The downy mildew resistance locus Pp523 is located on chromosome C8 of Brassica oleracea L.

We have previously constructed a genetic map of Brassica oleracea L. containing the Pp523 locus that confers downy mildew resistance to adult plants. In this work, 44 SSR markers of reference for the Brassica C genome chromosomes were added to the map, allowing the nine major linkage groups to be assigned to the nine chromosomes of B. oleracea. Locus Pp523 was located on chromosome C8, and a locus determining flower colour was mapped to chromosome C3. In comparison with the first version of the map, the new map is denser and more compact. The available genomic information on B. oleracea was enriched with the chromosome location of two phenotypic traits and 421 DNA markers (RAPD, ISSR, AFLP, SCAR, BAC-end derived STS, SSR and other PCR markers). Conversely, the genomic information on B. oleracea chromosome C8 is being used as an additional tool for the map-based cloning of Pp523, the first gene for adult plant resistance to downy mildew precisely located to a specific chromosome of this crop species.

The determination of sinigrin in Brassica, and investigations into the use of allyl-isothiocyanate as a nematicide

The goal of this thesis was to study factors related to the development of Brassica juncea as a sustainable nematicide. Brassica juncea is characterized by the glycoside (glucosinolate) sinigrin. Various methods were developed for the determination of sinigrin in Brassica juncea tissue extracts. Sinigrin concentrations in plant tissues at various stages of growth were monitored. Sinigrin enzymatically breaks down into allylisothiocyanate (AITC). AITC is unstable in aqueous solution and degradation was studied in water and in soil. Finally, the toxicity of AITC against the root-lesion nematode (Pratylenchus penetrans) was determined. A method was developed to extract sinigrin from whole Brassica j uncea tissues. The optimal time of extraction wi th boiling phosphate buffer (0.7mM, pH=6.38) and methanol/water (70:30 v/v) solutions were both 25 minutes. Methanol/water extracted 13% greater amount of sinigrin than phosphate buffer solution. Degradation of sinigrin in boiling phosphate buffer solution (0.13%/minute) was similar to the loss of sinigrin during the extraction procedure. The loss of sinigrin from boiling methanol/water was estimated to be O.Ol%/minute. Brassica juncea extract clean up was accomplished by an ion-pair solid phase extraction (SPE) method. The recovery of sinigrin was 92.6% and coextractive impurities were not detected in the cleaned up extract. Several high performance liquid chromatography (HPLC) methods were developed for the determination of sinigrin. All the developed methods employed an isocratic mobile phase system wi th a low concentration of phosphate buffer solution, ammonium acetate solution or an ion-pair reagent solution. A step gradient system was also developed. The method involved preconditioning the analytical column with phosphate buffer solution and then switching the mobile phase to 100% water after sample injection.Sinigrin and benzyl-glucosinolate were both studied by HPLC particle beam negative chemical ionization mass spectrometry (HPLCPB- NCI-MS). Comparison of the mass spectra revealed the presence of fragments arising from the ~hioglucose moiety and glucosinolate side-chain. Variation in the slnlgrin concentration within Brassica juncea plants was studied (Domo and Cutlass cuItivars). The sinigrin concentration in the top three leaves was studied during growth of each cultivar. For Cutlass, the minimum (200~100~g/g) and maximum (1300~200~g/g) concentrations were observed at the third and seventh week after planting, respectively. For Domo, the minimum (190~70~g/g) and maximum (1100~400~g/g) concentrations were observed at the fourth and eighth week after planting, respectively. The highest sinigrin concentration was observed in flower tissues 2050±90~g/g and 2300±100~g/g for Cutlass and Domo cultivars, respectively. Physical properties of AITC were studied. The solubility of AITC in water was determined to be approximately 1290~g/ml at 24°C. An HPLC method was developed for the separation of degradation compounds from aqueous AITC sample solutions. Some of the degradation compounds identified have not been reported in the literature: allyl-thiourea, allyl-thiocyanate and diallyl-sulfide. In water, AITC degradation to' diallyl-thiourea was favored at basic pH (9.07) and degradation to diallyl-sulfide was favored at acidic pH (4 . 97). It wap necessary to amend the aqueous AITC sample solution with acetonitrile ?efore injection into the HPLC system. The acetonitrile amendment considerably improved AITC recovery and the reproducibility of the results. The half-life of aqueous AITC degradation at room temperature did not follow first-order kinetics. Beginning with a 1084~g/ml solution, the half-life was 633 hours. Wi th an ini tial AITC concentration of 335~g/ml the half-life was 865 hours. At 35°C the half-life AITC was 76+4 hours essentially independent of the iiisolution pH over the range of pH=4.97 to 9.07 (1000~g/ml). AITC degradation was also studied in soil at 35°C; after 24 hours approximately 75% of the initial AITC addition was unrecoverable by water extraction. The ECso of aqueous AITC against the root-lesion nematode (Pratylenchus penetrans) was determined to be approximately 20~g/ml at one hour exposure of the nematode to the test solution. The toxicological study was also performed with a myrosinase treated Brassica juncea extract. Myrosinase treatment of the Brassica juncea extract gave nearly quantitative conversion of sinigrin into AITC. The myrosinase treated extract was of the same efficacy as an aqueous AITC solution of equivalent concentration. The work of this thesis was focused upon understanding parameters relevant to the development of Brassica juncea as a sustainable nematicide. The broad range of experiments were undertaken in support of a research priority at Agriculture and Agri-Food Canada.

Efecto de la aplicación de ácidos orgánicos de bajo peso molecular en la Biodisponibilidad de Pb y Zn en un sistema usando Brassica juncea.

Tesis (Maestría en Ciencias con Orientación en Procesos Sustentables) UANL, 2010.

Extracción y caracterización química,nutricional y funcional de proteinas de la semilla de Brassica campestris L.

Tesis (Doctorado en ciencias con Especialidad en alimentos) UANL

Biochemical characterization of a novel halide/bisulfide mythyltransferase purified from Brassica oleracea L.

Thèse diffusée initialement dans le cadre d'un projet pilote des Presses de l'Université de Montréal/Centre d'édition numérique UdeM (1997-2008) avec l'autorisation de l'auteur.

A survey of brassica vegetable smallholder farmers in the Gauteng and Limpopo provinces of South Africa

A study was taken to investigate the types of brassica vegetables mostly grown by smallholder farmers in two provinces of South Africa. Thirty-one smallholder vegetable farmers in the Gauteng province and Waterberg district in the Limpopo province were surveyed. In addition, the study also sought to establish the common diseases, the management strategies used and problems encountered by the farmers. Farmers were interviewed using a questionnaire with closed and open–ended questions. The results indicated that the smallholder farmers mostly grew cabbage (93.6%) as their main brassica crop followed by rape (41.2%). Thirty percent of farmers could not identify or name the predominant disease/s encountered in their fields. Major diseases encountered by farmers surveyed were an unknown disease/s (33.3%), black rot (26.7%), Alternaria leaf spot (6.7%) and white rust (6.7%). Smallholder farmers have inadequate technical information available especially relating to crop diseases, their identification and control. Farmers encountered challenges with black rot disease especially on cabbage, rape and kale and the disease was a problem during winter and summer. Generally, the smallholder farmers used crop rotation (74.2%) as a major practice to manage the diseases experienced. They rotated their brassica vegetables with other crops/vegetables like tomatoes, onions, beetroots and maize. Most of the farmers interviewed (61.3%) did not use chemicals to control diseases, whereas 38.7% of them used chemicals. This was mostly because they lacked information and knowledge, high costs associated with use of chemical fungicides and some were shifting towards organic farming. From the study it was noted that there was a need for technical support to improve farmers’ knowledge on disease identification and control within the surveyed areas.

Análisis del cultivo de brócoli (Brassica oleracea var Italica), manejo-reducción de pesticidas y posibilidades de aplicación del método circular de estudio en la Comunidad de Gatazo Zambrano, Cantón Colta, Provincia Chimborazo - Ecuador

La investigación propuesta pretende comprobar si la educación formal, la capacitación, la asistencia técnica son fundamentales para manejar adecuadamente los pesticidas y reducir sus residuos tóxicos en los agro-ecosistemas de pequeña producción. De hecho, a través de la encuesta proporcionada por el equipo técnico del proyecto Marco de Inversión en Desarrollo Rural y Reducción de Pobreza (MIRREP) se trabajó con indicadores socio-ambientales y económicos que permitieron reconocer que miembros de la comunidad son los más perjudicados por el mal uso de pesticidas. Para el efecto, se recopiló los datos desde el mes de julio hasta el mes de octubre del 2007. Los datos se compilaron en las encuestas prediseñadas de las que se obtuvo datos porcentuales y datos estadísticos levantados a través de Análisis de Correspondencia Múltiple (MCA) y Chi-Cuadrado de Pearson. A la vez, se introdujo el Método Circular de Estudio (SCM) como metodología de capacitación para el campesinado adulto con el fin de comprobar su efectividad, y generar y transmitir conocimientos respecto al uso de pesticidas y eliminación de sus residuos. Ahora bien, para analizar cual es la situación respecto al manejo de pesticidas se hizo observación directa, entrevistas (informales, formales), conferencias grupales y comunales para finalmente por medio del SCM establecer diálogos horizontales donde tanto técnicos como campesinos tienen el mismo derecho de participar y opinar, considerando sus opiniones tan valederas como las del personal técnico con el fin de enriquecerse las partes participantes en un verdadero proceso de transmisión y generación de conocimientos in situ. De esta forma, se ha generado un trabajo de investigación-acción enmarcado en la capacitación y valoración de las condiciones socio-ambientales y económicas con el fin de implementar programas y metodologías para el manejo y reducción de pesticidas. De hecho, contextualizar la realidad del campesinado respecto al manejo de pesticidas, evidenciarlo ante las autoridades de turno y proponer una alternativa de desarrollo rural valedera donde se reduzcan las múltiples externalidades negativas (sociales, económicas, y ambientales) es el valor agregado que este trabajo con el fin de reducir la pobreza a la que el campesinado se ha visto acogido por más de 2 décadas.

Use of airborne remote sensing to detect riverside Brassica rapa to aid in risk assessment of transgenic crops

High resolution descriptions of plant distribution have utility for many ecological applications but are especially useful for predictive modelling of gene flow from transgenic crops. Difficulty lies in the extrapolation errors that occur when limited ground survey data are scaled up to the landscape or national level. This problem is epitomized by the wide confidence limits generated in a previous attempt to describe the national abundance of riverside Brassica rapa (a wild relative of cultivated rapeseed) across the United Kingdom. Here, we assess the value of airborne remote sensing to locate B. rapa over large areas and so reduce the need for extrapolation. We describe results from flights over the river Nene in England acquired using Airborne Thematic Mapper (ATM) and Compact Airborne Spectrographic Imager (CASI) imagery, together with ground truth data. It proved possible to detect 97% of flowering B. rapa on the basis of spectral profiles. This included all stands of plants that occupied >2m square (>5 plants), which were detected using single-pixel classification. It also included very small populations (<5 flowering plants, 1-2m square) that generated mixed pixels, which were detected using spectral unmixing. The high detection accuracy for flowering B. rapa was coupled with a rather large false positive rate (43%). The latter could be reduced by using the image detections to target fieldwork to confirm species identity, or by acquiring additional remote sensing data such as laser altimetry or multitemporal imagery.

Characterization and origin of infection of Rhizoctonia solani associated with Brassica oleracea crops in the UK

An extensive study was conducted to determine where in the production chain Rhizoctonia solani became associated with UK module-raised Brassica oleracea plants. In total, 2600 plants from 52 crops were sampled directly from propagators and repeat sampled from the field. Additional soil, compost and water samples were collected from propagation nurseries and screened using conventional agar isolation methods. No isolates of R. solani were recovered from any samples collected from propagation nurseries. Furthermore, nucleic acid preparations from samples of soil and compost from propagation nurseries gave negative results when tested for R. solani using real-time PCR. Conversely, R. solani was recovered from 116 of 1300 stem bases collected from field crops. All the data collected suggested R. solani became associated with B. oleracea in the field rather than during propagation. Parsimony and Bayesian phylogenetic studies of ribosomal DNA suggested the majority of further classified isolates belonged to anastomosis groups 2-1 (48/57) and AG-4HGII (8/57), groups known to be pathogenic on Brassica spp. in other countries. Many R. solani isolates were recovered from symptomless plant material and the possibilities for such an association are discussed.

Use of airborne remote sensing to detect riverside Brassica rapa to aid in risk assessment of transgenic crops

High resolution descriptions of plant distribution have utility for many ecological applications but are especially useful for predictive modeling of gene flow from transgenic crops. Difficulty lies in the extrapolation errors that occur when limited ground survey data are scaled up to the landscape or national level. This problem is epitomized by the wide confidence limits generated in a previous attempt to describe the national abundance of riverside Brassica rapa (a wild relative of cultivated rapeseed) across the United Kingdom. Here, we assess the value of airborne remote sensing to locate B. rapa over large areas and so reduce the need for extrapolation. We describe results from flights over the river Nene in England acquired using Airborne Thematic Mapper (ATM) and Compact Airborne Spectrographic Imager (CASI) imagery, together with ground truth data. It proved possible to detect 97% of flowering B. rapa on the basis of spectral profiles. This included all stands of plants that occupied >2m square (>5 plants), which were detected using single-pixel classification. It also included very small populations (<5 flowering plants, 1-2m square) that generated mixed pixels, which were detected using spectral unmixing. The high detection accuracy for flowering B. rapa was coupled with a rather large false positive rate (43%). The latter could be reduced by using the image detections to target fieldwork to confirm species identity, or by acquiring additional remote sensing data such as laser altimetry or multitemporal imagery.

Simple sequence repeats reveal uneven distribution of genetic diversity in chloroplast genomes of Brassica oleracea L. and (n=9) wild relatives

Diversity in the chloroplast genome of 171 accessions representing the Brassica 'C' (n = 9) genome, including domesticated and wild B. oleracea and nine inter-fertile related wild species, was investigated using six chloroplast SSR (microsatellite) markers. The lack of diversity detected among 105 cultivated and wild accessions of B. oleracea contrasted starkly with that found within its wild relatives. The vast majority of B. oleracea accessions shared a single haplotype, whereas as many as six haplotypes were detected in two wild species, B. villosa Biv. and B. cretica Lam.. The SSRs proved to be highly polymorphic across haplotypes, with calculated genetic diversity values (H) of 0.23-0.87. In total, 23 different haplotypes were detected in C genome species, with an additional five haplotypes detected in B. rapa L. (A genome n = 10) and another in B. nigra L. (B genome, n = 8). The low chloroplast diversity of B. oleracea is not suggestive of multiple domestication events. The predominant B. oleracea haplotype was also common in B. incana Ten. and present in low frequencies in B. villosa, B. macrocarpa Guss, B. rupestris Raf. and B. cretica. The chloroplast SSRs reveal a wealth of diversity within wild Brassica species that will facilitate further evolutionary and phylogeographic studies of this important crop genus.

Glucosinolates in brassica vegetables: the influence of the food supply chain on intake, bioavailability and human health

Glucosinolates (GLSs) are found in Brassica vegetables. Examples of these sources include cabbage, Brussels sprouts, broccoli, cauliflower and various root vegetables (e.g. radish and turnip). A number of epidemiological studies have identified an inverse association between consumption of these vegetables and the risk of colon and rectal cancer. Animal studies have shown changes in enzyme activities and DNA damage resulting from consumption of Brassica vegetables or isothiocyanates, the breakdown products (BDP) of GLSs in the body. Mechanistic studies have begun to identify the ways in which the compounds may exert their protective action but the relevance of these studies to protective effects in the human alimentary tract is as yet unproven. In vitro studies with a number of specific isothiocyanates have suggested mechanisms that might be the basis of their chemoprotective effects. The concentration and composition of the GLSs in different plants, but also within a plant (e.g. in the seeds, roots or leaves), can vary greatly and also changes during plant development. Furthermore, the effects of various factors in the supply chain of Brassica vegetables including breeding, cultivation, storage and processing on intake and bioavailability of GLSs are extensively discussed in this paper.

The potential to intensify sulforaphane formation in cooked broccoli (Brassica oleracea var. italica) using mustard seeds (Sinapis alba)

Sulforaphane, a naturally occurring cancer chemopreventive, is the hydrolysis product of glucoraphanin, the main glucosinolate in broccoli. The hydrolysis requires myrosinase isoenzyme to be present in sufficient activity; however processing leads to its denaturation and hence reduced hydrolysis. In this study, the effect of adding mustard seeds, which has a more resilient isoform of myrosinase, to processed broccoli was investigated with a view to intensify the formation of sulforaphane. Thermal inactivation of myrosinase from both broccoli and mustard seeds was studied. Thermal degradation of broccoli glucoraphanin was investigated in addition to the effects of thermal processing on the formation of sulforaphane and sulforaphane nitrile. Limited thermal degradation of glucoraphanin (less than 12 %) was observed when broccoli was placed in vacuum sealed bag (sous vide) and cooked in a water bath at 100 ºC for 8 and 12 min. Boiling broccoli in water prevented the formation of any significant levels of sulforaphane due to inactivated myrosinase. However, addition of powdered mustard seeds to the heat processed broccoli significantly increased the formation of sulforaphane.

Shoot calcium and magnesium concentrations differ between subtaxa, are highly heritable, and associate with potentially pleiotropic loci in Brassica oleracea

Calcium (Ca) and magnesium (Mg) are the most abundant group II elements in both plants and animals. Genetic variation in shoot Ca and shoot Mg concentration (shoot Ca and Mg) in plants can be exploited to biofortify food crops and thereby increase dietary Ca and Mg intake for humans and livestock. We present a comprehensive analysis of within-species genetic variation for shoot Ca and Mg, demonstrating that shoot mineral concentration differs significantly between subtaxa (varietas). We established a structured diversity foundation set of 376 accessions to capture a high proportion of species-wide allelic diversity within domesticated Brassica oleracea, including representation of wild relatives (C genome, 1n = 9) from natural populations. These accessions and 74 modern F-1 hybrid cultivars were grown in glasshouse and field environments. Shoot Ca and Mg varied 2- and 2.3-fold, respectively, and was typically not inversely correlated with shoot biomass, within most subtaxa. The closely related capitata (cabbage) and sabauda (Savoy cabbage) subtaxa consistently had the highest mean shoot Ca and Mg. Shoot Ca and Mg in glasshouse-grown plants was highly correlated with data from the field. To understand and dissect the genetic basis of variation in shoot Ca and Mg, we studied homozygous lines from a segregating B. oleracea mapping population. Shoot Ca and Mg was highly heritable (up to 40). Quantitative trait loci (QTL) for shoot Ca and Mg were detected on chromosomes C2, C6, C7, C8, and, in particular, C9, where QTL accounted for 14 to 55 of the total genetic variance. The presence of QTL on C9 was substantiated by scoring recurrent backcross substitution lines, derived from the same parents. This also greatly increased the map resolution, with strong evidence that a 4-cM region on C9 influences shoot Ca. This region corresponds to a 0.41-Mb region on Arabidopsis (Arabidopsis thaliana) chromosome 5 that includes 106 genes. There is also evidence that pleiotropic loci on C8 and C9 affect shoot Ca and Mg. Map-based cloning of these loci will reveal how shoot-level phenotypes relate to Ca 21 and Mg 21 uptake and homeostasis at the molecular level.