Genome and transcriptome sequencing of vitis vinifera cv pinotage

Thesis (PhD)--Stellenbosch University, 2018.ENGLISH ABSTRACT: Examining the genetic basis of natural phenotypic variation, and the transfer of this knowledge to a breeding program for improved crop cultivars or livestock races, is a major goal for biological sciences. As grapevine (Vitis vinifera) is one of the most important crop plants in the world, research into its genetics is imperatave, both in terms of sustainable food production and the vast economic impact of the wine industry. Grapevine displays a great level of intraspecies phenotypic diversity in viticultural and oenological traits, between cultivars. Understanding this genetic diversity is an important step towards developing improved grapevine cultivars, but also the conservation of the important traditional cultivars. Vitis vinifera cv Pinotage is an artificial Pinot noir/ Cinsaut cross, created with the South African climate and growing conditions in mind. Today it is a commercial cultivar, used for the production of premium wines, deeply rooted in the South African wine culture and history. This study focused on the next-generation sequencing and bioinformatic analysis of the Pinotage genome and transcriptome. A de novo assembly strategy was followed to produce the first Pinotage draft genome sequence. Sequencing read data were also aligned to the available reference Pinot noir genome, and from this alignment the Pinotage/ Pinot noir variant density, determined. This was followed by a more in-depth focus on a number of functional gene clusters with more than 50% of their genes influenced by these variants. Furthermore, this is the first research to lend scientific support to the current wine trend of exclusive, superior wines produced from old vineyards. These old-vine wines are assumed to have a deeper character and more flavour. To explore the role of genetics and differential gene expression in this phenomenon, RNA-seq data were used to survey and compare the leaf and berry transcriptomes of young and old Pinotage vines, at harvest. Differential gene expression between young and old vines was studied, and the involvement of these genes in fruit ripening, discussed. A general trend towards delayed ripening in older vines was observed. This suggests that the berries remain attached to the vine for a longer period, thereby allowing more time for flavour compounds to accumulate. In the final part of the study, the Pinotage genome and transcriptome data were combined to identify Pinotage genes present in neither the reference Pinot noir PN40024 nor ENTAV115. These genes were classified as both structural and regulatory genes and it was shown that genes involved in the stress response network are a major gene class contributing to the genetic differences between Pinotage and Pinot noir. A plant species is constantly challenged by various biotic and abiotic stresses and it is an evolutionary investment to diversify genes responsible for stress response, to be able to efficiently overcome these stresses. The information generated in this study will aid in grapevine breeding programs for sustainable production of high quality wine in a changing environment.AFRIKAANSE OPSOMMING: Die ondersoek na die genetiese basis van natuurlike fenotipiese variasie, en die oordrag van hierdie kennis na 'n teelprogram vir verbeterde gewaskultivars of vee-rasse, is 'n belangrike doelwit vir biologiese wetenskappe. Aangesien wingerd (Vitis vinifera) een van die belangrikste gewasplante ter wêreld is, is navorsing in sy genetika noodsaaklik, beide in terme van volhoubare voedselproduksie en die wye ekonomiese impak van die wynbedryf. Wingerd vertoon 'n groot vlak van fenotipiese diversiteit in die spesie, in wingerd- en wynboukundige eienskappe, tussen kultivars. Om hierdie genetiese diversiteit te verstaan, is 'n belangrike stap in die ontwikkeling van verbeterde wingerdkultivars, maar ook die bewaring van die belangrike tradisionele kultivars. Vitis vinifera kultivar Pinotage is 'n kunsmatige Pinot noir/ Cinsaut kruising, geskep met die Suid-Afrikaanse klimaat en groeitoestande in gedagte. Vandag is dit 'n kommersiële kultivar, wat gebruik word vir die produksie van gehalte wyne, diep gewortel in die Suid-Afrikaanse wynkultuur en -geskiedenis. Hierdie studie het gefokus op die volgende-generasievolgordebepaling en bioinformatiese analise van die Pinotage-genoom en transkriptoom. 'n De novo-samestellingstrategie is gevolg om die eerste Pinotage konsep-genoomvolgorde te produseer. Opvolgingsleesdata is ook in lyn gebring met die beskikbare verwysings Pinot noirgenoom en vanaf hierdie belyning is die Pinotage/ Pinot noir-variantdigtheid bepaal, gevolg deur 'n meer in-diepte fokus op 'n aantal funksionele geen-groepe met meer as 50% van hul gene beïnvloed deur hierdie variante. Verder is dit die eerste navorsing wat wetenskaplike ondersteuning verleen aan die huidige wyn-tendens van eksklusiewe, uitstekende wyne geproduseer van ou wingerde. Hierdie ouwingerdwyne word veronderstel om 'n dieper karakter en meer geur te hê. Om die rol van genetika en differensiële geenuitdrukking in hierdie verskynsel te ondersoek, is RNSopeenvolgings- data gebruik om die blaar- en korrel transkriptome van jong en ou Pinotagewingerdstokke, tydens oestyd, te ondersoek en te vergelyk. Differensiële geenuitdrukking tussen jong en ou wingerdstokke is bestudeer, en die betrokkenheid van hierdie gene in rypwording word bespreek. 'n Algemene neiging tot vertraagde rypwording in ouer wingerde is waargeneem. Dit dui daarop dat die korrels vir 'n langer tydperk aan die wingerdstok bly, en dat meer geurverbindings in die korrels kan versamel. In die laaste gedeelte van die studie is die Pinotage-genoom en transkriptoomdata gekombineer om Pinotage variëteit-spesifieke gene te identifiseer, wat nie in die verwysing genoom Pinot noir PN40024 of ENTAV115 voorkom nie. Hierdie gene is geklassifiseer as beide strukturele en regulatoriese gene en dit is gewys dat gene wat betrokke is by die stresresponsnetwerk, 'n belangrike geenklas is wat bydra tot die genetiese verskille tussen Pinotage en Pinot noir. 'n Plantspesie word voortdurend uitgedaag deur verskeie biotiese en abiotiese stres en dit is 'n evolusionêre belegging om gene wat verantwoordelik is vir stresrespons te diversifiseer, om hierdie stres doeltreffend te oorkom. Die inligting wat in hierdie studie gegenereer is, sal van nut wees in wingerdbouprogramme vir die volhoubare produksie van hoë kwaliteit wyn in 'n veranderende omgewing