in interspecific progeny. Specific primers were designed for pBNBH35 in order to amplify the repetitive sequence by PCR. A cloned sub-fragment of 329 bp was confirmed by sequencing as part of pBNBH35. PCR and hybridisation techniques were used on an array of Brassica species to confirm that the pBNBH35 subfragment was Brassica B-genome specific. Fluorescence in situ hybridisation (FISH) in B nigra, B. juncea (AABB, 2n=36) and B. napus (AACC, 2n=38) showed that the pBNBH35 sub-fragment was present on all eight Brassica Bgenome chromosomes and absent from A- and C-genome chromosomes. The pBNBH35 repeat was localised to the centromeric region of each B-genome chromosome. FISH clearly distinguished the B-genome chromosomes from the A-genome chromosomes in the amphidiploid species B. juncea. This is the first known report of a B-genome repetitive marker that is present on all Brassica Bgenome chromosomes. ... The results suggest that novel B. napus genotypes have been generated containing introgressions of B-genome chromatin from B. juncea chromosomes. B. juncea morphology occurred in interspecific progeny with a chromosome complement similar to B. napus (2n = 38) and without the entire Bgenome present. It also is highly likely that recombination has occurred between the A-genome of the two Brassica species. This research has demonstrated that the secondary gene pool of B. napus may be accessed by selfing interspecific hybrids, and without sacrificing canola quality, if the B. juncea parent is near canola-quality. Interspecific progeny may be screened to enhance the proportion with B-genome positive signals. Some progeny with B. junceatype morphology had improved seed quality over the JN29 parent.

Cauliflower curd meristem activity (organogenic, plastochronic, phyllotactic) was analysed biometrically and confirmed that the curd is the product of a constant process of meristem production and branch ramification with little if any dominance between branch apices. A growth model based on curd branching pattern was developed and its mathematical expression enabled the estimation of the number of meristems carried by a curd at maturity to be over ten million which was previously widely underestimated. Analysis of the response to the in vitro culture of this meristematic tissues revealed that meristems are not predetermined to produce flower and that their shoot regeneration capacity is under several levels of control, the most important being explant physical property (size) and the culture system (nutrient supply). Optimisation of these parameters enabled the development of a low cost, semi-automated protocol for mass production of cauliflower propagules at an unprecedented scale with over 10000 propagules produced per curd. Micropropagules a few millimetres in length were produced, encapsulated in calcium alginate hydrogel, stored at 4°C for several months and used as an 'artificial seed' system of cauliflower propagation. The response to the procedure of micropropagule production is genotype-dependent with summer heading varieties being less reactive than winter heading varieties, this phenomenon was also associated with plasmalemma instability at the cellular (protoplast) level. Furthermore, this material was successfully cryopreserved in liquid nitrogen using a dehydration / vitrification method. The micropropagation protocol is of great interest when used as a regeneration system for experiments involving genetic manipulation such as genetic transformation. A preliminary study of genetic transformation by microprojectile bombardment, using the gus reporter gene, allowed transient expression in curd meristematic tissue. The fundamental and industrial implications for cauliflower breeding of the different protocols developed in this thesis are discussed.

Polyploidy is widely acknowledged as a widespread mechanism in the evolution and speciation of the majority of flowering plants. Allopolyploid forms through interspecific hybridization and whole genome duplication. While allopolyploids may display increased vigor relative to their progenitors, they can also face challenges to fertility following hybridization. Genetic changes in allopolyploids result from recombination between the hybridized subgenomes, which can influence phenotype and ultimately determine fitness of future generations. To study dynamic changes that follow allopolyploid formation, Brassica napus lineages were derived by hybridizing Brassica oleracea and Brassica rapa. Two lineages of B. napus were analyzed for genetic and phenotypic changes in the S2, S7, and S12 generations. Although these lineages were genetically identical at the time of hybridization, divergence was apparent by the S2 generation. There was a significant increase in sequence loss across generations within both lineages. Four of six generations from both lineages displayed no significant differences to each other in sequence loss relative to the parental generation. In both lineages, there was a bias towards losing sequences from the B. olereacea subgenome. Some individual plants showed novel phenotypes; however, there was no correlation between the examined genetic changes and selected phenotypes.

Variables for the accelerated ageing (AA) test, methods for reducing fungal contamination during the AA test, using the conductivity test as a vigour test, the effect of seed size on seed vigour and the relationship between laboratory test results and field perfonnance in selected Brassica spp were investigated. In the first experiment, three seed lots of turnip rape hybrid (B. rapa x campestris), turnip (B. campestris) and forage rape (B. napus); and seven seed lots of Asian rape (B. napus), six seed lots of Asian kale (B. oleraceae var. alboglabra L.) and five seed lots of choisum (B. rapa var. pekinensis) with germinations above 90% were aged at two different temperatures (41 and 42°C ± 0.3°C) and three ageing times (24, 48 and 72 ± 15 minutes). The second experiment was divided into three sections. In the first, the same seed lots and species were aged at one temperature (41°C) and time (72 h), but either 40 ml of saturated salts; KCl (83%RH), NaCl (76%RH), NaBr (55%RH); or distilled water (96%RH) were used as the ageing solutions. In the second, one turnip rape hyprid seed lot was aged at three temperatures (41, 42 and 45°C) and two times (72 and 96h), again using the three saturated salts and distilled water as ageing solutions. In the third, three turnip rape hybrid seed lots and three Asian kale seed lots were surface sterilised (1 % sodium hypochlorite) prior to ageing at one temperature (41°C) and time (72 h). In the third experiment, the same species and seed lots used in experiment one at their original seed moisture content (SMC) were tested for conductivity after soaking in deionised water for 4, 8, 12, 16, 20 and 24 h. They were then re-tested after the SMC had been adjusted to 8.5%. In the fourth experiment, three seed lots of forage rape and three seed lots of Asian kale were graded into three seed size categories; large (retained on a 2.0 mm screen), medium (retained on a 1.7 mm screen) and small (passed through a 1.7 mm screen). Graded seeds were then tested for standard germination, AA (41°C/48 h) and conductivity (measured at 16 and 24 h). In the final experiment, the relationships between laboratory tests for the six species (each consisting of three seed lots), field emergence from three sowings, and cold room emergence were evaluated. Both time and temperature influenced post-AA germination. Increasing the ageing period from 48 to 72 hours at 41°C, and 24 to 48 hours at 42°C resulted in decreased mean germination percentage for all species but not always clear separation of seed lots. While there were sometimes few differences between ageing at 41°C and 42°C, the former is preferred because it is already the temperature used for other species. For Asian rape, choisum and turnip, the previously recommended testing conditions of 41°C/72 h provided good seed lot separation, but for Asian kale and turnip rape hybrid, AA testing at 41°C/48 h provided better results. Seed moisture content after ageing ranged from 29-37% depending on species. Fungal growth on seeds during the ageing period appeared to reduce post-ageing germination in some seed lots . Substituting saturated salts for distilled water did not stress seed lots in the AA test, due to the lowered RH%, the exception being seed lots 1210 and 1296. For forage and Asian species, seed lot germination mostly remained above 90% when aged for 72 h at lowered RH%. Increasing the ageing duration from 72 to 96 hours resulted in some decreases in post-AA germination but no clear separation of seed lots. Surface sterilising the seeds prior to the AA test resulted in a lower incidence of contaminant fungi which was associated with a lower percentage of abnormal seedlings. The conductivity test was mostly able to identify vigour differences among forage and Asian vegetable brassica seed lots. Differences in conductivity readings were observed among seed lots in all species. Increasing the period of imbibition resulted in increased conductivity from most seed lots but radicle emergence occurred after 16-20 h of imbibition. Variation was observed in the time to reach 95% maximum of the imbibition curve for most species. Conductivity readings at 16 h would avoid possible influences of radicle emergence on results. Adjusting the SMC to 8.5% resulted in reduced variation in conductivity among replicates of seed lots, due to a reduction in imbibition damage. Seed size had a significant effect on both post-AA germination and conductivity results. In forage rape, large size seeds had higher post-AA germination cf. medium cf. small size seeds. In Asian kale, large size seeds had higher post-AA germination compared with small size seeds. For both forage rape and Asian kale, large size seeds had lower conductivity readings cf. small size seeds. The correlation analyses demonstrated significant relationships between AA testing and field emergence parameters (percentage emergence, emergence index and emergence rate). Significant relationships were also observed between conductivity testing and these field emergence parameters. Based on the correlation analysis, AA testing at 41°C/48 hand/or 42°C/48 h could be recommended to be used as an AA test for turnip and Asian rape; and 41°C/48 hand/or 41°C/72 h for Asian kale and choisum. Based on the correlation analysis, conductivity testing at 16 h can be used to predict the field emergence potential of forage and Asian vegetable seed lots. Vigour tests were consistently able to provide better indicators of field perfonnance than the standard germination test, although these relationships did vary with the different field sowings.

Sclerotinia stem rot caused by Sclerotinia sclerotiorum (Lib.) de Bary is an economic disease affecting canola (Brassica napus L). Since expression of sclerotinia stem rot symptoms shows much variability and the trait is quantitative in nature, reliable phenotypic evaluation methods for characterization are needed. The three major objectives of this dissertation were to: i) evaluate eight different inoculation methods to discriminate between S. sclerotiorum-resistant and susceptible B. napus germplasm; ii) develop breeding lines with resistance to multiple diseases, and; iii) to identify QTL associated with resistance to sclerotinia stem rot using association mapping (AM). The eight methods evaluated were the detached leaves, detached stems, petiole inoculation (PIT), straw-inoculation, stem-piercing with toothpick, mycelial spray (MSI), petal inoculation and oxalic acid assay. MSI and PIT can better discriminate between the isolates and germplasm. Breeding lines resistance to S. sclerotiorum, Leptosphaeria maculans, and Rhizoctonia solani were developed from a cross between two moderately sclerotinia stem rot resistant plant introductions (PI). F2 seedlings were screened for sclerotinia stem rot using PIT. Surviving plants were self pollinated and their progeny screened again. This process was repeated until the F6 generation. In addition, F5 seedlings were evaluated for their reaction to R. solani and F5 and F6 seedlings for their reaction to L. maculans. Eight lines were identified as moderately resistance to these three pathogens. The genomes of a group of 278 B. napus plant introductions were screened using Diversity Array Technology to detect QTL associated with resistance to sclerotinia stem rot. The population was classified into nine sub-populations and 32 significant markers each explaining between 1.5 and 4.6% of the variation were identified. Blastn search indicates that similar nucleotide sequences are distributed throughout the genomes of B. oleracea, B. rapa, and A. thaliana. Results of these studies suggest the PIT and MSI are reliable screening tools to evaluate materials for resistance to sclerotinia stem rot; materials identified as resistant to S. sclerotiorum were also moderately resistant against R. solani and L. maculans and could be valuable sources for canola improvement programs; and AM allowed us to identify QTL associated with resistance to sclerotinia stem rot.

Les insectes phytophages causent des dommages importants aux cultures. Ces insectes sont principalement contrôlés par l’utilisation d’insecticides mais les effets néfastes des composés utilisés sur la santé humaine et l’environnement ainsi que l’apparition de populations résistantes à ces composés imposent de trouver des stratégies de lutte alternatives. Sélectionner des plantes pour leur résistance aux insectes pourrait être une solution intéressante. Un frein majeur au développement de cette stratégie est le fait qu’un grand nombre de génotypes doit être criblé pour identifier des sources de résistance et que le phénotypage est souvent complexe et limite les possibilités de criblage à grande échelle. Pour lever ces verrous, il est important de comprendre les mécanismes à l’origine des résistances. Une fois ces mécanismes précisés dans le cadre de l’interaction entre une culture et un ravageur, l’identification de traits clés de cette interaction permet d’envisager l’utilisation de biomarqueurs de la résistance qui peuvent fortement simplifier le phénotypage. Ce type d’approche a été conduit au cours de cette thèse dans le cadre de l’interaction entre le colza (Brassica napus) et l’un de ses principaux ravageurs, le méligèthe (Brassicogethes aeneus). Des travaux antérieurs avaient mis en évidence l’existence de certains composés biochimiques présents dans le périanthe des boutons floraux et corrélés au niveau d’attaque de différents génotypes de colza. Ces résultats avaient été obtenus au laboratoire et il était nécessaire de les confirmer dans des conditions de culture réalistes. Le premier objectif de cette thèse était donc de développer une méthode permettant de cribler au champ différents génotypes de colza pour leur résistance au méligèthe afin de valider ou non le potentiel des composés biochimiques identifiés précédemment comme biomarqueurs mais aussi d’en identifier de nouveaux. Le second objectif de ce travail était de mieux comprendre l’écologie alimentaire du méligèthe afin d’identifier de nouvelles cibles potentielles pour la sélection de plantes résistantes. Nous avons mené plusieurs expérimentations sur le terrain qui nous ont permis de développer une méthode de crible au champ des génotypes de colza pour leur résistance au méligèthe. Cette méthode a mis en évidence l’existence de différences de résistance fortes et robustes entre génotypes parmi une gamme de 19 génotypes testés. Les biomarqueurs potentiels précédemment identifiés n’ont pas été validés au champ mais deux autres composés semblent influencer la résistance au méligèthe et pourraient être utilisés comme futurs biomarqueurs. Ces expériences au champ ont aussi permis de montrer que la biochimie des périanthes était très dépendante des conditions environnementales, ce qui pourra compliquer le travail de sélection. Par ailleurs, des expériences de développement réalisées en conditions contrôlées ont montré que le méligèthe utilisait le nectar présent dans les fleurs de colza mais que celui-ci ne semblait pas affecter son développement. Le pollen semble, jouer un rôle important sur son développement mais les larves parviennent à se développer sans son apport. Les expériences sur le comportement alimentaire du méligèthe adulte ont montré que cet insecte avait un patron d’attaque spécifique sur les inflorescences de colza en s’alimentant avant tout sur les boutons de petite taille (donc moins riches en pollen par rapport aux gros). Il semble que la disponibilité mais surtout l’accessibilité du pollen médiée par le périanthe joue un rôle essentiel dans le comportement de cet insecte. Ce travail a permis de mieux comprendre le comportement alimentaire du méligèthe et d’identifier des traits importants pour son développement. Ce travail montre que des résistances modérées existent au sein du colza et qu’elles pourraient être utilisées pour la sélection variétale. Il pointe aussi les limites des approches basées sur des biomarqueurs biochimiques
Herbivorous insects cause important yield losses to crops. These insects are mainly managed through insecticides but negative effects of used compounds on the human health and the environment as well as the development of resistant pest populations impose finding alternative strategies to manage them. Breeding plants for enhanced resistance to insects is an attractive strategy. This kind of strategy was already implemented to manage insects but examples of its utilisation remain rare. The main limitation of this strategy is that a large number of genotypes needs to be screened through generally complex phenotyping methods to identify resistances. To circumvent these issues, an approach based on the understanding of plant defence mechanisms and the utilisation of biochemical biomarkers linked to resistance could be interesting. During this PhD, this kind of approach was developed on the oilseed rape (Brassica napus) and one of its main pests, the pollen beetle (Brassicogethes aeneus). Previous studies identified chemical compounds present in the perianth of flower buds and correlated to oilseed rape resistance to pollen beetle. However these studies were carried out in the laboratory and need to be validated in field conditions. Moreover, information on pollen beetle feeding ecology are still lacking while they could help identifying new targets for breeding. The first objective of this PhD is to develop a method allowing to screen resistance to the pollen beetle in the field. This method will allow to confirm the potential biomarkers previously identified and to look for additional biomarkers. The second objective of the present work was to better understand key steps in the interaction between the pest and its host plant to identify potential new target traits for resistance. For this purpose, the importance of food sources present in flowers such as pollen and nectar on pollen beetle development and the factors impacting the feeding pattern of adults on the inflorescence were investigated. We conducted field experiments in two different sites and for two consecutive years and propose a method allowing to screen oilseed rape resistance to pollen beetle in the field. Using this method, we were able to identify genotypes with moderate levels of resistance among a set of 19 genotypes. Previously identified biochemical biomarkers were not correlated with plant resistance in the field but new markers were identified (i.e. quinic acid and arginine). Our field experiments also showed that plant composition is highly variable according to the environment and this variability could affect usefulness of these markers during plant breeding programs. Experiments under controlled conditions also showed that pollen beetle used nectar for feeding but that it did not seem to affect its development. Pollen, on the other hand, seemed to have a more important impact but was not indispensable to pollen beetle development. The study of the pollen beetle feeding behaviour showed that this insect has a surprising feeding pattern on oilseed rape inflorescences and that small buds are more used for feeding than large buds that contain more pollen. It seems that accessibility and to a lesser extend availability of feeding resource explain this pattern and that the perianth has a major role on this preference. These experiments allowed to better understand the pollen beetle feeding ecology and to identify plant traits important for its development. Our work showed that moderate levels of resistance are present in oilseed rape and could be used in breeding programs. Limitations of approaches based on biochemical biomarkers are discussed

While breeding for increased oil yield has generated new lines of spring canola (Brassica napus L.) for biodiesel production, emissions of N2O from fertilized canola fields threaten to undermine the climate change mitigation benefits of canola as a biodiesel alternative to conventional diesel. This study determined the response of N2O emissions to canola line and N treatment in a maritime setting (Truro, Nova Scotia). Tissue N uptake was measured to determine whether differences in N uptake between the lines could explain any observed effect of canola line. Nitrate Exposure (the summation of daily soil NO3- concentrations over a growing season, serving as an integrated measure of the exposure of soil biomass to nitrate over the growing season) was determined to investigate its potential as a predictor of N2O emissions. Four spring canola lines (‘Topaz’, ‘Sentry’, ‘Polo’, and 04C204, in order of increasing seed oil content) were paired with five N treatments (40, 60, 80, 100, and 120 kg N ha-1) in an incomplete two-factor factorial design over two growing seasons (2008 and 2009). N2O emissions were determined using a non-steady state vented chamber method. N2O emissions peaks closely followed increases in soil water content in both years, indicating that limited aerobicity was the trigger for N2O emissions events, and suggesting that denitrification was the predominant microbial process responsible for N2O emissions. The magnitude of average N2O emissions both years was considerably low when compared to other studies (0.55 and 0.56 kg N2O ha-1 in 2008 and 2009 respectively). Increasing N treatment resulted in significantly increased N2O emissions in 2008. Though the same trend was observed in 2009, it was not found to be significant. Differences in weed cover, soil C, soil N supplying capacity, and elevation between the sites may have contributed to the inability to detect an N2O emissions response to N treatment in 2009. Canola line had no effect on N2O emissions in either study year, though heavy competition by weeds significantly affected canola plant health and survival in 2009. Tissue N uptake increased with increasing N treatment, but did not change with choice of line, which is consistent with the observation of no N2O emissions response to line. Nitrate Exposure was found to be strongly correlated with N2O emissions in a linear relationship, supporting the conclusion that Nitrate Exposure can be a promising indicator of N2O emissions when they are limited by soil N. Finally, FluxPerOil, the ratio of N2O emissions per unit oil yield (kg N2O kg-1 oil) was found to decrease with decreased N treatment in 2008, though only very little, indicating a marginal abatement of N2O emissions at a significant cost of oil. FluxPerOil was unreliable in 2009 due to weeds compromising the line effect and therefore oil yield.

The ogu-INRA cytoplasmic male sterility (CMS) system is the global leader for the development of high quality hybrid canola and high erucic acid rapeseed (HEAR) cultivars of oilseed rape (Brassica napus L.). The largest challenge for plant breeders using this system is the development of high quality restorer lines (R-lines) due to tight linkage of the radish (Raphanus sativus L.) derived restorer gene PPR-B and elevated seed glucosinolate concentration. The purpose of this study was to identify improved quality restorer lines for hybrid cultivar development through both field studies and molecular marker development. In the first study 67 R-lines of different genetic backgrounds were screened over the course of two growing seasons and lines with significantly reduced glucosinolate concentration were identified. In the second study a new sequence characterized amplified region (SCAR) marker was successfully developed for the rapid screening of selections for the ogu-INRA CMS restorer gene PPR-B.

AFLP markers are widely used in breeding in some other crops, but their utilization in breeding of Brassica crops is not so frequent. AFLP markers are used for molecular characterization of particular varieties or genotypes and for evaluation of genetic diversity. The aim of this thesis was the application of this method in breeding of rapeseed and in comparative study of genetic variability of different oil seed rape cultivars of Czech, Czechoslovak and German origin. AFLP is based on selective amplification of genomic DNA. Technique consists of three basic steps: 1. restriction of genomic DNA on smaller fragments and ligation of adaptors; 2. selective amplification of fragments with specific primers and 3. electrophoresis of products and computer analysis (Vos et al., 1995). In this thesis 16 Czech (CZ) and Czechoslovak (CZS) and 16 German (G) cultivars of oilseed rape were compared. Two specific primers, EcoRI AGC and fluorescence labeled primer MseI ACC, were chosen for AFLP method. It was possible to distinguish group of CZ/CZS and G cultivars, which have origin in gene resources from Czech Republic and Germany. AFLP technique allows detection of very small differences in Brassica genome. The differences between studied cultivars exist, but they are not very distinct. They show on certain relationship and reduction of genetic basis of genetic resources, which are used in Central Europe.