Auswahl der wissenschaftlichen Literatur zum Thema „Grapes Genetics“

Two morphologically distinct types of grapes belonging to the subgenera Euvitis and Muscadinia in the genus Vitis are cultivated in the United States. The former is commonly called bunch grapes while the latter is usually called muscadine. Genetic diversity among these grapes was investigated using RAPD markers. Sixteen grape cultivars, with parentage including V. rotundifolia Michx., V. vinifera L., and several American Vitis species, were used for the RAPD analysis. A total of 156 RAPD markers was produced from 19 random primers, over 90% of which was polymorphic among the muscadine and the bunch grapes. Polymorphisms were lower within each subgenus. Relationships between these two subgenera were estimated based on band-sharing and cluster analysis. The average genetic distance between the bunch and the muscadine grape cultivars was 0.45. The results based on DNA analysis agree with isozyme data obtained from a separate study, which demonstrated that muscadine grapes share very few common alleles with American bunch grapes and European grapes.

Reduced expression of MADS-box gene AGAMOUS-LIKE11 (VviAGL11) is responsible for stenospermocarpic seedlessness in bunch grapes. This study is aimed to characterize the VviAGL11 orthologous gene (VroAGL11) in native muscadine grapes (Vitis rotundifolia) at the molecular level and analyze its divergence from other plants. The VroAGL11 transcripts were found in all muscadine cultivars tested and highly expressed in berries while barely detectable in leaves. RT-PCR and sequencing of predicted ORFs from diverse grape species showed that AGL11 transcripts were conservatively spliced. The encoded VroAGL11 protein contains highly conserved MADS-MEF2-like domain, MADS domain, K box, putative phosphorylation site and two sumoylation motifs. The muscadine VroAGL11 proteins are almost identical (99%) to that of seeded bunch cultivar, Chardonnay, except in one amino acid (A79G), but differs from mutant protein of seedless bunch grape, Sultanina, in two amino acids, R197L and T210A. Phylogenetic analysis showed that AGL11 gene of muscadine and other Vitis species formed a separate clade than that of other eudicots and monocots. Muscadine grape cultivar “Jane Bell” containing the highest percentage of seed content in berry (7.2% of berry weight) had the highest VroAGL11 expression, but almost none to nominal expression in seedless cultivars Fry Seedless (muscadine) and Reliance Seedless (bunch). These findings suggest that VroAGL11 gene controls the seed morphogenesis in muscadine grapes like in bunch grape and can be manipulated to induce stenospermocarpic seedlessness using gene editing technology.

Sugar accumulation is a critical event during grape berry ripening that determines the grape market values. Berry cells are highly dependent on sugar transporters to mediate cross-membrane transport. However, the role of sugar transporters in improving sugar accumulation in berries is not well established in grapes. Herein we report that a Sugars Will Eventually be Exported Transporter (SWEET), that is, VvSWEET10, was strongly expressed at the onset of ripening (véraison) and can improve grape sugar content. VvSWEET10 encodes a plasma membrane-localized transporter, and the heterologous expression of VvSWEET10 indicates that VvSWEET10 is a hexose-affinity transporter and has a broad spectrum of sugar transport functions. VvSWEET10 overexpression in grapevine calli and tomatoes increased the glucose, fructose, and total sugar levels significantly. The RNA sequencing results of grapevine transgenic calli showed that many sugar transporter genes and invertase genes were upregulated and suggest that VvSWEET10 may mediate sugar accumulation. These findings elucidated the role of VvSWEET10 in sugar accumulation and will be beneficial for the improvement of grape berry quality in the future.

Abstract A factorial study was designed to examine the effects of fruit maturities, juice extraction temperatures, storage time, and storage temperature on the quality of ‘Concord’ grape (Vitis labrusca L.) juice. Mature grapes produced juice with superior sensory quality and better objective color initially than less-mature grapes, but juice from grapes at both maturities had similar sensory quality after 18 months. The high extraction temperature (99°C) resulted in juice with better color initially than extraction at 60°, but juice extracted at 85° or 99° resulted in greater browning and loss of total anthocyanins during storage than extraction at 60°. High storage temperature (35°) greatly accelerated quality loss. Sensory color ratings correlated better with a ratio of absorbances at 520/430 nm (r = 0.95) and a/b (r = 0.92) than other objective color measurements.

Four new simple sequence repeat (SSR) loci (designated VVMD5, VVMD6, VVMD7, and VVMD8) were characterized in grape and analyzed by silver staining in 77 cultivars of Vitis vinifera. Amplification products ranged in size from 141 to 263 base pairs (bp). The number of alleles observed per locus ranged from 5 to 11 and the number of diploid genotypes per locus ranged from 13 to 27. At each locus at least 75% of the cultivars were heterozygous. Alleles differing in length by only 1 bp could be distinguished by silver staining, and size estimates were within 1 or 2 bp, depending on the locus, of those obtained by fluorescence detection at previously reported loci. Allele frequencies were generally similar in wine grapes and table grapes, with some exceptions. Some alleles were found only in one of the two groups of cultivars. All 77 cultivars were distinguished by the four loci with the exception of four wine grapes considered to be somatic variants of the same cultivar, 'Pinot noir', 'Pinot gris', 'Pinot blanc', and 'Meunier'; two table grapes that are known to be synonymous, 'Keshmesh' and 'Thompson Seedless'; and three table grapes, 'Dattier', 'Rhazaki Arhanon', and 'Markandi', the first two of which have been suggested to be synonymous. Although the high polymorphism at grape SSR loci suggests that very few loci would theoretically be needed to separate all cultivars, the economic and legal significance of grape variety identification requires the increased resolution that can be provided by a larger number of loci. The ease with which SSR markers and data can be shared internationally should encourage their broad use, which will in turn increase the power of these markers for both identification and genetic analysis of grape. Key words : grape, Vitis, microsatellite, simple sequence repeat, DNA typing, identification.

As a polymicrobial disease, sour rot decreases grape berry yield and wine quality. The diversity of microbial communities in sour rot-affected grapes depends on the cultivation site, but the microbes responsible for this disease in eastern coastal China, has not been reported. To identify the microbes that cause sour grape rot in this important grape-producing region, the diversity and abundance of bacteria and fungi were assessed by metagenomic analysis and cultivation-dependent techniques. A total of 15 bacteria and 10 fungi were isolated from sour rot-affected grapes. High-throughput sequencing of PCR-amplicons generated from diseased grapes revealed 1343 OTUs of bacteria and 1038 OTUs of fungi. Proteobacteria and Firmicutes were dominant phyla among the 19 bacterial phyla identified. Ascomycota was the dominant fungal phylum and the fungi Issatchenkia terricola, Colletotrichum viniferum, Hanseniaspora vineae, Saprochaete gigas, and Candida diversa represented the vast majority ofmicrobial species associated with sour rot-affected grapes. An in vitro spoilage assay confirmed that four of the isolated bacteria strains (two Cronobacter species, Serratia marcescens and Lysinibacillus fusiformis) and five of the isolated fungi strains (three Aspergillus species, Alternaria tenuissima, and Fusarium proliferatum) spoiled grapes. These microorganisms, which appear responsible for spoiling grapes in eastern China, appear closely related to microbes that cause this plant disease around the world.

Abstract Eight or 9 commercial cultivars from each of 4 grape classes were inoculated with the black rot fungus (Guignardia bidwellii f. sp. euvitis and f. sp. muscadinii) of bunch and muscadine grapes, respectively. The 4 classes consisted of 3 bunch grape classes (Vitis vinifera L., American hybrids, and French hybrids) and 1 muscadine class (V. rotundifolia Michx). Resistance was determined by leaf and petiole infection ratings (0−4) and average leaf and petiole lesion size (mm). Inoculation with Guignardia bidwellii f. sp. euvitis resulted in significantly larger average leaf lesion size on Vitis vinifera than on American and French hybrids (4.3 vs. 3.5). American hybrids had significantly higher (3.3 vs 2.3) leaf disease rating than French hybrids. Vitis rotundifolia received significantly lower disease rating then the 3 bunch grapes classes (0.4 vs. 2.9). A hypersensitive reaction was observed on leaves of most V. rotundifolia cultivars. Inoculation with Guignardia bidwellii f. sp. muscadinii resulted in significant differences in leaf infection ratings between French and American hybrids (0.8 vs. 1.6) and between V. rotundifolia and the 3 bunch grape classes (3.1 vs. 1.2). There were significant pathogen form × grape class and pathogen form × cultivar within grape class interactions.

Abstract Hand- and machine-harvested ‘Stover’ (a white Euvitis hybrid) and ‘Noble’ (a red Vitis rotundofolia cultivar) grapes were either treated with 100 mg SO2/kg (applied as potassium metabisulfite, K2S2O5) or not treated and held at ambient temperature (≈25°C) for 0, 24, and 48 hr after harvest. Raw fruit and wine quality from each treatment were evaluated. ‘Noble’ grapes of all treatments held for 24 hr had developed signs of microbial spoilage, and the machine-harvested grapes held without SO2 had the most. However, only the machine-harvested ‘Stover’ grapes held without SO2 had began to deteriorate during 24 hr. Grapes of both cultivars had deteriorated after 48 hr, but SO2 limited the deterioration of both machine- and hand-harvested grapes. In general, machine-harvested grapes deteriorated to a greater extent than hand-harvested grapes. Wines made from ‘Noble’ grapes that had been held for 0 or 24 hr had similar quality, but machine-harvested ‘Stover’ grapes held for 0 to 24 hr without SO2 produced a poorer wine than the other treatments. Wines from machine-harvested grapes held for 48 hr had poorer quality than wines from hand-harvested grapes. Postharvest additions of SO2 improved the quality of wine from machine-harvested but not hand-harvested fruit held for 48 hr.

Progress in grape breeding requires the exploitation of genetic variation among market classes, races and gene pools. Wild grapevines (Vitis vinifera ssp. sylvestris) are being endangered in their natural habitats and high priorities should be given to the wild germplasm. Turkey is one of the richest sources of wild grapevine and they mostly grown on forest trees on river basin. The present study was carried out to determine the amount of genetic variation and the degree of relatedness among 23 wild grape genotypes using 17 simple-sequence-repeat markers (SSR). Two international grape cultivars, Cabernet Sauvignon and Merlot are also included study. Number of alleles per locus of the 17 Simple Sequence Repeat (SSR) markers ranged from 3.0 to 14.0 and a total of 162 alleles with an average of 9.53 alleles per locus. The average expected and observed heterozygosity values were 0.773 and 0.781, respectively, which exhibited high level of genetic diversity in the wild grape germplasm. The unweighted pair group method with arithmetic mean analysis revealed three main genetic clusters that partially separated wild grape genotypes each other and. The international cultivars formed a out group. The high genetic diversity among native wild grapes from Coruh valley is suggesting that this area could be one of the centre of diversity of the specie. The results indicate a substantial genetic diversity in V. vinifera ssp. sylvestris and the need of exploring a wider area to increase the chance of finding a particular genotype.

Hydroxycinnamylated anthocyanins (or simply ‘acylated anthocyanins’) increase color stability in grape products, such as wine. Several genes that are relevant for anthocyanin acylation in grapes have been previously described; however, control of the degree of acylation in grapes is complicated by the lack of genetic markers quantitatively associated with this trait. To characterize the genetic basis of anthocyanin acylation in grapevine, we analyzed the acylation ratio in two closely related biparental families, Vitis rupestris B38 × ‘Horizon’ and ‘Horizon’ × Illinois 547-1, for 2 and 3 years, respectively. The acylation ratio followed a bimodal and skewed distribution in both families, with repeatability estimates larger than 0.84. Quantitative trait locus (QTL) mapping with amplicon-based markers (rhAmpSeq) identified a strong QTL from ‘Horizon’ on chromosome 3, near 15.85 Mb in both families and across years, explaining up to 85.2% of the phenotypic variance. Multiple candidate genes were identified in the 14.85–17.95 Mb interval, in particular, three copies of a gene encoding an acetyl-CoA-benzylalcohol acetyltransferase-like protein within the two most strongly associated markers. Additional population-specific QTLs were found in chromosomes 9, 10, 15, and 16; however, no candidate genes were described. The rhAmpSeq markers reported here, which were previously shown to be highly transferable among the Vitis genus, could be immediately implemented in current grapevine breeding efforts to control the degree of anthocyanin acylation and improve the quality of grapes and their products.

Thesis (MScAgric)--Stellenbosch University, 2002.
ENGLISH ABSTRACT: Grapevine supports one of the oldest industries in South Africa today, and is also of significant international importance. With increasing international trade and the transport of fruit and other grapevine-derived products between borders, it has become increasingly important for South African farmers and viticulturalists to ensure their products conform to strict international market requirements if they are to remain competitive. Such requirements include the correct and accurate identification of berries and wines according to cultivar. In light of this, 26 different wine, table grape and rootstock cultivars, as well as a number of clones from KWV's core germplasm collection were characterised at 16 microsatellite marker loci. Microsatellite markers are known for their high level of informativeness, reliability and reproducibility, and are widely used in the identification and characterisation of plant varieties, population analyses and forensic applications. Unique allelic profiles were obtained for all but two plants, which proved to be identical at all loci considered, and thus 'clones'. These profiles were collated to form a database, containing the DNA fingerprints of each sample at each locus. The relative levels of informativeness of each marker used were also determined, and compared with those found in the literature. Six markers proved to be highly informative, and are promising in the potential application of this technology to other cultivars. The applicability of microsatellite markers to such studies is confirmed; this approach could easily be extended to include any number of cultivars of national and international interest. The results of such an investigation would have important implications for both the farming and commercial industries alike.
AFRIKAANSE OPSOMMING: Wingerd ondersteun een van die oudste industriee in Suid-Afrika vandag, en is ook van groat intemasionale belang. Met die toenemende intemasionale ruilhandel en die vervoer van vrugte en ander wingerd produkte tussen grense, het dit toenemend belangrik geword vir SuidAfrikaanse wingerdboere om te. verseker dat hulle produkte voldoen aan die streng vereistes van die intemasional mark, indien hulle kompeterend wil bly. Hierdie vereistes sluit in die korrekte en akkurate identifisering van druiwe en wyn volgens kultivar. Met hierdie vereistes in ag geneem, is 26 verskillende wyn, tafeldruif en wortelstok kultivars, asook 'n aantal klone van die KWV se kern kiemplasma versameling, gekarakteriseer by 16 mikrosatelliet merker loki. Mikrosatelliet merkers word gekenmerk deur 'n hoe vlak van informatiwiteit, betroubaarheid en herhaalbaarheid en word wydverspreid gebruik in die identifisering en karakterisering van plant varieteite, populasie analises en forensiese toepassings. Unieke alleliese profiele is vir a1 die plante verkry, behalwe vir twee plante wat identiese resultate by alle loki opgelewer het en dus as "klone" beskou kan word. Hierdie profiele is bymekaar gevoeg om 'n databasis te vorm wat die DNA vingerafdrukke van elke monster by elke lokus bevat. Die relatiewe vlak van informatiwiteit van al die merkers is ook bepaal en vergelyk met merkers in die literatuur. Ses van die merkers blyk om hoogs informatief te wees en lyk belowend in die potensiele toepassing van hierdie tegnologie op ander kultivars. Die toepaslikheid van mikrosatelliet merkers op sulke studies is bevestig; hierdie benadering kan maklik aangepas word om enige aantal kultivars van nasionale en intemasionale belang in te sluit. Die resultate van s6 'n ondersoek sal belangrike implikasies inhou vir beide die boerdery en kommersiele industriee.

Thesis (MSc)--Stellenbosch University, 2013.
ENGLISH ABSTRACT: Metagenomic studies that make use of next-generation sequencing (NGS) generate large amounts of sequence data, representing the genomes of multiple organisms of which no prior knowledge is necessarily available. In this study, a metagenomic NGS approach was used to detect multiple novel mycoviral sequences in grapevine phloem tissue. Individual sequencing libraries of doublestranded RNA (dsRNA) from two grapevine leafroll diseased (GLD) and three shiraz diseased (SD) vines were sequenced using an Illumina HiScanSQ instrument. Over 3.2 million reads were generated from each of the samples and these reads were trimmed and filtered for quality before being de novo assembled into longer contigs. The assembled contigs were subjected to BLAST (Basic Local Alignment Search Tool) analyses against the NCBI (National Centre for Biotechnology Information) database and classified according to database sequences with which they had the highest identity. Twenty-six putative mycovirus species were identified, belonging to the families Chrysoviridae, Endornaviridae, Narnaviridae, Partitiviridae and Totiviridae. Two of the identified mycoviruses, namely grapevine-associated chrysovirus (GaCV) and grapevine-associated mycovirus 1 (GaMV-1) have previously been identified in grapevine while the rest appeared to be novel mycoviruses not present in the NCBI database. Primers were designed from the de novo assembled mycoviral sequences and used to screen the grapevine dsRNA used for sequencing as well as endophytic fungi isolated from the five sample vines. Only two mycoviruses, related to sclerotinia sclerotiorum partitivirus S and chalara elegans endornavirus 1 (CeEV-1), could be detected in grapevine dsRNA and in fungus isolates. In order to validate the presence of mycoviruses in grapevine phloem tissue, two additional sequencing runs, using an Illumina HiScanSQ and an Applied Biosystems (ABI) SOLiD 5500xl instrument respectively, were performed. These runs generated more and higher quality sequence data than the first sequencing run. Twenty-two of the putative mycoviral sequences initially detected were detected in the subsequent sequence datasets, as well as an additional 29 species not identified in the first HiScanSQ sequence datasets. The samples harboured diverse mycovirus populations, with as many as 19 putative species identified in a single vine. This indicates that the complete virome of diseased grapevines will include a high number of mycoviruses. Additionally, the complete genome of a novel endornavirus, for which we propose the name grapevine endophyte endornavirus (GEEV), was assembled from one of the second HiScanSQ sequence datasets. This is the first complete genome of a mycovirus detected in grapevine. Grapevine endophyte endornavirus has the highest sequence similarity to CeEV-1 and is the same virus that was previously detected in fungus isolates using the mycovirus primers. The virus was detected in two fungus isolates, namely Stemphylium sp. and Aureobasidium pullulans, which is of interest since mycoviruses are not known to be naturally associated with two distinctly different fungus genera. Mycoviral sequence data generated in this study can be used to further investigate the diversity and the effect of mycoviruses in grapevine.
AFRIKAANSE OPSOMMING: Metagenomiese studies, wat gebruik maak van volgende-generasie volgordebepalingstegnologie, het die vermoë om die genetiese samestelling van veelvoudige onbekende organismes te bepaal deurdat dit groot hoeveelhede data genereer. Die bogenoemde tegniek was in hierdie studie aangewend om aantal nuwe mikovirusse in die floëem weefsel van wingerd te identifiseer. Dubbelstring-RNS was gesuiwer vanuit twee druiwestokke met rolbladsiekte en drie met shirazsiekte en Illumina HiScanSQ instrument is gebruik om meer as 3.2 miljoen volgorde fragmente te genereer van elk van die monsters. Lae-kwaliteit volgordes was verwyder en die oorblywende kort volgorde fragmente was saamgestel om langer konstrukte te vorm wat met behulp van BLAST soektogte teen die NCBI databasis geïdentifiseer kon word. Ses-en-twintig mikovirus spesies, wat aan die families Chrysoviridae, Endornaviridae, Narnaviridae, Partitiviridae en Totiviridae behoort, was geïdentifiseer. Twee van die geïdentifiseerde mikovirusse, naamlik grapevine-associated chrysovirus (GaCV) en grapevine-associated mycovirus 1 (GaMV-1), was voorheen al in wingerd gekry terwyl die res nuwe mikovirusse is wat tans nie in die NCBI databasis voorkom nie. Inleiers was ontwerp vanaf die saamgestelde mikovirus basisvolgordes en gebruik om wingerd dubbelstring-RNS sowel as swamme wat vanuit die wingerd geïsoleer is te toets vir die teenwoordigheid van hierdie mikovirusse. Slegs twee mikovirusse, wat onderskeidelik verwant is aan sclerotinia sclerotiorum partitivirus S en chalara elegans endornavirus 1 (CeEV-1), kon deur middel van die inleiers in wingerd en swam isolate geïdentifiseer word. Twee addisionele volgordebepalingsreaksies, wat gebruik gemaak het van die Illumina HiScanSQ en ABI SOLiD 5500xl volgordebepalingsplatforms, was gebruik om die teenwoordigheid van mikovirusse in wingerd te bevestig. Groter hoeveelheid volgorde fragmente was geprodusser wat ook van hoër gehalte was as dié van die eerste volgordebepalingsreaksie. Twee-en-twintig mikovirus spesies kon weer geïdentifiseer word, sowel as 29 spesies wat nie in die eerste HiScanSQ basisvolgorde datastelle gevind was nie. Die wingerdstokke wat in hierdie studie ondersoek was, het hoë diversiteit van mikovirusse bevat aangesien daar tot 19 mikovirus spesies in enkele wingerdstok geïdentifiseer was. Dit is aanduiding dat volledige virus profiele van siek wingerdstokke aantal mikovirusse sal insluit. Die vollengte genoomvolgorde van voorheen onbekende endornavirus was saamgestel vanuit een van die tweede HiScanSQ volgorde datastelle. Dit is die eerste mikovirus wat in wingerd gevind word waarvan die volledige genoomvolgorde bepaal is en ons stel die naam grapevine endophyte endornavirus (GEEV) voor vir hierdie virus. Grapevine endophyte endornavirus is die naaste verwant aan CeEV-1 en is dieselfde virus wat voorheen in wingerd dubbelstring-RNS en swam isolate gevind was deur middel van die mikovirus inleiers. Swam isolate waarin GEEV gevind is, was geïdentifiseer as Stemphylium sp. en Aureobasidium pullulans. Dit is van belang dat GEEV in twee swam isolate gevind is wat aan verskillende genusse behoort aangesien hierdie verskynsel nog nie voorheen in die natuur gevind is nie. Mikovirus nukleiensuurvolgordes wat in hierdie studie bepaal was kan gebruik word in toekomstige studies om die verskeidenheid en impak van mikovirusse in wingerd verder te ondersoek.
National Research Foundation (NRF)
Stellenbosch University

Dissertation (PhD)--University of Stellenbosch, 2004.
ENGLISH ABSTRACT: Understanding the complex nature of grapevine molecular biology is of great importance for viticulturists. Progress in the elucidation of key events on a genetic level could provide further insight into the underlying cues responsible for the precise control of physiological and metabolic changes during a specific condition such as fruit development. The use and analysis of molecular ‘tools’, such as promoters controlling the site and level of gene activity, could assist in the understanding of grapevine biology and serve as a platform for the future design and development of recombinant DNA protocols and strategies for Vitis vinifera L. A high-throughput gene expression system, cDNA-AFLPs, was successfully used to analyse large-scale transcriptional activity during berry ripening. Candidate cDNA fragments were selected on the basis of desired expression patterns and/or known gene function for subsequent promoter isolation. From three candidate cDNAs selected, the promoter of a gene encoding vacuolar pyrophosphatase (V-PPase) was isolated for computational and comparative analyses. Promoter activity was evaluated on a transient level using the green fluorescent protein (GFP) reporter gene. Comparative integration has allowed for putative correlation of cis-elements, acting as receptors within promoter regions, to regulate V-PPase gene expression in response to development, environmental stress and tissue-specificity. In this study, integration of genetic data have advanced the understanding and transcriptional role of a key enzyme (V-PPase) during grape ripening. Although never a replacement for experimental verification, this integrative strategy of combining gene expression profiles with bioinformatics and regulatory data will greatly assist in further elucidation of various other key components and regulatory cues associated with grapevine molecular biology. This study has allowed us to use molecular tools that could assist in gaining further insight into genetic complexities and could serve as a platform for a more refined genetic manipulation strategy in Vitis vinifera L.
AFRIKAANSE OPSOMMING: Begrip van die komplekse aard van wingerd molekulêre biologie is van groot belang vir wingerdkundiges. Vooruitgang in die begrip van belangrike gebeurtenisse op ń genetiese vlak behoort verdere insig in die onderliggende instruksies vir die noukeurige beheer van fisiologiese en metaboliese veranderinge tydens ń spesifieke kondisie soos vrug rypwording te bevorder. Die gebruik en analise van molekulêre ‘instrumente’ soos promoters, wat die posisie en vlak van geen aktiwiteit beheer, kan bydra tot n beter begrip van wingerd biologie en sodoende dien as ń platform vir die toekomstige ontwerp en ontwikkeling van rekombinante DNS (deoksiribonukleiensuur) protokolle en strategieë vir Vitis vinifera L. ń Hoë-kapasiteit geen uitdrukkings sisteem, nl. kDNS-AFLPs (komplementêre deoksiribonukleiensuur- geamplifiseerde fragment lengte polimorfisme), is suksesvol gebruik vir die analise van grootskaalse transkripsionele aktiwiteit tydens druif rypwording. Kandidaat kDNS fragmente is geselekteer, gebaseer op verlangde uitdrukkings-patrone en/of bekende geen funksie vir daaropvolgende promoter isolering. Van drie geselekteerde kandidaat kDNS fragmente, is die promoter van ń geen wat vakuolêre pirofosfatase (V-PPase) kodeer geïsoleer vir rekenaar- en vergelykende analise. Promoter aktiwiteit is op ń nie-stabiele vlak deur die gebruik van ń groen-fluoresserende proteien (GFP) verklikker geen geëvalueer. Vergelykende integrering het dit moontlik gemaak om veronderstelde korrelasies van cis-elemente, wat as reseptore binne ń promoter area dien, en die regulering van V-PPase geen uitdrukking, in reaksie tot ontwikkeling, omgewings stres en weefsel-spesifisiteit, te maak. Tydens hierdie studie, het die integrering van genetiese data gehelp om die transkripsionele rol van ń belangrike ensiem (V-PPase) tydens druif rypwording beter te verstaan. Alhoewel dit nooit ń plaasvervanger vir eksperimentele bewyse sal wees nie, kan hierdie gëintegreerde strategie, wat die kombinasie van geen-uitdrukkingsprofiele met bioinformatika en regulatoriese data behels, grootliks bydra om verskeie ander belangrike komponente en regulatorieseaanwysings geassosieërd met wingerd molekulêre biologie te ontrafel. Hierdie studie het verdere insig in genetiese kompleksiteite verleen, en kan nou dien as ń platform vir ń meer presiese genetiese manipulering strategie in Vitis vinifera L.

Thesis (MSc)--University of Stellenbosch, 2003.
ENGLISH ABSTRACT: The genetic information of plants is found in the nucleus, the mitochondria, and the plastids. The DNA of plastids is comprised of multiple copies of a double-stranded, circular, prokaryoticallyderived genome of -150 kb. The genome equivalents of plastid organelles in higher plant cells are an attractive target for genetic engineering as high protein expression levels are readily obtained due to the high genome copy number per organelle. The resultant proteins are contained within the plastid organelle and the corresponding transgenes are inherited, in most crop plants, uniparentally, preventing pollen transmission of DNA. Plastid transformation involves the uniform modification of all the plastid genome copies, a process facilitated by homologous recombination and the non-Mendelian segregation of plastids upon cell division. The plastid genomes are in a continuous state of inter- and intra-molecular exchange due to their common genetic complement. This enables the site-specific integration of any piece of DNA flanked by plastid targeting sequences, via homologous recombination. The attainment of homoplasmy, where all genomes are transformed, requires the inclusion of a plastid-specific selectable marker. Selective pressure favouring the propagation of the transformed genome copies, as well as the random segregation of plastids upon cell division, make it feasible to acquire uniformity and hence genetic stability. From this, a complete transplastomie line is obtained where all plastid genome copies present are transgenic, having eliminated all wild-type genome copies. The prokaryotic nature of the chloroplast genetic system enables expression of multiple proteins from polycistronic mRNAs, allowing the introduction of entire operons in a single transformation. Expression cassettes in vectors thus include single regulatory elements of plastid origin, and harbour genes encoding selectable and screenable markers, as well as one or more genes of interest. Each coding region is preceded by an appropriate translation control region to ensure efficient translation from the polycistronic mRNA. The function of a plastid transformation vector is to enable transfer and stable integration of foreign genes into the chloroplast genomes of higher plants. The expression vector constructed in this research is specific for the transformation of the grape chloroplast genome. Vitis vinifera L., from the family, Vitaceae, is the choice species for the production of wine and therefore our target for plastid transformation. All chloroplast derived regulatory elements and sequences included in the vector thus originated from this species.
AFRIKAANSE OPSOMMING: Die genetiese inligting van plante word gevind in die kern, die mitochondria, en die plastiede. Die DNA van plastiede bestaan uit veelvuldige kopieë van 'n ~ 150 kb dubbelstring, sirkulêre genoom van prokariotiese oorsprong. Die genoomekwivalente van plastiede in hoër plante is 'n aantreklike teiken vir genetiese manipulering, aangesien die hoë genoom kopiegetal per organel dit moontlik maak om gereeld hoë vlakke van proteïenuitdrukking te verkry. Hierdie proteïene word tot die plastied beperk, en die ooreenstemmende transgene word in die meeste plante sitoplasmies oorgeërf, sonder die oordrag van DNA deur die stuifmeel. Plastied transformasie behels die uniforme modifikasie van al die plastied genoomkopieë, 'n proses wat deur homoloë rekombinasie en die nie-Mendeliese segregasie van plastiede tydens seldeling gefasiliteer word. As gevolg van die gemeenskaplike genetiese komplement, vind aanhoudende interen intra-molekulêre uitruiling van plastiedgenome plaas. Dit maak die setel-spesifieke integrasie, via homoloë rekombinasie, van enige stuk DNA wat deur plastied teikenvolgordes begrens word, moontlik. Vir die verkrying van homoplasmie, waar alle genome getransformeer is, word die insluiting van 'n plastiedspesifieke selekteerbare merker benodig. Seleksiedruk wat die vermeerdering van die getransformeerde genoomkopieë bevoordeel, en die lukrake segregasie van plastiede tydens seldeling, maak dit moontlik om genetiese stabiliteit en uniformiteit van die genoom te verkry. Dit kan op sy beurt tot die verkryging van 'n volledige transplastomiese lyn lei, waar alle aanwesige plastiedgenome transgenies is, en wilde tipe genoomkopieë geëlimineer is. Die prokariotiese aard van die chloroplas genetiese sisteem maak die uitdrukking van veelvuldige proteïene vanaf polisistroniese mRNAs moontlik, wat die toevoeging van volledige operons in 'n enkele transformasie toelaat. Uitdrukkingskassette in vektore bevat dus enkel regulatoriese elemente van plastied oorsprong, gene wat kodeer vir selekteerbare en sifbare merkers, asook een of meer gene van belang (teikengene). Voor elke koderingsstreek, is daar ook 'n toepaslike translasie beheerstreek om doeltreffende translasie vanaf die polisistroniese mRNA te verseker. Die funksie van 'n plastied transformasie vektor is om die oordrag en stabiele integrasie van transgene in chloroplasgenome van hoër plante moontlik te maak. Die uitdrukkingsvektor wat in hierdie studie gekonstrueer is, is spesifiek vir die transformasie van die druif chloroplasgenoom. Vitis vinifera L., van die familie Vitaceae, is die voorkeur species vir die produksie van wyn, en daarom die teiken vir plastied transformasie. Alle chloroplast-afgeleide regulatoriese elemente en volgordes wat in hierdie vektor ingesluit is, het huloorsprong vanaf VUis vinifera L.

Thesis (MSc)--Stellenbosch University, 2002.
ENGLISH ABSTRACT: Grapevine is worldwide an agronomically important crop. Traditionally selective breeding has been used to improve existing cultivars. In the last ten years, however, the advent of biotechnology has shortened these breeding programmes by producing transgenic grapevine. Because this new technology is aimed at the possible genetic manipulation of the ripening process in grape berries, it is important to elucidate all the mechanisms that may be involved in ripening. The aim of the present study was the identification of genes that play an important role during the ripening process in grape berries. This was achieved by investigation of putative differentially expressed genes in ripening Chardonnay berries isolated through subtractive hybridisation. Two subtraction libraries, representing early and late ripening stages were constructed. Four of the ten genes analysed exhibited expression during berry ripening. One of the four genes was expressed in a tissue and stage specific manner. Further characterisation of eight of the DNA and protein sequences revealed that the putative translation products of these clones had homologues that are involved in amongst others cell wall structure in other species. These included UDP-glucose dehydrogenase, which is involved in the synthesis of hemicellulose precursors. The remaining seven clones encoded putative stress response proteins. These included two heat shock proteins, a vacuolar pyrophosphatase and a protein involved in cell division. It is suggested that specific grape mRNAs accumulate in response to stresses such as the storage of high concentrations of sugars and rapid cell expansion. These processes occur rapidly during the ripening of berries. Accumulation of specific mRNAs can be attributed to part of the normal ripening developmental programme.
AFRIKAANSE OPSOMMING: Druiwe is wêreldwyd 'n belangrike landbougewas en kultivars word tradisioneel deur middel van tydsame selektiewe teling verbeter. Die tyd wat hieraan bestee word, kan verkort word deur die implementering van biotegnologie en die produksie van transgeniese duiwe. Omdat hierdie nuwe tegnologie op die moontlike genetiese manipulering van die rypwordingsproses in druiwe gemik is, is dit belangrik dat alle meganismes betrokke by rypwording ondersoek en verstaan word. Die doel van hierdie studie was om gene wat moontlik tydens die rypwordingsproses in druiwe 'n rol kan speel, te identifiseer. Hierdie doel is bereik deurdat differensieel uitgedrukte gene uit die kultivar Chardonnay geïsoleer is met behulp van verrykingsbiblioteke vanuit jong en volwasse druiwekorrels. Vier van die tien gene wat geanaliseer is, word uitgedruk tydens die rypwordingsproses. Verder het een van die vier gene weefsel- en rypwordingstadium- spesifisiteit getoon. Volledige karakterisering van agt van die DNA- en proteïenvolgordes het aangedui dat die proteïenprodukte van hierdie gene homoloog is aan volgordes wat onder andere by selwandstruktuur betrokke is. Dit sluit UDP-glukose dehidrogenase in, wat betrokke is by die sintese van hemi-sellulose boustene. Die ander sewe gene kodeer vir moontlike spanningsproteïene. Twee hitteskokproteïene, 'n vakuolêre pirofosfatase en 'n proteïen wat betrokke is by selverdeling is geïdentifiseer. Daar word voorgestel dat druiwe mRNA versamel in reaksie op spanningsituasies soos die berging van hoë konsentrasies suikers en selvergroting. Hierdie prosesse vind baie vinnig plaas tydens rypwording. Versameling van spesifieke mRNAs kan toegeskryf word as 'n normale deel van die rypwordingsproses.

Thesis (MSc)--University of Stellenbosch, 2004.
ENGLISH ABSTRACT: A number of proteins essential for the survival of a plant are encoded by the chloroplast genome. The characterization and sequencing of a number of algal and plant chloroplast genomes has facilitated researchers understanding of cellular functions and metabolism. Chloroplast DNA (cpDNA) has also been used to determine inter- and intraspecies evolutionary relationships and this organelle offers an alternative means of expressing foreign genes. Although a number of species' chloroplast genomes have been characterized and sequenced, no previous attempts of this kind have been made for a chloroplast genome of the family Vitaceae. In this study, attempts were made to characterize and partially sequence the chloroplast genome of Vilis vinifera. Chloroplast DNA was isolated from the Sultana and Sugra 1 cultivars and digested with restriction enzymes that produced cpDNA fragments of a suitable size for cloning. The fragments were shotgun-cloned into a plasmid vector and white colonies were screened by means of PCR and colony blotting. Three EcoRI-digested clones and one PstI-digested clone were obtained in this manner. Walking outwards from a previously sequenced grapevine rrn 16 gene region by means of PCR also allowed us to sequence a further -3310 bp region of the Sultana chloroplast genome. BAC clones containing V. vinifera cv L. Cabernet Sauvignon cpDNA inserts became available later in the project. It was decided to use these clones for further library construction instead of isolated cpDNA. The 5' and 3' end sequences of seven of the 24 BAC clones were obtained. These were compared to sequences found in the NCBI database to find - homologous chloroplast regions and determine the size of each BAC insert. One clone appeared to contain the entire grapevine chloroplast genome, apart from a 500 bp region. This clone was selected for further analysis. The BAC clone DNA was isolated and restriction-digested fragments were shotgun-cloned into a plasmid vector. White colonies were screened by isolating the plasmid DNA and digesting it with appropriate restriction enzy~es. The 5' and 3' ends of putative positive clones were sequenced and mapped onto the Atropa belladonna chloroplast genome. A total of 15 clones were obtained in this project. Five of these contain cpDNA isolated from grapevine leaves and 10 contain fragments sub-cloned from the BAC clone. The biggest problem encountered with both methods used for library construction was genomic DNA contamination. Genomic DNA either originated from the plant nuclear genome or from the bacterial host cells in which the BAC clones were maintained. Many of the clones screened contained genomic DNA, and these could only be identified and removed once the clones had been sequenced. Even when a commercial kit was used for BAC clone isolation, 31% of the clones screened contained genomic DNA. This kit was specifically designed for the isolation of genomic DNA-free large constructs. The clones obtained from the two strategies provided a good representation of the grapevine chloroplast genome. The only region not represented was the Small Single Copy (SSC) region. Approximately 40% of the grapevine chloroplast genome was covered by these clones. This provides a basis for further genome characterization, physical mapping and sequencing of the grapevine chloroplast genome.
AFRIKAANSE OPSOMMING: Die chloroplasgenoom kodeer VIr 'n hele aantal proteïene wat essensieel is VIr die voortbestaan van 'n plant. Die karakterisering en volgorde bepaling van 'n aantal alg en plant chloroplasgenome het dit. vir navorsers moontlik gemaak om sellulêre funksies en metabolisme van plante te ontrafel. Chloroplas DNA (cpDNA) is ook gebruik om intra- en interspecies evolusionêre verwantskappe vas te stel. Dié organel verskaf ook 'n alternatiewe manier vir die uitdrukking van transgene. Alhoewel die chloroplasgenome van 'n hele aantal species al gekarakteriseer is en die DNA volgorde daarvan bepaal is, is daar nog geen navorsing van bogenoemde aard op die chloroplasgenoom van die Vitaceae familie gedoen rue. In hierdie studie is beoog om die chloroplasgenoom van Vitis vinifera te karakteriseer en gedeeltelike volgordebepaling daarvan te doen. Chloroplas DNA is geïsoleer vanaf Sultana en Sugra 1 kultivars en restriksie-ensiem vertering is gedoen met ensieme wat cpDNA fragmente, met geskikte grootte vir klonering, produseer. Dié fragmente is in 'n plasmiedvektor gekloneer met die haelgeweer-metode en wit kolonies is gesif deur middel van PKR en die kolonieklad metode. Op hierdie manier is drie EcoRI-verteerde klone en een PstI-verteerde kloon verkry. Deur uitwaarts te loop, deur middel van PKR, vanaf 'n druif rrnl6 geenstreek, waarvan die volgorde voorafbepaal is, was dit vir ons moontlik om ook die volgorde te bepaal van 'n verdere ~3310 bp streek van die Sultana chloroplasgenoom. BAC klone wat V. vinifera cv L. Cabernet Sauvignon cpDNA fragmente bevat, het later in die projek beskikbaar geraak. Daar is besluit om hierdie klone, i.p.v. die geïsoleerde cpDNA, te gebruik vir verdere biblioteek konstruksie. Die 5' en 3' entpuntvolgordes van sewe uit die 24 BAC ~lone is verkry. Hierdie volgordes is vergelyk met volgordes in die NCB Idatabasis om homoloë chloroplas streke te identifiseer, en die grootte van elke BAC fragment te bepaal. Die het geblyk dat die hele druif chloroplasgenoom in een van die klone vervat is, behalwe vir 'n 500 bp streek. Die BAC-kloon DNA is geïsoleer en die restriksie-verteerde fragmente is in 'n plasmiedvektor gekloon d.m.V. die haelgeweer-metode. Wit kolonies is gesif deur die isolering van plasmied DNA en die vertering daarvan met geskikte restriksie-ensieme. Die volgorde van die 5' en 3' entpunte van skynbare positiewe klone is bepaal en gekarteer op die Atropa belladonna chloroplasgenoom. In hierdie studie is 'n totaal van 15 klone verkry. Vyf hiervan bevat cpDNA wat vanaf druifblare geïsoleer is, en 10 bevat fragmente wat vanaf die BAC-klone gesubkloneer is. Genorniese DNA kontaminasie was die grootste probleem wat ondervind is tydens beide metodes wat gebruik is vir biblioteek konstruksie. Genomiese DNA was afkomstig vanaf óf die plant nukleêre genoom óf die bakteriële gasheerselle waarin die BAC-klone gehou is. Baie van die klone wat gesif is, het genomiese DNA bevat, en dit kon eers geïdentifiseer en verwyder word nadat die volgorde van die klone bepaal is. Selfs al is 'n kommersiële produk vir BAC-kloon isolasie gebruik, het 31% van die gesifde klone steeds genomiese DNA bevat. Dié kommersiële produk is spesifiek vir die isolasie van groot konstrukte, wat genomiese DNA vry is, ontwerp. Die klone wat deur die twee strategeë verkry is, het 'n goeie verteenwoordiging van die druif chloroplasgenoom gegee. Die enigste streek wat die verteenwoordig is nie, was die Klein Enkelkopie (SSC) streek. Ongeveer 40% van die druif chloroplasgenoom is deur hierdie klone gedek. Dit verskaf 'n basis vir verdere genoomkarakterisering, fisiese kartering en volgordebepaling van die druif chloroplasgenoom.

Dissertation (PhD)--University of Stellenbosch, 2004.
152 Leaves printed single pages, preliminary pages i-xiv and 129 numberd pages. Includes bibliography. List of abbreviations.
ENGLISH ABSTRACT: Despite increased focus on ripening-related gene transcription in grapevine, and the large number of ripening-related cDNAs identified from grapes in recent years, the molecular basis of processes involved in grape berry ripening is still poorly understood. Moreover, little is known about the mechanisms involved in the ripening-related regulation of fruit-specific genes, since the isolation and characterisation of no ripening-related, fruit-specific promoter elements has been reported to date. This study was aimed at the isolation and characterisation of a fruit-specific, ripeningregulated gene from Vitis vinifera L. In the first phase of the work, gene transcription in ripening berries of Cabernet Sauvignon (a good quality wine cultivar) and Clairette blanche (a poor quality wine cultivar) were studied by Amplified Fragment Length Polymorphism analysis of complementary DNA (cDNA-AFLP analysis). Total RNA from immature (14-weeks post flowering, wpf) and mature (18-wpf) berries was used for the analysis. A total of 1 276 cDNA fragments were visualised, of which 175 appeared to be ripening related. Average pairwise difference of the fragments amplified from immature and mature Clairette and Cabernet berries, suggested that ripening-related gene transcription in these two phenotypically different cultivars is remarkably similar. Nevertheless, it was shown that seventy percent of the 175 ripening-related cDNA fragments were cultivar-specific. It was suggested that these differences should be targeted to identify genes related to the phenotypical differences between the two cultivars, but also to identify genes possibly involved berry quality. Moreover, the analysis illustrated the usefulness of cDNA-AFLPs for the analysis of ripening-related gene transcription during grape berry ripening. In the second phase of the work, one of the ripening-related cDNAs identified by the cDNA-AFLP analysis, was selected for further characterisation. This work highlighted the limitation placed on the isolation of a single specific sequence from a cDNA-AFLP gel, indicating the presence of multiple ripening-related genes in a single band excised from a cDNA-AFLP gel. Steps to overcome this limitation of cDNA-AFLP analysis to identify and clone a specific ripening-related gene, were implemented. In short, the band corresponding to the particular ripening-related cDNA was band was excised from the cDNA-AFLP polyacrylamide gel and re-amplified. Northern blot analysis using the re-amplified, uncloned product confirmed the ripening-related transcription demonstrated by cDNA-AFLP analysis. The re-amplified, uncloned product was then cloned. Sequence analysis of two randomly selected candidate clones revealed two distinctly different sequences, of which neither hybridised to messenger RNA from ripening grape berries. Furtheranalysis revealed an additional five cDNAs with terminal sequences corresponding to the selective nucleotides of the primers used for selective amplification, in the re-amplified, uncloned product. Of these, only two were abundantly expressed in ripening grape berries, accounting for the ripeningrelated transcription visualised by cDNA-AFLP analysis. All seven cDNAs identified from the particular excised band were shown to be ripening-regulated during berry development, although most were characterised by low levels of transcription during berry ripening. One of the clones, based on the relative high levels of the transcript and the initiation of gene transcription at the onset of véraison (10- to 12-wpf), was identified for isolation and characterisation of the full length coding sequence. In the third phase of the work, it was shown that this cloned sequence corresponded to a gene encoding a proline-rich protein (PRP) associated with ripening in Merlot and Chardonnay (mrip1, Merlot ripening-induced protein 1). It was shown that the gene is specifically transcribed in the fruit tissue, seed and bunchstems of grapes, from 10-wpf (véraison) to the final stages of berry ripening. The results showed that mrip1 encodes a distinct member of the plant PRP family. Most obvious is the central region of mrip1, which is comprised of eight consecutive repeats of 19 amino acid residues each. In comparison with other grapevine PRPs, mrip1 revealed single amino acid differences and deletion of one of the 19 amino acid residues repeats, all in the central region of mrip1. In situ hybridisation studies showed that accumulation of the mrip1 transcript in the ripening berry is limited to the mesocarp and exocarp cells of the ripening grape berry. No transcript with high sequences similarity to mrip1 could be detected in ripening strawberry or tomato fruit. Based on the properties and proposed function of PRPs, and the results obtained in this study, potential applications for the use of this gene in the control of cell wall architecture in fruits, were proposed. Furthermore, as manipulation of fruit properties in grape berries would be most important in the later stages of ripening, mrip1 was proposed an ideal candidate gene for the isolation of a fruit- and late-ripening-specific promoter to achieve transgene transcription in genetically modified grapevine. The final phase of the work was dedicated to the isolation and characterisation of the mrip1 promoter element. A 5.5 kb sequence corresponding to the mrip1 5’ untranslated (UTR) flanking region was isolated and characterised by sequence analysis. In the 2.8 kb sequence directly upstream of the mrip1 transcription initiation site, several putative cis-acting regulatory elements were identified. These include a spectrum of hormone-, light-, phytochrome-, sugar-and stressresponsive elements, as well as elements implicated in tissue-specific transcription. Analysis of the sequence further upstream (3.6 – 5.5 kb) of the mrip1 transcription initiation site (TIS), revealed the presence of another proline-rich protein directly upstream of mrip1. Sequence identity of this sequence (mprp2) to the mrip1 coding sequence was 88%. This information provided the first insight into the chromosomal organisation of grapevine PRPs. For functional analysis of the mrip1 promoter element, the 2.2 kb sequence directly upstream of the mrip1 TIS, was translationally fused to the sgfpS65T reporter gene. Functionality of the mrip1:sgfpS65T fusion was verified by transient expression in green pepper pericarp tissue, before introduction into tobacco by Agrobacteriummediated transformation. In transgenic tobacco, transcription of the mrip1:sgfpS65T fusion was developmentally-regulated and specific to the ovary and nectary-tissue of the developing flower. Whilst low in immature flowers, the green fluorescent protein (GFP) rapidly accumulated to the high level of expression visualised in the flower in full-bloom, followed by a decrease in the final stages of ovary development. These observations suggested that the 2.2 kb mrip1 promoter is functional and that this promoter region harbours cis-elements necessary for tissue- and developmental-specific regulation of GFP accumulation. It furthermore suggested that the transcriptional activation of mrip1 is mediated by developmental signals present in both grapevine berries and tobacco flowers. Results presented, suggest that the use of tobacco as heterologous system for the analysis of ripening-related promoters, can be more generally applied. Evidently, characterisation of the mrip1 promoter region contributes towards a better understanding of the regulatory mechanisms involved in non-climacteric fruit ripening, and forms a basis for future experiments defining the cis-acting elements necessary for tissue- and cell-specific gene regulation in fruit, more specifically in grapevine. Moreover, the mrip1 promoter is an ideal candidate for the ripening-related, tissue-specific regulation of transgene transcription in genetically modified grapevine.
AFRIKAANSE OPSOMMING: Ten spyte van toenemende fokus op rypwordings-verwante geentranskripsie in druiwe, en die groot aantal rypwordings-verwante komplimentere DNA (cDNA) fragmente wat gedurende die laaste paar jaar in druiwe geïdentifiseer is, word die molekulêre basis van prosesse betrokke by die rypwording van die druif, steeds swak begryp. Nog te meer, is baie min bekend oor die meganismes betrokke in the rypwordings-verwante regulering van vrugspesifieke gene, aangesien die isolering en karakterisering van nie een rypwordings-verwante, vrugspesifieke promoter tot dusver gerapporteer is nie. Die doel van hierdie studie was die isolering en karakterisering van ‘n vrugspesifieke, rypwordings-verwante geen uit druiwe (Vitis vinifera L). In die eerste fase van die werk, is geentranskripsie in rypwordende druiwekorrels van Cabernet Sauvignon (‘n goeie kwaliteit wyn kultivar) en Clairette blanche (‘n swak kwaliteit wyn kultivar) bestudeer deur middel van cDNA-AFLP vingerafdrukke. Totale RNA van onvolwasse (14-weke na blom vorming) en volwasse (18-weke na blom vorming) druiwekorrels was gebruik vir die analise. ‘n Totaal van 1 276 cDNA fragmente is gevisualiseer, waarvan 175 as rypwordings-verwant voorgekom het. Gemiddelde paarsgewyse verskille van die fragmente wat vanaf onvolwasse en volwasse Clairette en Cabernet druiwekorrels geamplifiseer is, het aangedui dat rypwordingverwante geentranskripsie in die twee kultivars, wat fenotipies baie van mekaar verskil, merkwaardig soortgelyk is. Nieteenstaande, is daar gewys dat sewentig persent van die 175 rypwordings-verwante cDNA fragmente, kultivar-spesifiek is. Daar is voorgestel dat hierdie spesifieke cDNAs verder geanaliseer word om gene betrokke by die fenotipiese verskille tussen die twee kultivars te identifiseer; maar ook om gene te identifiseer wat moontlik by die kwaliteit van die druiwekorrel betrokke is. Voorts, het die analise die bruikbaarheid van die cDNA-AFLP tegniek vir die karakterisering van rypwordings-verwante geentranskripsie in rypwordende druiwekorrels, geïllustreer. In die tweede fase van die werk, is een van die rypwordings-verwante cDNAs wat met die cDNAAFLP analise geïdentifiseer is, geselekteer vir verdere karakterisering. ‘n Aantal rypwordingsverwante cDNAs is in die enkele band wat uit die cDNA-AFLP gel gesny is, geïdentfiseer. Dit het die beperking wat geplaas word op die isolering van ‘n enkel, spesifieke cDNA uit die cDNA-AFLP gel, beklemtoon. Stappe om hierdie beperking te oorkom, en ‘n spesifieke rypwordings-verwante cDNA te identfiseer en te kloneer, is beskryf. In kort, die band oorstemmend met die spesifieke rypwordings-verwante cDNA, is uit die cDNA-AFLP poli-akrielamied gel gesny en gereamplifiseer. Noordelike klad analise waarin die ge-reamplifiseerde, ongekloneerde produk aspeiler gebruik is, het die rypwordings-verwante transkripsie soos deur cDNA-AFLP analise aangedui, bevestig. Die ge-reamplifiseerde, ongekloneerde produk is daarna gekloneer. Nukleotied volgorde bepaling van twee ewekansig geselekteerde kandidaat klone, het twee duidelik verskillende cDNAs aangetoon, waarvan nie een enige hibridisering met boodskapper RNA van rypwordende druiwekorrels getoon het nie. Verder analise het die teenwoordigheid van ‘n verder vyf cDNAs met terminale nukleotied volgordes ooreenstemmend met die selektiewe nukleotiede van die voorlopers wat gebruik is vir selektiewe amplifisering, aangetoon. Van hierdie, het slegs twee hoë vlakke van geentranskripsie in rypwordende druiwekorrels getoon; heel moontlik verteenwoordigend van die rypwordings-verwante geentranskripsie wat met die cDNA-AFLP analise gevisualiseer is. Die studie het gewys dat al sewe cDNAs rypwordings-verwant is, alhoewel die meeste van hierdie cDNAs baie lae vlakke van geentranskripsie tydens duiwekorrel rypwording getoon het. Gebaseer op relatief hoë vlakke van die transkrip, en die inisiering van geen transkripsie met die aanvang van vrugrypwording (véraison, 10- tot 12-weke na blomvorming), is een van die cDNAs geselekteer vir isolering en karakterisering van die vollengte koderings volgorde. In die derde fase van die werk, is dit aangetoon dat hierdie cDNA ooreenstem met ‘n geen wat vir ‘n proline-ryke proteïen (PRP), geassosieerd met vrugrypwording in Merlot en Chardonnay, kodeer. Hierdie geen is genoem Merlot rypwording-geïnduseerde proteïen 1 (mrip1). Die studie het verder aangetoon dat hierdie geen spesifiek in die weefsel van druiwekorrels, saad and stammetjies van die druiwetros getranskribeer word, vanaf 10-weke na blomvorming (véraison) tot 16-weke na blomvorming. Resultate het aangetoon dat mrip1 vir ‘n unieke lid van die plant PRP familie kodeer. Mees opvallend, is die sentrale gedeelte van mrip1, wat uit agt opeenvolgende herhalings van negentien aminosure elk bestaan. In vergelyking met ander druif PRPs, toon mrip1 enkel aminosuur verskille en ‘n delesie van een van die negentien aminosuur herhalings, alles in die sentrale gedeelte van mrip1. In situ hibridisering het getoon dat akkumulering van die mrip1 transkrip net in selle van die mesocarp en eksokarp van die rypwordende druif plaasvind. Geen transkip met hoë nukleotied gelyksoortigheid aan mrip1 kon in rypwordende aarbeie of tamatie vrugte aangetoon word nie. Gebaseer op die eienskappe en funksie van PRPs soos voorgestel in die literatuur, en die bevindinge van hierdie studie, is potensiële toepassings vir die gebruik van die geen in die beheer van selwand argitektuur in vrugte, voorgestel. Verder, aangesien die manipulering van vrugkwaliteit in die druif veral belangrik is vanaf die aanvang van vrugrypwording (véraison), is daar voorgestel dat mrip1 ‘n ideale kandidaat is vir die isolering van ‘n vrugspesifieke en rypwording-verwante promoter vir gebruik in geneties gemodifiseerde druiwe. Die laaste fase van die studie was gewy aan die isolering en karakterisering van die mrip1 promotor element. ‘n 5.5 kb fragment ooreenstemmend met die mrip1 5’ ongetransleerde area is geisoleer en gekarakteriseer deur middel van nukleotied volgorde bepaling. In die 2.8 kb area direk stroomop van die mrip1 transkripsie inisiasie punt (TIS), is verskeie moontlike cis-beherende regulatoriese elemente geïdentifiseer. Hierdie sluit in ‘n spektrum van hormoon-, lig-, fitochroom-, suiker- en stress-reagerende elemente, asook elemente geïmpliseer in weefselspesifieke geentranskripsie. Analise van die area verder stroomop (3.6 – 5.5 kb) van die mrip1 TIS, het die teenwoordigheid van ‘n ander PRP direk stroomop van mrip1 getoon. Nukleotied gelyksoortigheid van hierdie geen (MPRP2) aan die mrip1 koderingsgebied was slegs 88%. Hierdie inligting verskaf die eerste insig in die chromosomale organisasie van druif PRPs. Vir funksionele analise van die mrip1 promotor element, is die 2.2 kb area direk stroomop van die mrip1 TIS transkripsioneel verenig met die sgfpS65T merker geen. Funksionaliteit van die mrip1: sgfpS65T fusie is bevestig deur middel van kortstondige (transient) geenuitdrukking in die perikarp van groenrissie, voordat dit ingevoer is in tabak met Agrobacterium-bemiddelde genetiese transformasie. In transgeniese tabak was transkripsie van die mrip1:sgfpS65T fusie ontwikkelingsstadium-gereguleerd, en spesifiek in die ovarium en heuningsakkie (nektarium) van die ontwikkelende blomme. Terwyl die vlak van geenuitdrukking laag was in die jong blomme, het GFP baie vinnig akkumuleer tot die hoë vlakke wat in die blomme in volle-blom gevisualiseer is. Daarna het dit weer vinnig afgeneem tydens die finale stadiums van ovarium ontwikkeling. Hierdie waarnemings dui daarop dat die 2.2 kb mrip1 promotor element funksioneel is en dit al die nodige cis-beherende regulatoriese element bevat wat nodig is vir weefsel- en ontwikkelingsstadium-spesifieke regulering van GFP akkumulering. Dit dui verder daarop dat transkripsionele aktivering van mrip1 beheer word deur ontwikkelingsstadium seine teenwoordig in beide die druif en tabakblomme. Hierdie resultate stel voor dat tabak meer algemeen gebruik kan word as heteroloë sisteem vir die analise van rypwording-verwante promotors. Duidelik dra die karakterisering van die mrip1 promoter element by tot ‘n beter begrip van die regulatoriese meganismes betrokke by die rypwordingsproses van nie-klimateriese vrugte, en vorm die basis vir toekomstige eksperimente waarin die cis-beherende regulatoriese elemente vir vrug- en sel-spesifieke geen regulering, meer spesifiek die druif, bepaal sal word. Meer nog, is die mrip1 promotor ‘n ideale kandidaat vir weefsel-spefieke en rypwording-verwante regulering van transkripsie van die transgeen in geneties gemodifiseerde druiwe.

Thesis (MSc)--University of Stellenbosch, 2000.
ENGLISH ABSTRACT: Grapevine leafroll is one of the most damaging viral diseases that affect many viticultural regions of the world. Numerous reports over the last few years have associated closterovirus-like particles with leafroll disease. To date, eight serologically distinct closteroviruses have been isolated from leafroll infected vines, of which grapevine leafroll associated closterovirus-3 (GLRaV-3) is the best characterized. Virus resistance in transgenic plants based on the expression of a virusderived gene is known as pathogen-derived resistance. The viral coat protein (CP) gene, which expresses a structural protein responsible for coating the virus particles, was used in the first demonstration of virus-derived resistance. Coat protein-mediated resistance is currently the most feasible and most widely used method to obtain virus resistance in crop plants. The CP gene of a South African isolate of GLRaV-3 infected grapevine was isolated, cloned and sequenced. Double stranded RNA (dsRNA) was extracted from GLRaV-3 infected material and a high molecular weight band, of -18 kb was identified from infected vines. The dsRNA was used as a template in a reverse transcription PCR together with GLRaV-3 CP gene specific primers for the amplification of the GLRaV-3 CP gene (975 bp). The GLRaV-3 CP gene was cloned into the pGem®-T Easy vector. Clones hosting the CP gene in the sense (pLR3CP+) and antisense (pLR3CP-) orientations respectively were obtained. The sequence obtained from these two clones showed 99.26 % similarity to the only other GLRaV-3 CP nucleotide sequence available. The GLRaV-3 CP gene was excised from pLR3CP+ and pLR3CP- and subcloned into a plant expression vector, pCAMBIA 3301 in the sense (pCamBLR3CP+) and antisense (pCamBLR3CP-) orientations respectively, therefore enabling sense and antisense gene expression in transgenic plants. The GLRaV-3 CP gene was also subcloned from pCamBLR3CP+ into another plant expression vector, pCAMBIA 2301 in the sense orientation and designated as pCVSLR3CP+. These three constructs were given to Dr. M. Vivier (Institute for Wine Biotechnology, Stellenbosch) for grapevine transformation experiments. Two of these constructs, pCamBLR3CP+ and pCamBLR3CP- as well as pCAMBIA 3301 were used to transform Nicotiana tabacum by Agrobacterium tumefaciens-mediated transformation. Plants were selected for their ability to withstand the herbicide, Basta. This resistance is due to the presence of a plant selectable marker gene on each of these constructs, known as the bar gene. PCR with GLRaV-3 CP gene specific primers showed no amplification of the GLRaV-3 CP gene in the plants transformed with pCamBLR3CP+ and pCamBLR3CP-. Southern blot analysis with the GLRaV-3 CP gene as hybridization probe showed no signal for these plants, thus confirming the PCR results. PCR with bar gene specific primers showed no amplification of the bar gene in the plants infected with pCAMBIA 3301. The plants transformed with pCamBLR3CP+ and pCamBLR3CP- were also screened for the presence of the bar gene. Three of the eight plants tested showed amplification of the -560 bp bar gene. This result suggests that these plants were transformed with pCAMBIA 3301 (vector without the ligated GLRaV-3 CP gene) and not pCamBLR3CP+ or pCamBLR3CP- as had been expected. This project provides preliminary work for the subsequent transformation of grapevine with the GLRaV-3 CP gene, in an attempt to impart virus resistance.
AFRIKAANSE OPSOMMING: Wingerd rolblaar is een van die mees beskadigende virale siektes wat baie wingerd areas in die wêreld aantas. In Aantal verslae oor die afgelope jare het closterovirus partikels met wingerd rolblaar geassosieer. Tot hede, is agt serologiese onderskeibare closterovirusse geïsoleer vanuit geaffekteerde wingerde, waarvan wingerd rolblaar geassosieerde closterovirus-3 (GLRaV-3) die beste gekarakteriseerd is. Virus bestandheid in transgeniese plante gebaseer op die uitdrukking van gene afkomstig vanaf virusse, staan bekend as patogeen-afgeleide weerstand. Die virale kapsule protein (CP) geen vervaardig In strukturele protein wat verantwoordelik is vir die bedekking van die virus partikel. Dié geen was gebruik in die eerste demonstrasie van patogeen-afgeleide weerstand. Kapsuul protein-bemiddelde weerstand is tans die mees praktiese en algemene gebruikte metode om virus weerstand in plant gewasse te verkry. Die CP geen van In Suid Afrikaanse isolaat van GLRaV-3 geïnfekteerde wingerde is geïsoleer, gekloneer en die volgorde is bepaal. Dubbelstring RNA (dsRNA) was uit GLRaV-3 geïnfekteerde materiaal geëkstraheer en In hoë molekulêre gewig band van -18 kb is geïdentifiseer. Die dsRNA is gebruik as In templaat vir In omgekeerde transkripsie PKR saam met GLRaV-3 CP geen spesifieke inleiers vir die amplifikasie van die GLRaV-3 CP geen (975 bp). Die GLRaV-3 CP geen is gekloneer in die pGem®-T Easy vektor. Klone met die CP geen in die sin (pLR3CP+) en teensin (pLR3CP-) oriëntasies respektiewelik is verkry. Die volgorde wat verkry is vanuit hierdie twee klone dui op In 99.26 % ooreenstemming met die enigste ander GLRaV-3 CP geen volgorde wat beskikbaar is. Die GLRaV-3 CP geen is uit pLR3CP+ en pLR3CP- gesny en is gesubkloneer in In plant ekspressie vektor, pCAMBIA 3301 in die sin (pCamBLR3CP+) en teensin (pCamBLR3CP-) oriëntasies respektiewelik, wat die sin en teensin geen ekspressie in transgeniese plante in staat stel. Die GLRaV-3 CP geen was ook gesubkloneer vanaf pCamBLR3CP+ in In ander plant ekspressie vektor, pCAMBIA 2301 in die sin orientasie en is as pCVSLR3CP+ benoem. Hierdie drie konstruksies is aan Dr. M. Vivier (Instituut vir Wyn Biotegnologie, Stellenbosch) gegee vir wingerd transformasie eksperimente. Twee van hierdie konstruksies, pCamBLR3CP+ en pCamBLR3CP- asook pCAMBIA 3301 is gebruik om Nicotiana tabacum deur middel van Agrobacterium tumefaciens-bemiddelde transformasie te transformeer. Plante is geselekteer vir hul vermoë om die onkruiddoder, Basta, te weerstaan. Die teenwoordigheid van die plant selekteerbare merker geen, bar, op elke konstruksie lui tot dié weerstand. Die plante wat getransformeer is met pCamBLR3CP+ en pCamBLR3CP- is deur PKR saam met die GLRaV-3 CP geen spesifieke inleiers getoets, en geen amplifikasie van die GLRaV-3 CP geen is getoon nie. Southern blot analise met die GLRaV-3 CP geen as hibridisasie peiler het geen sein gewys vir hierdie plante nie, wat die PKR resultate bevestig. Die plante wat getransformeer is met pCAMBIA 3301 is deur PKR saam met die bar geen spesifieke inleiers getoets, en geen amplifikasie van die bar geen is getoon nie. Die plante wat getransformeer is met pCamBLR3CP+ en pCamBLR3CP- is ook getoets vir die teenwoordigheid vir die bar geen. Drie van die agt plante wat getoets is, het amplifikasie van die -560 bp bar geen getoon. Hierdie onverwagte resultate stel voor dat dié plante met pCAMBIA 3301 (vektor sonder die geligeerde GLRaV-3 CP geen) en nie met pCamBLR3CP+ en pCamBLR3CPgetransformeer is nie. Hierdie projek verskaf voorlopige werk vir die daaropvolgende transformasie van wingerd met die GLRaV-3 CP geen in 'n poging om virus bestandheid te verskaf.

Thesis (MSc)--University of Stellenbosch, 2004.
ENGLISH ABSTRACT: Viruses are some of the most important pathogens of grapevines. There are no effective chemical treatments, and no grapevine- or other natural resistance genes have been discovered against grapevine infecting viruses. The primary method of grapevine virus control is prevention by biological indexing and molecular- and serological screening of rootstocks and scions before propagation. Due to the spread of grapevine viruses through insect vectors, and in the case of GRSPaV the absence of serological screening, these methods of virus control are not always effective. In the past several methods, from cross-protection to pathogen derived resistance (PDR), have been applied to induce plant virus resistance, but with inconsistent results. In recent years the application of post-transcriptional gene silencing (PTGS), a naturally occurring plant defense mechanism, to induce targeted virus resistance has achieved great success. The Waterhouse research group has designed plant transformation vectors that facilitate specific virus resistance through PTGS. The primary focus of this study was the production of virus specific transformation vectors for the introduction of grapevine virus resistance. The Waterhouse system has been successfully utilised for the construction of three transformation vectors with the pHannibal vector as backbone. Each vector contains homologous virus coat protein (CP) gene segments, cloned in a complementary conformation upstream and downstream of an intron sequence. The primary vector (pHann-SAScon) contains complementary CP gene segments of both GRSPaV and GLRaV-3 and was designed for the introduction of multiple-virus resistance. For the construction of the primary vector the GRSPaV CP gene was isolated from RSP infected grapevines. A clone of the GLRaV-3 CP gene was acquired. The second vector (pHann- LR3CPsas) contains complementary CP gene segments of GLRaV-3. The third vector (pHann-LR2CPsas) contains complementary CP gene segments of GLRaV-2. The cassette containing the complementary CP gene segments of both GRSPaV and GLRaV-3 was cloned into pART27 (pART27-HSAScon), and used to transform N tabacum cv. Petit Havana (SRI), through A. tumefaciens mediated transformation. Unfortunately potential transformants failed to regenerate on rooting media; hence no molecular tests were performed to confirm transformation. Once successful transformants are generated, infection with a recombinant virus vector (consisting of PYX, the GFP gene as screenable marker and the complementary CP gene segments of both GRSPaV and GLRaV-3) will be used to test for the efficacy of the vectors to induce resistance. A secondary aim was added to this project when a need was identified within the South African viticulture industry for GRSPaV specific antibodies to be used in serological screening. To facilitate future serological detection of GRSPaV, the CP gene was isolated and expressed with a bacterial expression system (pETI4b) within the E. coli BL2I(DE3)pLysS cell line. The expressed protein will be used to generate GRSPaV CP specific antibodies.
AFRIKAANSE OPSOMMING: Virusse is van die belangrikste patogene by wingerd. Daar bestaan geen effektiewe chemiese beheer nie, en geen wingerd- of ander natuurlike weerstandsgene teen wingerdvirusse is al ontdek nie. Die primêre metode van beheer t.o.v. wingerdvirusse is voorkoming deur biologiese indeksering, en molekulêre- en serologiese toetsing van onderstokke en entlote voor verspreiding. As gevolg van die verspreiding van wingerdvirusse deur insekvektore, en in die geval van GRSPa V die tekort aan serologiese toetsing, is dié metodes van virusbeheer nie altyd effektief nie. In die verlede is metodes soos kruis-beskerming en patogeen-afgeleide weerstand (PDR) gebruik om virusweerstand te induseer, maar met inkonsekwente resultate. In onlangse jare is post-transkripsionele geenonderdrukking (PTGS), 'n natuurlike plantbeskermingsmeganisme, met groot sukses toegepas om geteikende virusweerstand te induseer. Die Waterhouse-navorsingsgroep het planttransformasievektore ontwerp wat spesifieke virusweerstand induseer d.m.v. PTGS. Die vervaardiging van virus spesifieke tranformasievektore vir die indusering van wingerdvirusweerstand was die primêre doelwit van hierdie studie. Die Waterhouse-sisteem was gebruik vir die konstruksie van drie transformasievektore, met die pHannibal vektor as basis. Elke vektor bevat homoloë virus kapsiedproteïen (CP) geensegmente, gekloneer in 'n komplementêre vorm stroom-op en stroom-af van 'n intronvolgorde. Die primêre vektor (pHann-SAScon) bevat komplementêre CP geensegmente van beide GRSPaV en GLRaV-3, en was ontwerp vir die indusering van veelvoudige-virusweerstand. Die CP-geen van GRSPa V was vanuit RSP-geïnfekteerde wingerd geïsoleer, vir die konstruksie van die primêre vektor. 'n Kloon van die GLRa V-3 CP-geen was verkry. Die tweede vektor (pHann-LR3CPsas) bevat komplementêre CP geensegmente van GLRaV-3. Die derde vektor (pHann-LR2CPsas) bevat komplementêre CP geensegmente van GLRa V-2. Die kasset bestaande uit die komplementêre CP geensegmente van beide GRSPaV en GLRaV-3, was gekloneer in pART27 (pART27-HSAScon), en gebruik om N tabacum cv. Petit Havana (SRI) te transformeer d.m.v. A. tumefaciens bemiddelde transformasie. Ongelukkig het potensiële transformante nie geregenereer op bewortelingsmedia nie; gevolglik was geen molekulêre toetse gedoen om transformasie te bevestig nie. Na suksesvolle transformante gegenereer is, sal infeksie met 'n rekombinante-virusvektor (bestaande uit PYX, die GFP geen as waarneembare merker en die komplementêre CP geensegmente van beide GRSPa V en GLRa V-3) gebruik word om die effektiwiteit van die vektore as weerstandsinduseerders te toets. 'n Sekondêre doelwit is by die projek gevoeg toe 'n behoefte aan GRSPaV spesifieke teenliggame binne die Suid-Afrikaanse wynbedryf geïdentifiseer is, vir gebruik in serologiese toetsing. Om toekomstige serologiese toetsing van GRSPa V te bemiddel, was die CP-geen geïsoleer en in 'n bakteriële uitdrukkingsisteem (PETI4b) uitgedruk, in die E. coli BL21(DE3)pLysS sellyn. Die uitgedrukte proteïne sal gebruik word vir die vervaardiging van GRSPa V CP spesifieke antiliggame.

The textbook outlines the cytological foundations of heredity, the regularities of Mendelian genetics, the chromosomal theory of heredity, the basics of molecular genetics and genetic engineering, the patterns of inheritance during distant hybridization, polyplodia and mutagenesis. Special attention is paid to the heterosis and genetics of populations. A significant part of the data is presented in the form of figures, diagrams, graphs and tables that facilitate the understanding of the material presented. Meets the requirements of the federal state educational standards of higher education of the latest generation. For students of higher educational institutions studying in agronomic specialties.

It is shown that the value of the water use efficiency at the leaf level, in terms of CO2 assimilation and transpiration, exceeds the value obtained from the ratio of yield to evapotranspiration for both considered crops (soybeans and grapes). The differences between the assessments of the water use efficiency in terms of CO2 assimilation and in terms of yield are greater in soybeans than in grapes.

Text generation is the task of generating natural language, and producing outputs similar to or better than human texts. Due to deep learning’s recent success in the field of natural language processing, computer generated text has come closer to becoming indistinguishable to human writing. Genetic Algorithms have not been as popular in the field of text generation. We propose a genetic algorithm combined with text classification and clustering models which automatically grade the texts generated by the genetic algorithm. The genetic algorithm is given poorly generated texts from a Markov chain, these texts are then graded by a text classifier and a text clustering model. We then apply crossover to pairs of texts, with emphasis on those that received higher grades. Changes to the grading system and further improvements to the genetic algorithm are to be the focus of future research.

In field experience, the influence of two-fold processing of table grape plants was studied by the Gibber-ellin, Zircon and Epin extra growth regulators on the structure of the bunch, yield and saccharinity of berry juice depending on the hydrothermal conditions of the growing season. It was established that the treatment of grape plants of the Great variety by growth regulators allows to reduce the negative influence of adverse ex-ternal factors and increase the productivity and quality of grape berries. Under more humidified conditions, treatment of Epin Extra plants (3.2 ml/l) contributes to an increase in yield by 82%, in dry conditions - Zir-conom (0.6 ml/l) by 1.5 times compared to control. The use of growth regulators contributes to a greater ac-cumulation of sugar in the juice of berries.

The primary imperative of the sustainable development of the wine sector is to obtain organic pro-ducts, with low consumption of resources, in conditions of increased economic efficiency and the use of tec-hnological links that contribute to reducing energy consumption and protecting the environment. As a result of crossing Vitis vinifera L. with Muscadinia rotundifolia Michx. interspecific vine genotypes have been obtai-ned and identified that can be multiplied by pruning, without grafting, thus obtaining rhizogenic propagating material that contributes to reducing the costs of setting up vineyards. They were approved as table grape vari-eties: "Malena", "Nistreana" and "Algumax" and grape varieties for fresh consumption and processing: "Au-gustina", "Alexandrina" and "Amethyst". By creating plantations, it will contribute to the extension of the area to the northern limit of vine cultivation and obtaining products derived from the "organic" category.

The article presents data on the structural elements of the spike and the biochemical content of the grains in newly created common wheat winter genotypes, cultivated in extreme drought conditions of 2020. It was concluded that the lines and cultivars created by hybridization and individual selection of segregating populations are well adapted to drought and have a high nutritional value of grains which is of great practical interest.

The original objectives are listed below: 1. Design vectors for constitutive expression of endochitinase from Trichoderma harzianum strain P1. Design vectors with signal peptides to target gene expression. 2. Extend transformation/regeneration technology to other cultivars of importance in the U.S. and Israel. 3. Transform cultivars with the endochitinase constructs developed as part of objective 1. A. Characterize foliar powdery mildew resistance in transgenic plants. Background of the topic Conventional breeding of grapevines is a slow and imprecise process. The long generation cycle, large space requirements and poor understanding of grapevine genetics prevent rapid progress. There remains great need to improve existing important cultivars without the loss of identity that follows from hybridization. Powdery mildew (Uncinula necator) is the most important fungal pathogen of grapevines, causing economic losses around the world. Genetic control of powdery mildew would reduce the requirement for chemical or cultural control of the disease. Yet, since the trait is under polygenic control, it is difficult to manipulate through hybridization and breeding. Also, because grapevines are heterozygous and vegetatively propagated cultivar identity is lost in the breeding process. Therefore, there is great need for techniques to produce transgenic versions of established cultivars with heterologous genes conferring disease resistance. Such a gene is now available for control of powdery mildew of grapevines. The protein coded by the Endochitinase gene, derived from Trichoderma harzianum, is very effective in suppressing U. necator growth. The goal of this proposal is to develop transgenic grapevines with this antifungal gene, and to test the effect of this gene on resistance to powdery mildew. Conclusions, achievements and implications Gene transfer technology for grape was developed using commercial cultivars for both wine and table grapes. It paved the way for a new tool in grapevine genetic studies enabling the alteration of specific important traits while maintaining the essential features of existing elite cultivars. Regeneration and transformation technologies were developed and are currently at an advanced stage for USA wine and Israeli seedless cultivars, representing the cutting edge of grape genetic engineering studies worldwide. Transgenic plants produced are tested for powdery mildew resistance in greenhouse and field experiments at both locations. It is our ultimate goal to develop transgenic grapes which will be more efficient and economical for growers to produce, while also providing consumers with familiar products grown with reduced chemical inputs.

Genetic analysis and mapping in grapes has been difficult because of the long generation period and paucity of genetic markers. In the present work, chromosome linkage maps were developed with RAPD, RFLP and isozyme loci in interspecific hybrid cultivars, and RAPD markers were produced in a V. vinifera population. In three cultivars, there were 19 linkage groups as expected for a species with 38 somatic chromosomes. These maps were used to locate chromosome regions with linkages to important genes, including those influencing powdery mildew and botrytis bunch rot resistance; flower sex; and berry shape. In V. vinifera, the occurrence of specific markers was correlated with seedlessness, muscat flavor and fruit color. Polymorphic RAPD bands included single copy as well as repetitive DNA. Mapping procedures were improved by optimizing PCR parameters with grape DNA; by the development of an efficient DNA extraction protocol; and with the use of long (17- to 24-mer) primers which amplify more polymorphic loci per primer. DNA fingerprint analysis with RAPD markers indicated that vinifera cultivars could be separated readily with RAPD profiles. Pinot gris, thought to be a sort of Pinot noir, differed by 12 bands from Pinot noir. This suggests that while Pinot gris may be related to Pinot noir, it is not likely to be a clone. The techniques developed in this project are now being further refined to use marker-assisted selection in breeding programs for the early selection of elite seedlings. Furthermore, the stage has been set for future attempts to clone genes from grapes based upon map locations.

Grapevine is perhaps the most widely grown fruit crop. To understand the genetic make-up so as to improve the yield and quality of grapes and grape products, researchers in Europe have recently sequenced the genomes of Pinot noir and its inbred. As expected, function of many grape genes is unknown. Functional genomics studies have become the major focus of grape researchers and breeders. Current genetic approaches for gene function studies include mutagenesis, crossing and genetic transformation. However, these approaches are difficult to apply to grapes and takes long periods of time to accomplish. It is thus imperative to seek new ways for grape functional genomics studies. Virus-induced gene silencing (VIGS) offers an attractive alternative for this purpose and has proven highly effective in several herbaceous plant species including tomato, tobacco and barley. VIGS offers several advantages over existing functional genomics approaches. First, it does not require transformation to silence a plant gene target. Instead, it induces silencing of a plant gene through infection with a virus that contains the target gene sequence, which can be accomplished within a few weeks. Second, different plant genes can be readily inserted into the viral genome via molecular cloning and functions of a large number of genes can be identified within a short period of time. Our long-term goal of this research is to develop VIGS-based tools for grapevine functional genomics, made of the genomes of Grapevine virus A (GVA) from Israel and Grapevine rupestris stem pitting-associated virus (GRSPaV) from Canada. GVA and GRSPaV are members of the Flexiviridae. Both viruses have single-stranded, positive sense RNA genomes, which makes them easy to manipulate genetically and excellent candidates as VIGS vectors. In our three years research, several major breakthroughs have been made by the research groups involved in this project. We have engineered a cDNA clone of GVA into a binary vector that is infectious upon delivery into plantlets of micropropagated Vitis viniferacv. Prime. We further developed the GVA into an expression vector that successfully capable to silence endogenous genes. We also were able to assemble an infectious full-length cDNA clones of GRSPaV. In the following sections Achievements and Detailed description of the research activities, we are presenting the outcome and results of this research in details.

Original objectives . 1. Test anti-fungal gene products for activity against Uncinula necator, Aspergillus niger, Rhizopus stolonifer and Botrytis cinerea. 2. For Agrobacterium transformation, design appropriate vectors with gene combinations. 3. Use biolistic bombardment and Agrobacterium for transformation of important cultivars. 4. Characterize gene expression in transformants, as well as level of powdery mildew and Botrytis resistance in foliage of transformed plants. Background The production of new grape cultivars by conventional breeding is a complex and time-consuming process. Transferring individual traits via single genes into elite cultivars was proposed as a viable strategy, especially for vegetatively propagated crops such as grapevines. The availability of effective genetic transformation procedures, the existence of genes able to reduce pathogen stress, and improved in vitro culture methods for grapes, were combined to serve the objective of this proposal. Effective deployment of resistance genes would reduce production costs and increase crop quality, and several such genes and combinations were used in this project. Progress The efficacy of two-way combinations of Trichoderma endochitinase (CHIT42), synthetic peptide ESF12 and resveratrol upon the control of growth of Botrytis cinerea and Penicillium digitatum were evaluated in vitro. All pairwise interactions were additive but not synergistic. Per objective 2, suitable vectors with important gene combinations for Agrobacterium transformation were designed. In addition, multiple gene co-transformation by particle bombardment was also tested successfully. In New York, transformation work focused on cultivars Chardonnay and Merlot, while the technology in Israel was extended to 41B, R. 110, Prime, Italia, Gamay, Chardonnay and Velika. Transgenic plant production is summarized in the appendix. Among plants developed in Israel, endochitinase expression was assayed via the MuchT assay using material just 1-5 days after co-cultivation. Plants of cv. Sugraone carrying the gene coding for ESF12, a short anti-fungal lytic peptide under the control of the double 358 promoter, were produced. Leaf extracts of two plants showed inhibition zones that developed within 48 h indicating the inhibitory effect of the leaf extracts on the six species of bacteria. X fastidiosa, the causal organism of Pierce's disease, was very sensitive to leaf extracts from ESF12 transformed plants. Further work is needed to verify the agricultural utility of ESF12 transformants. In New York, some transformants were resistant to powdery mildew and Botrytis fruit rot. Major conclusions, solutions, achievements and implications The following scientific achievements resulted from this cooperative BARD project: 1. Development and improvement of embryogenesis and tissue culture manipulation in grape, while extending these procedures to several agriculturally important cultivars both in Israel and USA. 2. Development and improvement of novel transformation procedures while developing transformation techniques for grape and other recalcitrant species. 3. Production of transgenic grapevines, characterization of transformed vines while studying the expression patterns of a marker gene under the control of different promoter as the 35S CaMV in different part of the plants including flowers and fruits. 4. Expression of anti-fungal genes in grape: establishment of transgenic plants and evaluation of gene expression. Development of techniques to insert multiple genes. 5. Isolation of novel grape specific promoter to control the expression of future antimicrobial genes. It is of great importance to report that significant progress was made in not only the development of transgenic grapevines, but also in the evaluation of their potential for increased resistance to disease as compared with the non engineered cultivar. In several cases, increased disease resistance was observed. More research and development is still needed before a product can be commercialized, yet our project lays a framework for further investigations.

Botrytis cinerea is the postharvest pathogen of many agricultural produce with table grapes, strawberries and tomatoes as major targets. The high efficiency with which B. cinerea causes disease on these produce during storage is attributed in part due to its exceptional ability to develop at very low temperature. Our major goal was to understand the genetic determinants which enable it to develop at low temperature. The specific research objectives were: 1. Identify expression pattern of genes in a coldenriched cDNA library. 2. Identify B. cinerea orthologs of cold-induced genes 3. Profile protein expression and secretion at low temperature on strawberry and grape supplemented media. 4. Test novel methods for the functional analysis of coldresponsive genes. Objective 1 was modified during the research because a microarray platform became available and it allowed us to probe the whole set of candidate genes according to the sequence of 2 strains of the fungus, BO5.10 and T4. The results of this experiment allowed us to validate some of our earlier observations which referred to genes which were the product of a SSH suppression-subtraction library. Before the microarray became available during 2008 we also analyzed the expression of 15 orthologs of cold-induced genes and some of these results were also validated by the microarray experiment. One of our goals was also to perform functional analysis of cold-induced genes. This goal was hampered for 3 years because current methodology for transformation with ‘protoplasts’ failed to deliver knockouts of bacteriordopsin-like (bR) gene which was our primary target for functional analysis. Consequently, we developed 2 alternative transformation platforms, one which involves an air-gun based technique and another which involves DNA injection into sclerotia. Both techniques show great promise and have been validated using different constructs. This contribution is likely to serve the scientific community in the near future. Using these technologies we generated gene knockout constructs of 2 genes and have tested there effect on survival of the fungus at low temperature. With reference to the bR genes our results show that it has a significant effect on mycelial growth of the B. cinerea and the mutants have retarded development at extreme conditions of ionic stress, osmotic stress and low temperature. Another gene of unknown function, HP1 is still under analysis. An ortholog of the yeast cold-induced gene, CCH1 which encodes a calcium tunnel and was shown to be cold-induced in B. cinerea was recently cloned and used to complement yeast mutants and rescue them from cold-sensitivity. One of the significant findings of the microarray study involves a T2 ribonuclease which was validated to be cold-induced by qPCR analysis. This and other genes will serve for future studies. In the frame of the study we also screened a population of 631 natural B. cinerea isolates for development at low temperature and have identified several strains with much higher and lower capacity to develop at low temperature. These strains are likely to be used in the future as candidates for further functional analysis. The major conclusions from the above research point to specific targets of cold-induced genes which are likely to play a role in cold tolerance. One of the most significant observations from the microarray study is that low temperature does not induce ‘general stress response in B. cinerea, which is in agreement to its exceptional capacity to develop at low temperature. Due to the tragic murder of the Co-PI Maria R. Davis and GopiPodila on Feb. 2010 it is impossible to deliver their contribution to the research. The information of the PI is that they failed to deliver objective 4 and none of the information which relates to objective 3 has been delivered to the PI before the murder or in a visit to U. Alabama during June, 2010. Therefore, this report is based solely on the IS data.

Reactome is a database of human biological pathways manually curated from the primary literature and peer-reviewed by experts. To evaluate the utility of Reactome pathways for predicting functional consequences of genetic perturbations, we compared predictions of perturbation effects based on Reactome pathways against published empirical observations. Ten cancer-relevant Reactome pathways, representing diverse biological processes such as signal transduction, cell division, DNA repair, and transcriptional regulation, were selected for testing. For each pathway, root input nodes and key pathway outputs were defined. We then used pathway-diagram-derived logic graphs to predict, either by inspection by biocurators or using a novel algorithm MP-BioPath, the effects of bidirectional perturbations (upregulation/activation or downregulation/inhibition) of single root inputs on the status of key outputs. These predictions were then compared to published empirical tests.

Dust particles carry microbial and chemical signatures from source regions to deposition regions. Dust and its occupying microorganisms are incorporated into, and can alter, snowpack physical properties including snow structure and resultant radiative and mechanical properties that in turn affect larger-scale properties, including surrounding hydrology and maneuverability. Microorganisms attached to deposited dust maintain genetic evidence of source substrates and can be potentially used as bio-sensors. The objective of this study was to investigate the impact of dust-associated microbial deposition on snowpack and microstructure. As part of this effort, we characterized the microbial communities deposited through dust transport, examined dust provenance, and identified the microscale location and fate of dust within a changing snow matrix. We found dust characteristics varied with deposition event and that dust particles were generally embedded in the snow grains, with a small fraction of the dust particles residing on the exterior of the snow matrix. Dust deposition appears to retard expected late season snow grain growth. Both bacteria and fungi were identified in the collected snow samples.