724 research outputs found
Fictionality and Literature: Core Concepts Revisited
No full textAuthor / Henrik Zetterberg-Nielsen -- Narrator / Sylvie Patron -- Plot / Wendy Veronica Xin -- Character / H. Porter Abbott -- Consciousness / Maria Mäkelä -- Metaphor / Greta Olson -- Paratext / Louise Brix Jacobsen -- Intertextuality / Rikke Andersen Kraglund -- Metafiction and metalepsis / Richard Walsh -- The novel / Catherine Gallagher and Simona Zetterberg-Nielsen -- Poetry / Lasse R. Gammelgaard -- Literary nonfiction / James Phelan -- Ethics / Jakob Lothe -- Social justice / Susan S. Lanser.Item embargoed for five year
Season 3 Episode 2: The Neighborhood Church
No full textWhat do you know about the church closest to your house? Chances are, not much. Today most churches have forgotten the art of connecting with their neighborhoods. They are quick to leave city neighborhoods for bigger, better buildings in the suburbs. Eric Jacobsen, author of Sidewalks in the Kingdom: New Urbanism and the Christian Faith, and Sister Maureen Geary of the Catholic Diocese of Grand Rapids offer their ideas on why. Shirley Hoogstra hosts. Episode #302
Resistance and susceptibility to late blight in Solanum: gene mapping, cloning and stacking
No full textThe potato late blight disease, caused by the oomycete Phytophthora infestans, is a major threat for potato production worldwide. To breed potato varieties with durable resistance against P. infestans, it is necessary to combine two or more resistance (R) genes. Single R genes are easily overcome by the rapidly evolving pathogen, whereas the presence of several R genes could probably prevent gain of virulence from a single mutation in the pathogen. The large gene pool available within wild potato species offers sufficient possibilities to identify new and diverse R genes conferring resistance to P. infestans (Rpi). Map-based cloning is the most suitable strategy to isolate such new Rpi genes. The objective of this research was mapping, if possible, followed by cloning of Rpi genes from wild Solanum species. Resistance to P. infestans occurring in four different wild Solanum species were mapped in a major R gene cluster on chromosome 11. Natural stacking of three R genes located on different chromosomes was identified in a natural hybrid. In addition, we initiated studies on another type of defense system that is not based on the typical R genes, namely the response of Solanum to INF1 elicitin. <br/
Dissection of the major late blight resistance cluster on potato linkage group IV
No full textPotato is consumed worldwide and represents the fourth most important staple food crop after rice and wheat. Potato cultivars display a large variety of color, shape, taste, cooking properties and starch content but are all derived from the same species; Solanum tuberosum. Potato breeding is an economic important activity for international breeding companies, but also plays an important role in breaking the circle of poverty for small farmers. In the Andean region, most farmers use many different potato genotypes combined with farming practices transmitted orally over thousands of years. The most prominent menace to potato production is Late Blight caused by the oomycete Phytophthora infestans which destroys leaves, stems and tubers. Differences of breeding methods between the potato grown in South America and in the rest of the world is related to differences in the consequences of Late Blight infection. In the 19th, century, entire potato fields in Ireland were devastated while in South America P. infestans proliferation was readily inhibited. This difference is found in the biodiversity reserve such as that of the Chiloé archipelago in Chile where local people cultivate about 200 varieties of native potato. Obviously, the genetic diversity of cultivated native potato acts as a shield against this versatile pathogen. Inspired by this model to solve the problems raised by the extensive use of potato monoculture, growers and breeders need to maintain genetic diversity in the European staple food crops. In exploring the South American native potato collection, Solanum demissum and later on Solanum bulbocastanum appeared to be a source of resistance genes (Rpi) to P. infestans. The S. demissum Rpi genes were transmitted to potato breeding clones by traditional introgression breeding. However the fading of their ability in providing effective resistance against Late Blight infection was witnessed within a decade. In the pursuit to provide a hopefully more durable protection in existing potato cultivars, plant breeding scientists proposed to directly introduce South American native potato Rpi genes in modern potato varieties by using a so-called cisgenic approach. This in contrast to transgenic plants which can contain genes which have originated from non related genera or even different kingdoms. Breeding of cisgenic plants is on its way to public acceptance because of its inherent resemblance to natural crossing and because efforts are made by the scientific community to explain the principles of cisgenesis. Lessons were learned from the flexibility of P. infestans to overcome the effect of newly introduced Rpi genes and, therefore, efforts are still ongoing to discover and clone new Rpi genes from native potatoes. With this in mind, a new family of Rpi genes represented by Rpi-blb3, Rpi-abpt, R2, R2-like and Rpi-mcd1.1 were characterized in clones derived from S. bulbocastanum, S. demissum, S. edinense and S. microdontum. We accomplished in this research the physical isolation of these genes, the molecular characterization of their functionality and the allelic distribution in the Petota collection. Rpi-blb3, Rpi-abpt, R2, R2-like and Rpi-mcd1.1 belong to the potato linkage group IV and all contain signature sequences characteristic of LZ-NBS-LRR proteins. The closest known R gene so far is RPP13 from Arabidopsis thaliana which shares an amino-acid sequence similarity of 35%. The LRR domains of Rpi-blb3, Rpi-abpt, R2 and R2-like proteins are highly homologous, whilst LZ and NBS domains are more polymorphic with those of R2 being the most divergent. All four Rpi genes recognize the recently identified RXLR effector protein PiAVR2 which is secreted by P.infestans in the cytoplasm of plant cells during the infection process. Unlike Rpi-blb3, Rpi-abpt, R2 and R2-like , the S. microdontum resistance gene Rpi-mcd1.1 does not interact with PiAVR2 and provides a different resistance spectrum. Rpi-mcd1.1 shares 90% nucleotide identity with Rpi-blb3 and polymorphic nucleotides are mainly located in the LRR region. The S. bulbocastanum haplotypes of Rpi-blb1, Rpi-blb2 and Rpi-blb3 were discovered in several Mexican diploid as well as polyploid species closely related to S. bulbocastanum. These three resistance genes occurred in different combinations and frequencies in S. bulbocastanum accessions and their distribution is confined to Central America. A selected set of genotypes was tested for their response to the avirulence effectors IPIO-2, Avr-blb2 and Pi-Avr2, which interact with Rpi-blb1, Rpi-blb2 and Rpi-blb3, respectively, as well as by disease assays with a diverse set of isolates. Using this approach some accessions could be identified that contain novel, yet unknown, Late Blight resistance factors in addition to the Rpi-blb1, Rpi-blb2 and Rpi-blb3 genes Analysis of the sequences obtained in different allele mining strategies suggests an evolution of the major late blight locus on linkage group IV through recombination and point mutations. By making use of the sequence information provided by the alleles, we identified the repeats and amino acids in the LRR domain which are specific for PiAVR2 recognition. Finally, we discussed the results described in this thesis in a potato/ P. infestans co-evolution context. <br/
Identification, characterization and high-resolution mapping of resistance genes to Phytophthora infestans in potato
No full textPotato ( Solanum tuberosum L.) is one of the most important crops in the world. The oomycete Phytophthora infestans (Mont. de Bary) is the causal agent of late blight which is the most devastating disease of the cultivated potato. It causes economic losses of several billion US dollars in crop production and protection annually worldwide. To control this disease, the application of enormous amounts of chemicals is required, which is expensive and environmentally unsafe. Also, late blight is increasingly more difficult to control because the pathogen is developing tolerance to the chemicals and new strains of the pathogen are evolving. Therefore, introduction of resistance genes from wild Solanum species into potato cultivars is considered as the most promising and environmental-safe approach to achieve durable late blight resistance.Late blight resistance exists in a wide range of Solanum wild species of which 75 % have a diploid constitution (2n=2x=24) although the majority of the cultivated potato is tetraploid (2n=4x=48). Therefore, several different approaches such as the use of 2n gametes, colchicine treatment, intraspecific hybridization, somatic hybridization, embryo rescue and gene transformation are used to overcome the difficulties caused by different ploidy levels. Of these approaches, gene transformation is the most controlled and time-efficient method to introduce resistance into the cultivated potato. However, gene identification and gene isolation are the first prerequisites for this approach.During the last century, most effort for late blight resistance was focused on the wild species S. demissum which contains at least 11 race-specific resistance genes ( R1 - R11 ). However, these resistances were rapidly overcome by new virulent pathogens which evolved. Therefore, new sources of late blight resistance are required and several resistances from different wild species such as S. microdontum , S. bulbocastanum , S. berthaultiiandS . pinnatisectumwere characterized. In the present research, we investigated both foliage and tuber blight resistances in different mapping populations derived from different sources of Solanum species.The pathogen P. infestans can infect both foliage and tuber of potato. To study tuber blight resistance, a wounded tuber assay was developed and the correlation between tuber and foliage blight resistance in four different mapping populations CE, SHRH, RH94-076 and RH4X-103, in which foliage blight resistances were identified, was examined (Chapter 2). Tuber blight resistance was found to be correlated with foliage blight resistance in three populations CE, SHRH and RH94-076, while it was independent in the RH4X-103 population. Further investigation to explore late blight resistances using molecular markers and resistance assays on both foliage and tuber with four different isolates was focused on the RH4X-103 population. Three specific resistance genes in foliage, tuber or both were segregating. The tuber blight resistance locus was closely linked to marker GP179 and co-segregating with the R1 -derived marker 76_2s on chromosome 5. The R1 gene functioned both as a foliage and tuber blight R gene, whereas the Rpi-abpt and R3a genes acted only on foliage.S. bulbocastanumis one of the most promising wildSolanumspecies possessing late blight resistance. ABPT material, which is a quadruple hybrid created by bridge crosses using the fourSolanumspecies;S. a caule,S. b ulbocastanum,S. p hurejaandS. t uberosum,to overcome incompatibility betweenS. bulbocastanumandS. tuberosumwere used to introduce late blight resistance. A tetraploid mapping population (RH4X-103) containing the late blight resistance geneRpi-abptwas created by a 4x-2x cross and was originally derived from ABPT material (Chapter 3). Resistance assay and AFLP marker analysis allowed localization of theRpi-abptlocus on chromosome 4. The origin of the resistance was confirmed by the analysis ofRpi-abptlocus-linked AFLP markers and the resistance assay of ABPT-related wild species accessions. An extended population of 1383 offspring was screened to construct a high-resolution map where the Rpi-abpt locus was consequently localized in a 0.5 cM interval between flanking AFLP markers and was co-segregating with one AFLP marker. The translated protein sequence of the co-segregating AFLP marker appeared to be highly homologues to several disease resistance proteins.The investigation of the race-specific R2-like gene was performed in a diploid mapping population (Chapter 4) primarily used for mapping nematode resistance. R2-like was phenotypically indistinguishable from the S. demissum -derived R2 gene although S. demissum is not directly involved in the pedigree of the population andR2-likeis expected to originate from another species. The R2-like locus was placed in a 0.4 cM interval, flanked by two AFLP markers on chromosome 4 using the extended population of 1586 offspring. Four AFLP markers were also identified to be co-segregating with the R2-like locus.A further late blight R gene ( Rpi-blb3)on chromosome 4 was identified in an intraspecific hybrid population derived from S. bulbocastanum (Chapter 5). The Rpi-blb3 locus was localized in a 0.9 cM interval between flanking AFLP markers and co-segregated with one AFLP marker in a population of 1396 offspring. According to the results of AFLP marker allele similarity tested by all co-segregating markers with four different R genes Rpi-abpt , R2-like , Rpi-blb3 and R2 , the four R genes appeared to be genetically very close and could be members of the same R gene cluster although the four R genes showed different race-specificities and origins.Additional interesting scientific results came from the 4x-2x population (RH4X-103) used for late blight resistance. In this population, the male parent produced unreduced 2n pollen by FDR (First Division Restitution), allowing the localization of centromeres by using half-tetrad analysis (Chapter 6). 130 male parent-derived AFLP markers were analyzed and the genetic linkage group and location of 95 AFLP markers was determined by comparing them with the reference markers of the ultra high density (UHD) map of potato. Depending on the heterozygosity (simplex) or homozygosity (nulliplex or duplex) of the AFLP markers of the offspring, the position of centromeres and the genetic distance of the AFLP markers from the centromere were determined.In FDR 2n pollen, all the parental heterozygous loci from centromere to first crossover are expected to be heterozygous (Aa) resulting in simplex genotype (Aaaa) of their offspring, when they are combined with homozygous recessive gametes (aa) from the female parent. In contrast, a single crossover between the locus and centromere releases 50 % homozygous and 50 % heterozygous gametes in FDR.The centromeres of 10 chromosomes were accurately localized.The centromere positions were compared with those, which were putatively identified in the UHD map by the fact that markers are dense due to suppression of recombination, and proved to be identical. The centromere position of two chromosomes could not precisely be determined by the half-tetrad analysis approach due to the absence of the 100 % heterozygous markers. However, their positions could be inferredbased on the combination of the results between the increasing or decreasing rate of heterozygosity in the half-tetrad analysis and marker density in the UHD map.Additional investigation of three chromosomes with sufficient numbers of markers proved that only one crossover occurs per chromosome arm, as a result of interference of recombination via centromere and telomere.In conclusion, three differentRgenes againstP. infestansin foliage were identified and the loci were accurately determined. These R loci are genetically very close and could be allelic. Our research for tuber blight resistance showed thatRgenes are not always active in both foliage and tuber. The results of the research described in this thesis demonstrate a great potential for further research. This will be directed towards the isolation of the R genes which can be used to introduce resistance into cultivated potato and to explore tuber blight resistance using a genetic transformation approach
Innovations in microspore embryogenesis in Indonesian hot pepper (Capsicum annuum L.) and Brassica napus L.
No full textHot pepper (Capsicum annuumL.) is the most important vegetable inIndonesia, but the yield is low, and the breeding programs are confined to the conventional methods and not efficient. To improve the efficiency of the breeding programs by speeding up the production of homozygous lines, studies were aimed at the introduction of haploid technology, which includes the regeneration and the production of doubled haploid plants from gametes. This technique is well developed in the model speciesBrassica napusL. via microspore culture. The results of various investigations involving both applied and fundamental aspects on microspore embryogenesis are presented in this thesis. The main results of the applied part deal with the development of an efficient shed-microspore culture protocol for the production of doubled haploid plants in Indonesian hot pepper (C. annuum), and its implementation under the local conditions of Indonesia. With regard to the more fundamental part, we presented for the first time an entirely new developmental pathway of embryogenesis including suspensor formation in microspore culture ofB. napuscv. Topas that mimics zygotic embryogenesis from early stages of development onwards. These results will have significant impact for practical application in hot pepper breeding programs as well as for further fundamental research on unraveling of early plant embryogenesis
Spectral Wave Dissipation by Vegetation: A new frequency distributed dissipation model in SWAN
No full textClimate change puts under pressure existing and future coastal interventions. Growing threats like sea-level rise and intensity of storms require solutions to be adaptable and resilient. Nature-based solutions have shown to tackle these challenges while providing social, environmental, and economic benefits. The role of vegetation in coastal protection is increasingly recognized. Aquatic vegetation reduces erosion, storm surge, and incoming wave height. Large-scale modeling of waves with spectral wave models such as SWAN is indispensable for the design of coastal structures and the assessment of flood risk. Wave dissipation due to vegetation can be modeled in SWAN as increased bottom friction (implicit modeling) or as an additional dissipation function (explicit modeling). The second assumes that vegetation can be represented as rigid cylinders or plates (canopies) with different properties. While some studies concluded that implicit modeling reproduces the spectral evolution of field measurements more closely, others concluded the opposite.Within the BE-SAFE project, field campaigns measured the spectral energy distribution over salt marshes in the Dutch Wadden Sea during several winter storms. The vegetated foreshore in front of the coastal dike got submerged over 2 m of water during high tide and storm surge. The measurements deployed wave gauges over the study transect, which was defined between the pioneer zone marsh edge and the near-dike location (300 m behind the salt marsh). Calibrating the implicit and explicit models in SWAN brought the modeled total wave energy decay closer to the measurement. Nevertheless, the spectral shape, which describes the energy distribution over frequencies, still showed significant and not yet understood differences near the dike.A methodology was executed to investigate the mechanisms that could reduce the spectral mismatch between the SWAN wave model and measurements over vegetation. First, the literature highlighted possible mechanisms that could be incorporated for this purpose. Next, a new frequency-distributed explicit dissipation model of Jacobsen et al. (2019) was implemented in SWAN and compared to implicit and explicit models using lab and field measurements.The results showed that the newly implemented model accurately captures the physics and the change of spectral shapes for all experimentally tested wave conditions and submergences. In contrast, the existing implicit and explicit dissipation models in SWAN reproduce the spectral evolution only under certain circumstances. In the validation and comparison to the field measurements with a much larger water depth than the vegetation height, the model of Jacobsen et al. (2019) correctly captured the vegetation's physical representation and the dissipation on the wind-sea frequencies. Nevertheless, the amount of energy on low frequencies was largely underpredicted by all frequency-distributed models. Therefore, the model of Jacobsen et al. (2019) was modified to include flexibility in a frequency-dependent reduction factor that reproduced the energy decay of the measurements in all frequency regions. Other mechanisms that could be responsible for the mismatch before and over the marsh are the redistribution of energy by non-linear triad interactions, generation of infra-gravity waves, and near-shore currents caused by horizontal variations on the vegetation properties.The present research provides the range of conditions in which the tested explicit and implicit energy dissipation functions in SWAN are able to simulate the spectral evolution over rigid canopies and flexible salt-marsh vegetation. A new version of SWAN includes a new frequency-distributed explicit model that performed more accurately than existing models for rigid canopies. The physical insights from the research contributed to developing additional versions of SWAN, which performed closely to the energy distribution of the measurements over deeply submerged and flexible salt marsh vegetation species. References:Jacobsen, McFall, Van der A (2019). A frequency distributed dissipation model for canopies. Coastal Engineering, 150, 135-146
Genetical metabolomics in apples (Malus x domestica Borkh)
No full text The aim of this thesis was finding genes that control the production of potentially health beneficial metabolites in apple fruits. The approach was genetic mapping of secondary metabolites such as phenolic compounds in an F1 progeny, leading to the detection of genetic loci that controlled these metabolites. At these genetic loci candidate genes were identified, using the whole genome sequence of apple, and it was investigated whether the expression of these candidate genes in the F1 progeny correlated with the metabolite levels. The cultivated apple (Malus x domestica Borkh) is among the most diverse and ubiquitously cultivated fruit species. It belongs to the family of Rosaceae which includes many commercial fruit species such as pear, strawberry, cherry, peach, apricot, almond, black cherry, and crab apple. Apple has a haploid chromosome number of 17. It is a self-incompatible and highly heterozygous crop. The breeding is further hampered by the long juvenile period which makes breeding in this crop a very slow process. The saying “An apple a day keeps the doctor away” has encouraged many researchers to search for the “magic” ingredients found in apple. Due to the beneficial role of apple phenolics, it is also called as a “new agrochemical crop”. Apple possesses many health beneficial properties for human beings as it is a rich source of phenolic compounds.It has been associated with reducing the risks of certain diseases such as cancers, particularly prostate, liver, colon, and lung cancers, cardiovascular diseases, coronary heart diseases, asthma, type-2 diabetes, thrombotic stroke, and ischemic heart disease. The second chapter of this thesis describes the construction of genetic linkage maps of the parents of a segregating population derived from the cross between the cultivars ‘Prima’ and ‘Fiesta’. For this purpose the already available linkage maps, as described in this chapter, were made denser by inclusion of 240 Diversity Array Technology (DArT) markers. Thus the total number of markers for ‘Prima’ and ‘Fiesta’ integrated map reached to 820. DArT-markers are hybridization based dominant DNA-markers. DArT provides a high-throughput whole genome genotyping platform for the detection and scoring of hundreds of polymorphic loci without any need for prior sequence information. This is the first report on DArT in horticultural trees. Genetic mapping of DArT markers in two mapping populations and their integration with other marker types showed that DArT is a powerful high throughput method for obtaining accurate and reproducible marker data, at low cost per data point. This method appears to be suitable for aligning the genetic maps of different segregating populations. Sequencing of the marker clones showed that they are significantly enriched for low copy, gene rich regions. Chapter 3 describes metabolic diversity of Malus. Wild germplasm was compared to advanced breeding selections and to the segregating F1 population from the cross between the cultivars ‘Prima’ and ‘Fiesta’. The metabolic profiles were analyzed by means of liquid chromatography-mass spectrometry (LC-MS). LC-MS is an analytical chemistry technique that combines the physical separation capabilities of liquid chromatography with the mass analysis capabilities of mass spectrometry. This resulted in the detection of 418 putative metabolites in the peel and 254 in the flesh. Fruits from 23 wild species, eight advanced selections and the segregating F1 population were analyzed. The data were subjected to Principle Components Analysis (PCA). Variance analysis of the first PC showed that genetic variation accounted for 96.6 % in peel and 97.4 % in flesh of the total metabolic variation. Technical variation accounted for 1.4 % and 0.8%, while environmental variation accounted for 2.0% and 1.8% in peel and flesh respectively. The genetic variation between wild genotypes was very large, compared to the advanced selections and the F1 progeny. Only 8 % of the genetic variation of the first principle component was captured by the advanced selections. This indicates strong genetic erosion during breeding. This genetic erosion was mainly caused by reduction of the levels of several flavonoids including catechin, epicatechin and procyanidins. PCA of the F1 progeny of the ‘Prima’ x ‘Fiesta’ cross showed a clear 3:1 Mendelian segregation of metabolites. These metabolites were 4.2 fold less in both peel and flesh in progeny that had inherited the recessive alleles of a gene at the top of Linkage Group16 (LG16) from the heterozygous parents. We found a separate group of 11 metabolites in peel and 12 in flesh. These metabolites were putatively identified as glycosylated forms of b-glycols: R-octane-1, 3-diol and its unsaturated form R-5-(Z)-octene-1, 3-diol which have a potential role in controlling infection by microorganisms and influence the aroma of some ciders. The levels of these metabolites were up to 50 fold more abundant in some progeny compared to both parents. Genetic mapping showed that this strong increase was caused by one locus at the top of LG8, in progeny that had inherited only the recessive alleles of that locus from the heterozygous parents. This research illustrates not only the strong genetic erosion in apple breeding regarding metabolic diversity, and strong reduction of flavonoids in some progeny, but also shows that inbreeding can lead to a strong increase of metabolites that were present at much lower levels in both parents and advanced selections. This loss and gain of metabolites was especially observed in case of accumulation of recessive alleles during inbreeding. The genetic factors controlling metabolite composition were studied in more detail in Chapter 4. We investigated the genetic factors of the quantitative variation of these potentially beneficial compounds (Chapter 3, 4), by combining the genetic maps (Chapter 2) with the LC-MS data for thesegregating F1 population from the cross ‘Prima’ x ‘Fiesta’. This resulted into metabolite quantitative trait loci (mQTLs). When using the software MetaNetwork, 669 significant mQTLs were detected: 488 in the peel and 181 mQTLs in the flesh. Four linkage groups (LGs) i.e. LG1, LG8, LG13 and LG16 were found to contain mQTL hotspots, mainly regulating metabolites that belong to the phenylpropanoid pathway. These include various metabolites i.e. sinapate hexoside, coumaroyl hexoside, phloridzin, quinic acids, phenolic esters, kaempferol glycosides, quercetin glycosides, cyanidin pnetoside, flavan-3-ols (catechin, epicatechin), and procyanidins. The genetics of annotated metabolites was studied in more detail using MapQTL®. It was found that quercetin conjugates had mQTLs on LG1 and LG13. The most important mQTL hotspot with the largest number of metabolites was, however, detected at the top of LG16: mQTLs for 32 peel-related and 17 flesh-related phenolic compounds. The metabolites that mapped in the mQTL hotspot on LG16 all belong to the phenylpropanoid pathway of secondary metabolites. These compounds showed a monogenic Mendelian inheritance in a 3:1 segregation ratio. Procyanidins dimer II was used as a representative of the numerous compounds that mapped at the LG16 mQTL hotspot. By means of graphical genotyping of this monogenic trait, a genetic window could be made in which the gene that caused the mQTL hotspot should reside. We located structural genes involved in the phenolic biosynthetic pathway, using the genetic map together with the published whole genome sequence of apple. The structural gene leucoanthocyanidin reductase (MdLAR1) was detected in the mQTL hotspot window on LG16, as were seven transcription factor genes. To our knowledge, this is the first time that a QTL analysis was performed on such a high number of metabolites in an outbreeding plant species. The expression of the candidate genes found in the mQTL window on LG16 was studied and discussed in Chapter 5. qPCR was used for this purpose and it was found that the expression of only the structural gene MdLAR1 was strongly positively correlated with the metabolite procyanidin dimer II content. Neither the expression profiles of other structural genes of the phenylpropanoid pathway, the transcription factor genes at the mQTL hotspot, nor of transcription factor genes outside the mQTLs hotspot, showed any significant correlation with the procyanidin dimmer II content that mapped at the mQTL hotpot. This indicates that MdLAR1 was the gene, which caused this mQTL hotspot (Chapter 5). The progeny that had inherited one or two copies of the dominant alleles (Mm, MM) showed on the average a 4.4 and 11.8 fold higher expression level of MdLAR1 respectively, compared to the progeny that had inherited the recessive alleles only (mm). This led to a 4.0 fold increase of procyanidin dimer II level at the ripe stage. Strikingly, at the mQTL hotspot at the top of LG16, there is also a locus that controls acidity of the ripe fruits. However, the dominant alleles for acidity appeared to be in repulsion to the dominant alleles for high metabolite levels (Chapter 6). This shows that acidity is controlled by another gene than the metabolite levels. The combination of the genetic position based on the whole apple genome sequence, annotation of potential genes, and expression profiling indicated that the malic acid transporter gene MdALMT2 was responsible for the clear differences in malic acid content and pH in mature apple fruits of the segregating F1 population. The genetic inheritance of at least one dominant allele (MaMa/Mama) of this gene sufficed for a three-fold increase of the malic acid concentration and a reduction of the pH from 4 to 3 in ripe apples, compared to the presence of only the lower expressed recessive allele (mama). This malic acid transporter gene is located at the top of LG16.Malic acid is the predominant organic acid associated with the pH in apple fruits. It is synthesized in the cytoplasm and transported into the cell vacuole. The concentration of malic acid in the cell vacuole determines the pH of the cell. pH is very important for the overall taste of many fruits, including apple, and has profound effects on the organoleptic quality of apples. The pH of mature apples was genetically mapped on LG16 in the segregating population from the cross ‘Prima’ x ‘Fiesta’. To our knowledge, this is the first time that the genetic segregation of the pH in apple is assigned to a specific gene. Further, this gene has not been reported yet in conjunction to pH of apples or other fruits. After cloning of the MdALMT2 gene, it can be used for, proof of principle, influencing the acidic of existing varieties either by silencing this gene in more acidic cultivars or by inserting this gene into the low acidic cultivars. Another step would be to develop an allele specific molecular marker for selection (Marker Assisted Selection) of the acidity of fruits already at seedling stage, five years before the trees carry fruits. In another study, a dominantly mutated allele of the transcription factor gene MdMYB10,including its upstream promoter, coding region and terminator sequence, was introduced by transformation into apple, strawberry and potato plants. The dominantly inherited mutant allele of MdMYB10 from apple induces anthocyanin production throughout the plant, also at the early stage after transformation. The aim was to determine whether MdMYB10 could be used as a visible selectable marker for plant transformation as an alternative to chemically selectable markers, such as kanamycin resistance. After transformation, the color of calli, shoots and well-growing plants were evaluated. Red and green shoots were harvested from apple explants and examined for the presence of the MdMYB10 gene by PCR analysis. Red shoots of apple explants always contained the MdMYB10 gene but not all MdMYB10 containing shoots were red. Strawberry plants transformed with the MdMYB10 gene showed anthocyanin accumulation in leaves and roots. No visible accumulation of anthocyanin could be observed in potato plants grown in vitro, even the ones carrying the MdMYB10 gene. However, acid methanol extracts of potato shoots or roots carrying the MdMYB10 gene contained up to four times higher anthocyanin content than control plants. Therefore, anthocyanin production as a result of the dominant MdMYB1010 gene can be used as a selectable marker for apple, strawberry and potato transformation, replacing kanamycin resistance gene such as nptII. We reported this MdMYB10 as a cisgenic selectable marker gene for apple transformation (Chapter 7). The results from all experimental chapters have been discussed in a broader sense in the general discussion (Chapter 8). The future prospectives and potential challenges in the genetical metabolomics are also highlighted. The approaches we developed in the current thesis could be used not only for developing potentially a more healthy and improved apple but can also be applied for the genetical metabolomics studies in other important crops. </p
Towards consumer-friendly cisgenic strawberries which are less susceptible to Botrytis cinerea
No full textThis thesis describes the development of genetically modified (GM) strawberries which are less susceptible to fruit rot caused by the fungus Botrytis cinerea. To achieve Botrytis resistance, a polygalacuronase inhibiting protein (PGIP) gene has been isolation from strawberry and was characterised. It was shown that the natural expression of this gene is probably insufficient to restrict colonisation of the fruit by Botrytis . Next to this, the activity of different promoter sequences has been tested in strawberry. A fruit-specific promoter from the strawberry Expansin gene was selected as a suitable candidate to provide enhanced expression of the strawberry PGIP gene in strawberry fruits. Furthermore, the development of a transformation method is described which enables the elimination of selection genes. Selection genes are essential for the production of transgenic plants, but have no function after the GM plants have been produced. After introduction of the combined Expansin promoter and PGIP gene in GM strawberries and subsequent removal of the selection gene, the ultimate GM strawberry plant will contain no gene sequences from foreign species. For this reason, these plants are called cisgenic rather than transgenic. The improved acceptance of such cisgenic strawberries by consumers and producers of strawberries is discussed
Biotechnology for cocoa pod borer resistance in cocoa
No full textThe cocoa tree ( Theobroma cacao L.) produces the beans that are the source of cacao, the basis for chocolate production, and an important commodity crop in South America, West Africa, and Southeast Asia.Cocoa Pod Borer (CPB,( Conopomorpha cramerella)has been the single most important limiting factor for cacao production in Southeast Asia.So far, there has been no single cost effective and environmentally safe way to control this pest.This thesis describes the first steps in a biotechnological approach to the control of CPB through the production of transgenic cocoa trees, which produce an insecticidal toxin from Bacillus thuringiensis (Bt) in their pod wall, making them resistant to CPB. Selection of a CPB-active toxin, optimization of its expression, making the expression tissue-specific and optimization of cocoa transformation and regeneration are described. Moreover, the isolation of two transcription factor genes involved in flower timing and architecture is described
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