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Genetic Identification of Cultivars and Marker Analysis in Olives (Olea

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Genetic Identification of Cultivars and Marker Analysis in Olives (Olea t:i\ Sl i P 15 "u.01 Genetic ldentifïcation of Cultivars and Marker LIL'ì, Analysis in Olives (Olea europaeø L.) Genet Teshome Mekuria B.Ag.Sc., Grad. Dip. (Nat. Res.) submitted in fulfilment of the requirements for the degree of Doctor of Philosophy Department of Horticulture, Viticulture and Oenology waite ^äï:'uiï:ffi rn s ritute July 2001 ( i!, rål¡l , . :' -.'^.* t' i'ri,;;fi:r' :1. ! /' Olive, OIea europaea sativa Table of Contents I Abstract iii Declaration iv Acknowledgments List of Tables vi List of Figures Chapter One General introduction 1 I 1.1 Introduction 1.2 Botanical classification of olives 1 1.2.1 Cultivated olives 2 1.2.2 Wild olives ^J 1.2.3 Ferals or escapes J 1.3 Olive cultivation 4 1.3.1 V/orld 4 1.3.1.1 History 4 1.3.1.2 Major producing areas 5 1.3.1.3 Economics 6 1.3.2 Australia 1 1.3.2.1 History 1 1.3.2.2 Major producing areas 8 1.3.2.3 Economics 9 1.4 Olive diseases 9 1.4.1 Overview 9 1.4.2 Olive leaf sPot disease 10 1.5 Techniques for cultivar identification 11 1.5.1 MorPhologY t2 T2 1.5.2 Gene Products 1.5.2.1 Isozyme T2 1.5.2.2 Restriction fragment length polymorphism (RFLP) I4 1.5.2.3 Microsatellite t4 1.5.2.4 Amplified fragment length polymorphism (AFLP) 15 1.5.2.5 Random amplified polymorphic DNA (RAPD) 16 1.6 Techniques for marker analYsis t] 1.6.1 Overview t7 1.6.2 Sequence tagged site t9 t.7 Summary 20 1.8 Thesis aims 2l Chapter Two General materials and methods 22 2.t Leaf material 22 2.2 Leaf storage and handling 22 2.3 DNA isolation 22 2.4 DNA amplification 23 24 2.5 Separation of PCR Products 2.5.1 Agarose gels (ethidium bromide stained) 24 2.5.2 Polyacrylamide gels (silver stained) 25 2.6 Data analysis 26 Chapter Three Genetic similarities between accessions of commercial cultivars 28 3.1 Abstract 28 3.2 Introduction 28 3.3 Materials and methods 30 3.3.1 Leaf material 30 3.3.2 DNA isolation and PCR amplification 32 3.3.3 Primer screening 32 3.3.4 Data analysis 32 3.4 Results 33 3.4.I Primer screening and amplification characters 33 3.4.2 DNA amplification between duplicate DNA extracts 34 3.4.3 Accessions of cultivars with similar designated names 34 3.4.3.1 Genetic similarities between accessions of Manzanillo 34 3.4.3.2 Genetic similarities between accessions of Kalamata and Calamata 36 3.4.3.3 Genetic similarities between accessions of Verdale 38 3.4.3.4 Genetic similarities between accessions of SA Verdale 38 3.4.3.5 Genetic similarities between accessions of Wagga Verdale 38 3.4.3.6 Genetic similarities between accessions of Verdale, SA Verdale, and Wagga Verdale 39 3.4.3.7 Genetic similarities between accessions of Nevadillo and Nevadillo Blanco 4T 3.4.3.8 Genetic similarities between accessions of Picual 4t 3.4.3.9 Genetic similarities between accessions of Corregiola and Corregiolo 42 3.4.3.10 Genetic similarities between accessions of Frantoio, Frantoja, and Frantojol 42 3.4.4 Accessions of cultivars with suspected synonyms 43 3.4.4.1 Genetic similarities between accessions of Nevadillo, Nevadillo Blanco, and Picual 43 3.4.4.2 Genetic similarities between accessions of Corregiolo, Corregiola, Frantoio, Frantoj a, and Frantoj ol 45 3.4.5 Identification of some misnamed cultivars 47 3.5 Discussion 48 3.5.1 Amplification characters of primers and reproducibility of results 48 3.5.2 Genetic similarities between accessions of cultivars with similar designated names 40 3.5.3 Genetic similarities between suspected synonyms 53 3.6 Summary 54 Chapter Four Genetic diversity within an isolated olive population in relation to feral spread 5õ 4.1 Abstract 56 4.2 Introduction 56 4.3 Materials and methods 58 4.3.1 Allocation of trees to putative groups within the population 58 4.3.2 Leaf material 58 4.3.3 DNA isolation and amplification 61 4.3.4 Separation of PCR products 6I 4.3.5 Data analysis 61 4.3.6 Measurement of tree age 62 4.4 Results 63 4.4.1 Genetic assessment 63 4.4.1.1 Genetic similarities between and within the putative groups 63 4.4.I.2 Genetic similarities between and within the molecular clusters 64 4.4.2 Assessment of tree age 69 4.4.2.1 Age of trees within the putative groups 69 4.4.2.2 Age of trees within the molecular clusters 73 4.5 Discussion 76 4.6 Summary 81 Chapter Five Genetic markers tinked to olive leaf spot resistance and susceptibility 82 5.1 Abstract 82 5.2 Introduction 82 5.3 Materials and methods 84 5.3.1 Segregating population 84 5.3.2 Leafmaterial 85 5.3.3 Bulked segregant analysis 85 5.3.3.1 Preparation of bulked DNA samples 85 5.3.3.2 Primer screening 86 5.3.3.3 Confirmation of markers linked to olive leaf spot 86 5.3.3.4 Data analysis 86 5.4 Results 87 5.5 Discussion 95 5.6 Summary 97 Chapter Six Sequence tagged site for the RAPD marker linked to leaf spot resistance in olives 98 6.1 Abstract 98 6.2 Introduction 98 6.3 Methodology 99 6.3.I Isolation and purification of the RAPD marker 99 6.3.2 DNA cloning and sequencing 102 6.3.2.1 DNA cloning t02 6.3.2.2 Small scale preparation of plasmid DNA r02 6.3.2.3 Preparation of cloned DNA for sequencing 103 6.3.2.4 Design and analysis of sequence specific primers 104 6.3.2.5 Purification of PCR products amplified with sequence specific primers 105 6.4 Results 106 6.4.1 Isolation and purification of the RAPD marker 106 6.4.2 Cloning and sequencing of the RAPD marker 101 6.4.3 Analysis of sequence specific primers 108 6.4.3.1 STS primers designed using PRIMER computer pfogram 108 6.4.3.2 The STS primers selected by visual observation 110 6.4.3.3 The STS primers designed using NetPrimer computer program 110 6.4.3.4 Analysis of the STS primer pair G7F and G7R tt2 6.5 Discussion t20 6.5.1 Isolation and purification of the RAPD marker 120 6.5.2 Cloning and sequencing of the RAPD marker, and analysis of the STS 120 6.5.2.1 Testing of the STS marker produced by the primer pair G7F and G7R t21 6.5.2.2 Comparison of results obtained with the lO-mer primer, OPA1 l and the STS primer pair, G7F and G7R 122 6.6 Summary 123 Chapter Seven General discussion L24 References cited 130 Appendix 145 Poster and papers from the thesis research 145 Abstract the f,'st introduction to South olive cultivation started in Australia in the early 1g00s, with government support, the industry Australia in 1g36. Despite numerous attempts, and much in South Australia is ideal for has never been commercially successful, although the climate many olive groves were growing olives that produce high quality oil. Over the years, original names, have been lost' abandoned, and records about the cultivars, including many olive trees that now extend Natural outcrossing has led to the development of feral or escape over much of southern Australia. have emigrated to Since 1950, many migrants from regions bordering the Mediterranean to over $A100 million per Australia creating a demand for orive products that now amounts been planted in annum. To cater for this increased demand, numerous olive groves have the olive industry depends Australia, many financed by large investors. However, the future of Other approaches on a number of factors, including the correct identification of cultivars. to the local include the selection of trees from the feral population that are adapted markers to improve environment and have high quality oil, and the development of molecular the research described in the efhciency of breeding programs. These topics were the focus of this thesis. such as Kalamata, The results from cultivar identification showed that while some cultivars, and Mission picual, and Manzanillo, exhibited little genetic variation, others such as Verdale could be considered as showed such a high level of genetic variation that some accessions the Verdale group were separate cultivars. It appears likely that some of the accessions within misnaming of cultivars was either misnamed or that errors were made during propagation. The fingerprint obtained clearly demonstrated for the accession of Calamata from Davis. The DNA In another for this accession could not be distinguished from that obtained for Manzanillo. as Manzanillo' The instance, an accession of Mission also produced the same DNA fingerprint that is now used DNA furgerprints developed in this study have formed the basis for a database for a commercial identification service to growers' to a large extent on the For any crop species, the gains that can be made from selection depend trees was estimated genetic variability of the population. The genetic variability of feral olive years old, consisting of an original by selecting an isolated population that was about 70 - 80 Forty five individual trees were grove, with a few planted progeny, and numerous feral trees' genetic similarities found between the 45 selected and their DNA fingerprints compared. The of high genetic diversity in the individuals varied from 6JVo to ggvo, confirming the existence were significantly different indicating gene pool.
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