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Analysis of rRNA genes in variets Brassica napus
Dofková, Květoslava ; RNDr.Roman Matyášek, CSc. (referee) ; Kovařík,, Aleš (advisor)
Brassica napus (AACC, 2n = 38) is an allotetraploid species derived from the parentel diploid species Brassica rapa (AA, 2n = 20) and Brassica oleracea (CC, 2n = 18). The aim of thesis was to carry out the genetic and epigenetic analysis of high-copy rRNA genes (or rDNA) in several varieties of hybrid species B. napus. The experiments involved determining the ratio of parental genes in hybrids, sequencing and methylation analysis of the promoter region of rDNA. Using Southern hybridization, it was revealed significant variability in the number of parental rDNA units between each variety. Data from sequence analysis were in good agreement with the results of Southern blot. Genetic recombination between parental rDNA units was revealed in one variety by DNA sequencing of promotor region. To study methylation, bisulfite sequencing was performed. It was found out that rDNA units of B. rapa origin have a higher value of methylation than units originated from B. oleracea.
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Optimisation of rapeseed in vitro cultures used for CRISPR/Cas9 technology
HARENČÁK, Jan
Plant transformation using Agrobacterium tumefaciens bacteria (Rhizobium radiobacter, according to new nomenclature) is widely used method for plant genome editing. However, different oilseed rape varieties do not always have the same in vitro regeneration capacity, which is crucial for transformation. This work aims to optimize regeneration protocol for selected varieties of oilseed rape. Of the six varieties tested, only two regenerated successfully - Arabella and Obelix. The created protocol can be used for regeneration without transformation, but it is also possible to implement specific steps in it that enable transformation using A. tumefaciens.
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Mapping of clubroot resistance genes by associative transcriptomics
HEJNA, Ondřej
The current high pathogenic pressure on agriculture crops is the task for breeders. One of the serious problems that modern breeding is struggling to address is clubroot. This disease is caused by the soil biotrophic pathogen Plasmodiophora brassicae. At present, this pathogen presents serious problems for most cruciferous crops worldwide. The possibilities for controlling this disease are very limited. From an economic point of view, the most effective option is growing resistant or tolerant varieties against this pathogen. To date, several genetic sources of resistance for breeding have been identified. However, the application of these resources inbreeding is problematic, because there is a lack of knowledge of the basis of the resistance and lack of understanding of causal genes or proteins. In this study, the resistance of the B. napus genotypes to clubroot was evaluated. An Associative Transcriptomics approach was used to identify markers and causal genes associated with resistance to clubroot. In assessing the response of a genetic diversity panel comprising 250 accessions of B. napus, the mixed pathotype ECD 17/31/31 was used. The result of this testing finds 46 genotypes with disease index (DI) greater than 80. Such varieties can be considered resistant. For 245 genotypes, RNAseq transcriptome data were obtained for use in two associative analyses. In the first assay, SNP association analysis was performed with 256,397 SNP markers distributed across the genome of B. napus. The result of this analysis revealed 86 SNP markers located in 9 different regions on chromosomes A01, A02, A03, A08, C02 and C07. Resistant loci were named according to their location as BnA01_0308, BnA02_0265, BnA02_0286, BnA03_0186, BnA03_0263, BnA08_0009, BnCO02_0414, BnCO07_0238 and BnCO07_0421. Altogether, 392 genes were found in these regions. For the second association analysis, transcript abundance data of 53,889 genes in the B. napus genome were used. The analysis itself evaluated the degree of association between expression profiles and DI profile. Based on this analysis, 19 associated genes were revealed. In the last step, annotation analyzes GO, InterPro, and annotations were performed based on similarity to Arabidopsis thaliana genes. Overall, 82 candidate genes were selected to be involved in clubroot resistance. These results provide useful information both for breeding by identifying associated SNP markers useful for breeding resistant varieties and for understanding the elements of resistance in B. napus.
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Distribution of S-alleles in Brassica genus
ŽALUDOVÁ, Václava
Self-inkompatibility (SI) is a system how to prevent self-pollinaton in population and enable gene-flow and diversification. SI determines one polymorphic locus S-locus which contains several genes responsible for SI reaction. One of genes lying on the S-locus is the SLG gene and according sequence similarity SLGs are devided into two groups. First group is considered to be domminant and the second recessive. Incompatible lines and five cultivars of Brassica napus, different SLG alleles were cloned and sequenced. Thirty-one sequences of different SLG clones were obtained and phylogenetic tree deduced.Three main groups of SLG sequences are obvious and it seems to have different allelic specificity. Two groups included majority of sequences from SI lines and cultivars suggest two S-alleles very common in Brassica napus. These results were compared with NBCI with different species of Brassica and also with Raphanus sapivus. After cluster analysis assessment was phylogenetic tree created, witch shows a great differences between different SLG classes, but slight differences between Brassica species.
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Analysis of abiotic stress induced genes in rape
HOŠTIČKOVÁ, Irena
Breeding for abiotic stress tolerance is one of main topics in plant breeding. Oilseed rape breeding programs were for a long time focused on morphological and physiological parameters. In this thesis few experiments focused on identification of genes involved in abiotic stress reaction were performed using RT-qPCR (quantitative reverse transcription PCR). Simultaneously SPR (surface plasmon resonance) method were used as modern optical method facilitating very low native protein concentration even in presence of other substances. This method facilitates quantification of concrete proteins by binding them to specific antigen and in oilseed rape research it was not used by now. ERD10 protein was identified by SPR as protein involved in cold stress reaction (or acclimation). The results show ERD10 accumulation in standard conditions affects dynamics of its accumulation change during cold stress. In case we are searching for genotypes great in acclimation ability even during short and warm autumn SPR method should be suitable method for fast, easy and relatively cheap screening of large number of genotypes in breeding collections. Also genes LTI78, RCI2A, NRP1 and two genes for hypothetical proteins were analysed. Their relative expression during cold stress was markedly increased too. Very little is known about these genes and proteins nowadays therefor it will be interesting topic of our oncoming experiment. Relative expression of genes picked according to MALDI-TOF/TOF analysis results was also tested in microspore embryo regenerants stressed by simulated drought. Genes for lactoylglutathione lyase I, phospholipase D 1 and peroxiredoxin antioxidase were tested. In tolerant cultivar was markedly decreased gene expression of peroxiredoxin antioxidase in standard conditions and early stress. These gene will be subject for next research as potential marker for more tolerant genotypes selection.
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Use of molecular markers for studying genetic diversity of breeding materials of rapeseed
ČÍŽKOVÁ, Pavla
This thesis is based on the use of ISSR method for studying genetic diversity of oilseed rape. Altogether, 180 genotypes were evaluated. Frozen leaves of oilseed rape from three breeding station called: 1/ breeding station Selgen a.s., 2/ OSEVA PRO s.r.o. and 3/ breeding station Slapy u Tábora were used as imput material. DNA isolation was performed by Williams, who modified CTAB method for oilseed rape. We selected for ISSR 4 UBC which showed high variability and stability. The analysis was performed by gel electrophoresis. This thesis is also focused on comparison of gel electrophoresis and chip electrophoresis. In this thesis We show a matrix of simmilarity between varieties. The results were also provided back to breeders as a basis for further breeding.
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The physiological and proteomic characterisation of winter oilseed rape upon abiotic stress
Urban, Milan ; Vítámvás, Pavel (advisor) ; Hnilička, František (referee) ; Valentová, Olga (referee)
- Ph.D. thesis - Milan Urban, 2017 In some years, the agricultural production of oilseed rape, an important crop in the Czech Republic, is - besides biotic stress - facing the problem of damage caused by frost or drought. Together with special attention paid to proteins revealing responses between crop genotypes with differential abiotic stress tolerance levels we reviewed possible applications of proteomic results in crop breeding programs aimed at an improvement of crop stress tolerance (paper 1). For first original result, cold temperature was imposed upon non-vernalized plants in the stage of leaf rosette. The article (paper 2) shows a significant correlation between frost tolerance (FT), dehydrin (DHN) accumulation, and photosynthetic acclimation in five cultivars (cvs). Newly, the specific DHN D97 was shown to accumulate and other DHNs were shown to have qualitative differences in accumulation. These results imply that proper FT assessment is based on rapid photosynthetic acclimation together with higher accumulation of protective compounds. Drought stress (paper 3) was imposed in the water- demanding stem prolongation phase before flowering, because late-spring drought before and during flowering decreases the yield and seed quality significantly. This paper newly describes two water-uptake...
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Hereditary and non-hereditary factors affecting the quality of rapeseed oil
Zemanová, Kateřina ; Sedláková, Vladimíra (advisor) ; Bečka, David (referee)
This literature review study quality of rapeseed oil in details, depending on heritable and non-heritable factors. Consumption of rapeseed oil is growing every year and in addition to use in human nutrition is also used in animal feed industry or as fuel for motor vehicles. The rapeseed oil has especially a large representation of oleic acid. Furthermore, it is linoleic acid and linolenic acid and undesirable erucic acid, whose quantity is considerably reduced. It is important to mention glucosinolate content, as antinutritional substances, which is reduced together with erucid acid which is known as "00" rapeseed oil. All these components are described in details in this review. The content of fatty acids in rapeseed oil may be influenced both genetic factors and environmental factors. Selection plays a significant role in breeding. Suitably selecting the correct genotypes along with environmental influences can reach the desired target. An important role is played by the synthesis of fatty acids, where in using a desaturation enzymes of fad series, there is desaturation of stearic acid to oleic acid, linoleic acid and linolenic acid. With QTL method, it can be detected genetic background of quantitative traits. Major genes responsible for the quality of rapeseed oil are located on the chromosome A, namely A1, A3, A5 and A8, which are mentioned in the work.
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Influence of white and yellow flower rape on incidence of natural enemies of pests
Křížek, Stanislav ; Kazda, Jan (advisor) ; Daniel, Daniel (referee)
The insect society living in oilseed rape is large. Change of the crop characteristics affects both harmful species and beneficious ones. The latter group consists of a lot of members of order Hymenoptera. This work focuses on the evaluation of impact of white oilseed rape cultivation on the insect society in comparison with two yellow winter varieties. Newly bred winter oilseed rape line variety Witt was compared to two hybrid varieties of yellow rape; Sherpa and DK Sensei. The experiment was carried out in 2015 and its plots were placed at Ruzyně at Crop Reserach Institute (VÚRV, v. v. i.) and also at the Experimental Unit of Czech University of Life Sciences (ČZU) in Uhříněves. The insects were collected by sweeping net, soil larvae traps and soil emergence traps. The occurrence of serious pests (pollen beetle Brassicogethes aeneus, brassica pod midge Dasineura brassicae, cabbage seed weevil Ceutorhynchus obstrictus, stem flea beetle Psylliodes chrysocephalus and cabbage flea beetles Phyllotreta sp.) was recorded. Dominant parasitoid species were Tersilochus heterocerus, Platygaster subuliformis, Trichomalus perfectus, Mesopolobus morys and Omphale clypealis. The level of pest mortality was evaluated. Numbers of species identified by key were processed using generalized linear model method in R statistical software. The results confirm suitability of the assessed varieties in the white oilseed rape cultivation system, notably in the combination of Sherpa and Witt. They give an insight into parasitoid community of oilseed rape in Czech republic, which is useful for management of serious pest species.
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