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Investigations of potato (Solanum tuberosum) have been hampered by its complicated genetics and high genetic load. This dissertation applies genome reduction techniques to investigate a broad swath of genomic and physiological phenomena. It begins with the presentation and evaluation of a protocol to characterize endoreduplication within potato tubers, demonstrating substantial variation between tissue types and among wild species which may facilitate research into the genesis and growth of these starchy underground stems. Next, we transitioned to explore the distribution and consequences of a specific class of transposable element, Miniature Inverted Transposable Elements (MITEs), showing that they comprise approximately 5% of the potato genome, occur more frequently in genes with stress-related functions, and may be associated with changes, especially decreases, in gene expression. We then combined homology and sparsity based approaches to predict recent MITE activity, identifying five families as especially active. Finally, we expose the gene underlying the potato flower color locus, a homolog of AN2, while showing the effects it exerts on the flavonoid biosynthesis and fruit ripening pathways. This region was shown to be particularly dynamic, replete with MITEs and structural variants which we hypothesize to be the ultimate cause of differences in AN2 expression within the germplasm we examined. While the separate topics of this dissertation are quite disparate, each addresses an important topic in potato genetics, the in-depth study of which is only possible through the utilization of genomic reduction approaches to acquire homozygous genotypes for study and currently available genomic resources. / Ph. D. / Despite their humble appearance and routine consumption, potatoes have a complex genetic structure and a life cycle capable of both sexual reproduction through flowers, fruit and seed, and asexual reproduction through the tubers which also comprise the edible product. From an agronomic perspective, one of the most important qualities of a potato tuber is size, a feature influenced by genetics and environment. Cell-to-cell variation for the amount of DNA per cell, one component that influences tuber size, is known to occur, yet our ability to measure DNA content in starchy tuber cells has been obscured by debris generated through routine preparation techniques. We present and evaluate a new method for measuring the DNA content of potato tuber cells, which provides reliable results across a range of different potato varieties and species. ‘Jumping genes’ also known as transposons, first reported in maize but now known to occur in most advanced plant and animal species, have been found to comprise ~5% of the recently sequenced potato genome. We show that a particular class of transposons is more likely to occur adjacent or actually in certain types of genes, such as those which confer resistance to disease, where they may have meaningful effects on how those genes operate. We then proceed to predict the current activity of the various families of these jumping genes to understand how they continue to alter the genetic landscape of potato. Finally we identify a particular gene which dictates flower color in potato (purple vs. white). We demonstrate that several transposons occur in some forms of the flower color gene. Originally we hypothesized that transposons were associated with the turning off of the purple flower color form; however, on closer examination, we could express the white flower form in transgenic plants that were originally white-flowered and convert them to have purple flowers, demonstrating that even the white flower form was functional. While the separate topics of this dissertation are quite disparate, each addresses an important topic in potato genetics, the in-depth study of which is only possible through the availability of the special strains of potatoes with reduced chromosome number and the publication of the potato genome.

Thesis (PhD)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Anthocyanins are naturally occurring pigments responsible for the colour of many natural products, including grapes and wine. These pigments are important to the food industry and have been recognised for their nutritional value since they play an important role in the reduced risk of various chronic diseases in humans. Anthocyanins also play an important role in the aesthetic perception and quality of red wine. However, due to the large structural diversity of grape-derived anthocyanins and the many derivatives formed from these during wine ageing, the accurate analysis of wine pigments is extremely challenging. Reversed-phase liquid chromatography (RP-LC) is mostly used for anthocyanin analysis, although the technique often provides insufficient resolving power for complex mixtures of anthocyanins. In addition, the lack of commercially available standards and identical mass spectral characteristics hampers identification of these compounds. The coupling of multiple orthogonal separation systems in comprehensive 2-dimensional liquid chromatography (LC×LC) offers a more powerful approach for the separation of complex mixtures. The current work therefore focussed on exploring the potential of LC×LC for the improved analysis of anthocyanins and derived pigments in natural products and wine. The first part of this work focussed on developing a hydrophilic interaction chromatography (HILIC) method as an alternative to RP-LC for the anthocyanin analysis. Following extensive optimisation, the method proved suitable for the analysis of a diverse range of anthocyanins in natural products. Significantly, it also showed alternative selectivity compared to RP-LC. The optimised HILIC method was then used in combination with RP-LC to develop an off-line LC×LC approach for anthocyanins. For this purpose, half-minute fractions of the HILIC effluent were collected and reinjected onto a RP-LC column. The off-line HILIC×RP-LC method demonstrated exceptionally high resolving power, as measured in terms of the practical peak capacity, with many compounds separated in two dimensions that co-eluted in 1-dimensional HPLC. Interestingly, group-type separation was also observed based on the degree and/or nature of glycosylation and acylation of anthocyanins. In the final part of the work, a systematic approach was used for the development and optimisation of and on-line HILIC×RP-LC method by using a 10-port switching valve to automatically transfer fractions between the two columns. This method was then coupled to high resolution mass spectrometry (MS) to allow the detailed investigation of anthocyanins and derived products in wine. Ninety four pigments were identified in one- and six-year old Pinotage wines based on HILIC×RPLC separation in combination with accurate mass MS data and fragmentation information. Significant differences in especially the content of derived pigments were observed between the wines. In summary, the methods developed in this work provide the means to improve anthocyanin analysis, and therefore also show promise for the detailed investigation of these important compounds and their alteration in natural products and their derived commodities. / AFRIKAANSE OPSOMMING: Antosianiene is natuurlike pigmente wat verantwoordelik is vir die kleur van baie natuurlike produkte, insluitende dié van druiwe en wyn. Hierdie pigmente is belangrik vir die voedsel industrie en word gereken vir hul voedingswaarde aangesien hulle 'n belangrike rol speel in die verlaagde risiko van verskeie chroniese siektes onder die mens. Antosianiene speel ook 'n belangrike rol in die estetiese persepsie en kwaliteit van rooiwyn. Desnieteenstaande, as gevolg van die groot strukturele diversiteit van druifgeproduseerde antosianiene en die vele chemiese afgeleides wat uit hulle gevorm kan word tydens wyn bereiding en veroudering, is die akkurate analise van natuurlike wyn-pigmente uiters uitdagend. Omgekeerde-fase vloeistofchromatografie (RP-LC) word meestal gebruik vir die analise van antosianiene. Dié tegniek bied egter dikwels onvoldoende skeidingsvermoë vir komplekse mengsels van antosianiene en verwante molekules. Verder belemmer die onbeskikbaarheid van kommersiële standaarde en identiese massa spektrale eienskappe die identifikasie van hierdie verbindings. Die kombinasie van verskillende ortogonale skeidings meganismes in omvattende 2- dimensionele vloeistofchromatografie (LC×LC) bied egter 'n baie kragtiger benadering vir die skeiding van komplekse mengsels. Die huidige werk fokus dus op die ontginning van die potensiaal van LC×LC vir die verbeterde ontleding van antosianiene en verwante afgeleide pigmente in natuurlike produkte en wyn. Die eerste deel van hierdie werk het gefokus op die ontwikkeling van 'n hidrofiliese interaksie chromatografiese (HILIC) metode as ʼn alternatief vir RP-LC analise van antosianiene. Na uitgebreide optimisering, is gevind dat die metode geskik is vir die ontleding van 'n verskeidenheid van antosianiene in natuurlike produkte. Van groot belang is dat dit ook alternatiewe selektiwiteit in vergelyking met RP-LC demonstreer. Hierdie geoptimiseerde HILIC metode word dan voorts gebruik in kombinasie met RP-LC vir die ontwikkeling van ʼn af-lyn LC×LC benadering vir die analise van antosianiene. Hiervoor is half-minuut fraksies van die HILIC uitvloei opgevang en her-ingespuit op 'n RP-LC kolom. Dié af-lyn HILIC×RP-LC metode toon buitengewoon hoë skeidingsvermoë, gemeet in terme van die bereikbare praktiese piek kapasiteit, met baie verbindings wat geskei is in die twee dimensies wat saam elueer in 1-dimensionele HPLC. Interessant genoeg is groep-tipe skeiding ook waargeneem gebaseer op die graad en / of aard van glukosilasie en asilering van die antosianiene. In die laaste deel van die werk, is 'n sistematiese benadering gevolg vir die ontwikkeling en optimisering van ʼn aan-lyn HILIC×RP-LC deur gebruik te maak van 'n 10-poort oorskakelingsklep wat fraksies outomaties oordra tussen die twee kolomme. Die bogenoemde metode is ook verder gekoppel aan hoë resolusie massaspektrometrie (HR-MS) om ʼn gedetailleerde ondersoek van antosianiene en hulle afgeleide verbindings in wyn moontlik te maak. Vier en negentig pigmente is in een- en ses jaar oue Pinotage wyne geïdentifiseer gebaseer op HILIC×RP-LC skeiding in kombinasie met akkurate massa MS data en fragmentasie inligting. Beduidende verskille in veral die inhoud van antosianien-afgeleide pigmente is tussen die wyne waargeneem. Ter samevatting, die metodes ontwikkel in hierdie werk baan die weg om antosianien ontleding te verbeter en stel gevolglik die moontlikheid van selfs meer gedetailleerde studies van hierdie belangrike verbindings in natuurlike produkte in die vooruitsig.

Master of Science / Department of Food Science / Weiqun Wang / We previously selected a purple-fleshed sweetpotato p40 clone that has been shown to protect against colorectal cancer in a murine model. This study is to identify anthocyanins by using HPLC/MS-MS and assess the stability during various coking conditions. P40 possesses a high content of anthocyanins up to 13 mg/g dry matter. Total 12 acylated anthocyanins with caffeic, ferulic, and p-hydrobenzoic acid have been identified on either cyanidin or peonidin bases. The top three major anthocyanins are cyanidin 3-caffeoyl-p-hydroxybenzoyl sophoroside-5-glucoside, peonidin 3-caffeoyl sophoroside-5-glucoside, and cyanidin 3-(6'' -caffeoyl-6''-feruloylsophoroside)-5-glucoside, which account for an half of the total anthocyanin contents. Seven non-, mono-, or di-acylated cyanidin species and five mono- or di-acylated peonidin species contribute for 69% and 31% of total anthocyanins, respectively. Over 80% of total anthocyanins measured by acid hydrolysis were cyanidin derivatives. Therefore, as a cyanidin-predominated variety, p40 is unique when compared with other reported purple-fleshed sweetpotatoes that usually contain more peonidin than cyanidin. While baking does not impact overall contents of anthocyanins, steaming, high pressure cooking, microwaving, and frying significantly reduce 20% of total anthocyanin contents. Mono-acylated anthocyanins show a higher resistance against heat than di- and non-acylated. Among of which, cyaniding 3-p-hydroxybenzoylsophoroside-5-glucoside exhibits the best thermal stability. Better understanding of dietary anthocyanins and their stabilities may lead to the development of a functional anthocyanin-enriched sweetpotato product for health benefits.

Doctor of Philosophy / Department of Human Nutrition / Weiqun Wang / Anthocyanins are flavonoid pigments that account for the purple color in many plant foods. It has been investigated that anthocyanins’ predominant occurrences in human diet and their health beneficial activities such as antioxidant, anti-inflammatory, and anti-carcinogenetic effects. Based on those scientific evidences, anthocyanins are now recognized as potential therapeutic compounds. Particularly, the chemopreventive effect of anthocyanins has been widely studied by many researchers in nutrition. However, their bioactivities are diverse due to different chemical structures of anthocyanins from different sources. In this study, we discuss the chemopreventive activity of anthocyanins from purple sweet potato. Previously, we selected a purple-fleshed sweetpotato clone, P40, crossbred seeds obtained from the International Potato Center in Lima, Peru. We hypothesized that anthocyanins enriched P40 may provide health beneficial activities in cancer prevention. For the first part of this study, we analyzed nutrient compositions, dietary fiber content, anthocyanins contents, total phenolics contents and total antioxidant activity. Even thought P40 presents similar composition and amount of nutrients with the control cultivars, white-fleshed O’Henry and yellow-fleshed NC Japanese, HPLC-MS analysis confirmed that it possesses much higher anthocyanin content even up to 7.5g/kg dry matter. Also, dietary fiber, particularly soluble dietary fiber content, total phenolics content, and total antioxidant capacity of P40 were significantly higher. For the second part of the study, we tested the potential anticancer characteristic of P40 cultivar in human colonic SW480 cancer cells and in azoxymethane-induced aberrant crypt foci in mice. Treatment with 0 – 40 μM of peonidin-3-glucoside or P40 extract containing corresponding amount of anthocyanins resulted in inhibition of cell growth in a dose-dependent manner. Interestingly, even though the patterns of growth inhibition were similar in the two treatment groups, the cells treated with P40 extract tend to survive significantly less than those treated with peonidin-3-glucoside. Cell cycle analysis confirmed that the growth inhibition was not due to cytotoxicity, but cytostatic mechanism with increased number at the G1 phase of the cell cycle. The cell cycle arrest was also significantly correlated with the anthocyanin contents in P40 cultivar when compared with the white-fleshed O’Henry and yellow-fleshed NC Japanese controls. After Azoxymethane (AOM) or saline injected mice were fed basal AIN-93M diet or diets containing 10~30% of P40, 20% O’Henry or 20% NC Japanese for 6 weeks, aberrant crypt foci (ACF) multiplicity was significantly inhibited by 10~30% P40 diet. Imunohistochemistry results of colonic mucosa showed that the expression level of apoptosis marker, caspase-3, was significantly induced in the mice treated with 10~20% P40 diet. Also, PCNA expression level, which is proliferation marker, was significantly inhibited by the 30% P40 diet. These findings indicated that consuming a purple sweet potato, P40, may prevent colon cancer by modulating antioxidant status, inducing apoptosis, and reducing cell proliferation.

O interesse em buscar fontes naturais para compostos antioxidantes aumentou os estudos sobre frutas, principalmente referente a presença das antocianinas. O camu-camu é um fruto oriundo da região amazônica que vem ganhando destaque nas indústrias de cosméticos, medicamento e alimentos por ser uma fonte rica em antocianinas, ácido ascórbico e minerais. O objetivo do trabalho foi avaliar a incorporação das antocianinas presentes na casca do camu-camu em filmes de desintegração oral à base de gelatina e amido. Os filmes foram produzidos pela técnica casting a partir de blendas de gelatina e amido, com diferentes concentrações (0:100; 25:75; 50:50; 75:25; 100:0), plastificante sorbitol e com a adição de diferentes concentrações do extrato aquoso da casca de camu-camu (0, 10, 15 e 20 g/100g solução filmogênica). Foram analisadas as propriedades mecânicas pelo do teste de tração, cor e opacidade, ângulo de contato, tempo de desintegração, FTIR e MEV. Nos filmes com adição do extrato de camu-camu foram realizadas, adicionalmente, análises de pH de superfície, antocianinas totais e estudo da estabilidade. Com relação as propriedades mecânicas, verificou-se que as alterações nas concentrações de amido e gelatina (AMI:GEL) apresentou diferenças significativas para todos os parâmetros estudados (tensão na ruptura, elongação na ruptura e módulo elástico). O aumento da concentração de gelatina elevou a tensão de ruptura e o aumento da concentração de amido produziu filmes mais frágeis e quebradiços. A adição do extrato apresentou diminuição na tensão de ruptura e no módulo elástico, e aumento na elongação dos filmes. Das análises de cor e opacidade observou-se que com maiores concentrações de amido e do extrato, aumentam os valores da luminosidade, da croma a* e da opacidade, enquanto para a croma b* ocorre uma diminuição. Os ângulos de contato dos filmes diminuíram significativamente com o aumento da concentração de amido e aumentaram com maiores concentrações de extrato. Os filmes com extrato, apresentaram tempo de desintegração inferior, sendo observados tempos maiores com o aumento da concentração de gelatina. Com relação a estabilidade, os filmes apresentaram elevada perda das quantidades de antocianinas para todas as formulações analisadas. Todos os filmes analisados apresentaram incorporação eficiente do extrato, dessa forma, conclui-se que a produção de filmes de desintegração oral com extrato de camu-camu é uma alternativa eficiente para o aproveitamento dos compostos funcionais presentes da casca. / The interest in finding natural sources for antioxidants increased studies on fruit, especially regarding the presence of anthocyanins. Camu-camu is a fruit originating in the Amazon region that is gaining prominence in the cosmetics, medicine and food to be a rich source of anthocyanins, ascorbic acid and minerals. The objective was to evaluate the incorporation of anthocyanins present in the shell of the camu-camu in films orally disintegrating based gelatin and starch. The films were produced by casting from blends of gelatin and starch with different concentrations (0: 100, 25:75, 50:50, 75:25, 100: 0), plasticizer sorbitol and with the addition of different concentrations the aqueous extract of camu-camu peel (0, 10,15 and 20 g / 100 g film solution). The mechanical properties were analyzed using the tensile test, color, and opacity, contact angle, disintegration time, FTIR and SEM. In the films with the addition of camu-camu extract were performed, additionally, surface pH analysis, anthocyanins and stability. Regarding the mechanical properties, it has been found that changes in starch and gelatin concentrations (AMI:GEL) showed significant differences for all the studied parameters (stress, elongation and elastic modulus). The increase in the gelatin concentration to increased breakdown voltage and increased concentration of starch produced more fragile and brittle films. The addition of the extract showed a decrease in breakdown voltage and elastic modulus, and increased elongation of the films. Analysis of the color and opacity was observed that with higher concentrations of starch and the extract, increases the brightness values of a* chroma and opacity as to chroma b* is decreased. The contact angles of the films decreased significantly with increasing starch concentration and increased with higher extract concentrations. The films to extract showed disintegration time lower, longer times being observed with increasing concentration of gelatin. Regarding stability, the films showed high loss of the amounts of anthocyanins for all formulations analyzed. All samples show efficient incorporation films extract thus concluded that the production of the orally disintegrable films with camu-camu extract is an effective alternative to the use of compounds of the functional shell.