Genetic characterisation and breeding of wine yeasts

Van der Westhuizen, T. J. (Theunes Johannes) (1990)

Thesis (MSc)--Stellenbosch University, 1990.

Thesis

ENGLISH ABSTRACT: To remain competitive in the market place, the South African wine industry will have to direct well-planned yeast strain-development programmes. However, the winemaker can only benefit from the extensive biochemical and molecular information of the yeast cell and the impressive arsenal of genetic techniques available, if the wine industry defines its requirements in genetic terms. The successful application of these genetic and recombinant deoxyribonucleic acid (DNA) techniques in breeding programmes depends on the availability of rapid and reliable techniques to differentiate between parental and hybrid strains. Ten strains of Saccharomyces cerevisiae used for commercial production of wine in South Africa, were characterised by electrophoretic banding patterns of total soluble cell proteins, DNA restriction fragments and chromosomal DNA. Variations in the protein and DNA profiles of strains N6, N21, N66, N76, N95 and N97 were apparent in the number, position and intensity of the bands. Strains N93 and N181 originated from the same culture and, as expected, displayed the same characteristic protein, DNA restriction fragment and chromosomal banding patterns. Similar protein and DNA profiles were also obtained for killer strain N96 and strain N91. Strain N91 is a derivative of strain N96, cured of the K2 killer character. Results obtained by electrophoretic fingerprinting and karyotyping corresponded well, indicating that these techniques are valuable in the identification and quality control of industrial wine yeasts. The value of electrophoretic fingerprinting and karyotyping was also demonstrated in a breeding programme. The aim of this breeding programme was to obtain hybrids that combine the desired oenological characteristics of strains N76 and N96, and of strains N96 and N181. The protein banding patterns of hybrids USM21, USM22 and USM23 were identical and contained a combination of prominent unique bands present in the profiles of parental strains, N76 and N96H (N96H is a haploid derived from N96). The DNA restriction fragment profiles of hybrids USM21, USM22 and USM23 contained slight variations, whereas their profiles were quite different from those of their parental strains, N76 and N96H. The contour clamped homogeneous electric field (CHEF) karyotypes of hybrids USM21, USM22 and USM23 were identical but differed from those of their parental strains, N76 and N96H. The protein profiles of hybrid USM30 and its parental strains, N96H and N181, were similar, whereas their DNA restriction fragment banding patterns and CHEF karyotypes showed discrete differences. In conclusion, protein and DNA fingerprinting techniques were found to be valuable in selecting four hybrid killer strains after mass spore-cell mating. These four killer hybrids contain desirable oenological properties long sought after by the South African wine industry. Fermentation trials and evaluation of these hybrids were conducted independently by the Deparment of Oenology, University of Stellenbosch and by Stellenbosch Farmers' Winery and they have now been released for commercial wine production.

AFRIKAANSE OPSOMMING: Om mededingend in die handel te bly, sal die Suid-Afrikaanse wynbedryf weloorwoe gisras-ontwikkelingsprogramme moet loads. Die wynmaker sal egter slegs voordeel kan trek uit die omvattende biochemiese en molekul...Lre inligting oor die gissel en die indrukwekkende arsenaal van genetiese tegnieke wat beskikbaar is, indien die wynbedryf sy vereistes in genetiese terme definieer. Die suksesvolle toepassing van hierdie genetiese en rekombinante deoksiribonuklei"ensuur (DNA) tegnieke in telingsprogramme sal afhang van die beskikbaarheid van vinnige en betroubare tegnieke om tussen ouerlike en hibried-rasse te onderskei. Tien rasse van Saccharomyces cerevisiae wat vir kommersiele wynproduksie in Suid-Afrika gebruik word, is met behulp van elektroforetiese bandpatrone van totale oplosbare selprotei"ene, DNA-restriksiefragmente en chromosomale DNA gekarakteriseer. Variasies in die protei"en- en DNA-profiele van rasse N6, N21, N66, N76, N95 en N97 het geblyk uit die aantal, posisie en intensiteit van die bande. Rasse N93 en N181 het uit dieselfde kultuur ontstaan en het, soos verwag, dieselfde karakteristieke protei"en-, DNA-restriksiefragmenten chromosomale bandpatrone getoon. Soortgelyke protei"en en DNA profiele is ook vir killerras N96 en ras N91 verkry. Ras N91 is 'n variant van ras N96 wat die K2 killerkenmerk verloor het. Resultate wat met behulp van elektroforetiese vingermerking en kariotipering verkry is, het goed ooreengestem en dui daarop dat hierdie tegnieke waardevol is vir die identifisering en beheer van industriele giste. Die waarde van elektroforetiese vingermerking en kariotipering in telingsprogramme is ook gedemonstreer. Die doel van hierdie telingsprogram was om hibriede te kry waarin die gewenste kenmerke van rasse N76 en N96, en van rasse N96 en N181, gekombineer is. Die protei"en-bandpatrone van hibriede USM21, USM22 en USM23 was identies en het 'n kombinasie van prominente unieke bande, teenwoordig in die profiele van hul ourlike rasse, N76 en N96H (N96H is 'n haploi"de afstammeling van N96), bevat. Die DNArestriksiefragment- profiele van hibriede USM21, USM22 en USM23 toon geringe onderlinge verskille, maar hul profiele het wesenlik van die van hul ouerlike rasse, N76 en N96H, verskil. Die kontoergeklampde-homogene-elektriese-veld (CHEF) elektroforetiese kariotipes van hibriede USM21, USM22 en USM23 was identies, maar het verskil van die van hul ouerlike rasse, N76 en N96H. Die protei"enprofiele van hibried USM30 en sy ouerlike rasse, N96H en N181, was soortgelyk, terwyl hul DNA-restriksiefragment-bandpatrone en CHEF-kariotipes diskrete verskille getoon het. Ten slotte is gevind dat prote'ien- en DNAvingermerkingstegnieke waardevol was in die seleksie van vier hibried-killerrasse na massa spoor-sel paring. Hierdie vier killerhibriede beskik oor gewenste wynkundige eienskappe waarna die Suid-Afrikaanse wynbedryf reeds lank soek. Fermentasie-proewe en evaluering is onafhanklik deur die Departement Wynkunde, Universitiet van Stellenbosch en deur Stellenbosch-Boerewynmakery gedoen en hulle is nou vir kommersiele wynproduksie vrygestel.

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