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Marker-Assisted Breeding for Papaya Ringspot Virus Resistance in Carica Papaya L

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Marker-Assisted Breeding for Papaya Ringspot Virus Resistance in Carica Papaya L Marker-Assisted Breeding for Papaya Ringspot Virus Resistance in Carica papaya L. Author O'Brien, Christopher Published 2010 Thesis Type Thesis (Masters) School Griffith School of Environment DOI https://doi.org/10.25904/1912/3639 Copyright Statement The author owns the copyright in this thesis, unless stated otherwise. Downloaded from http://hdl.handle.net/10072/365618 Griffith Research Online https://research-repository.griffith.edu.au Marker-Assisted Breeding for Papaya Ringspot Virus Resistance in Carica papaya L. Christopher O'Brien BAppliedSc (Environmental and Production Horticulture) University of Queensland Griffith School of Environment Science, Environment, Engineering and Technology Griffith University Submitted in fulfilment of the requirements of the degree of Master of Philosophy September 4, 2009 2 Table of Contents Page No. Abstract ……………………………………………………… 11 Statement of Originality ………………………………………........ 13 Acknowledgements …………………………………………. 15 Chapter 1 Literature review ………………………………....... 17 1.1 Overview of Carica papaya L. (papaya)………………… 19 1.1.1 Taxonomy ………………………………………………… . 19 1.1.2 Origin ………………………………………………….. 19 1.1.3 Botany …………………………………………………… 20 1.1.4 Importance …………………………………………………... 25 1.2 Overview of Vasconcellea species ……………………… 28 1.2.1 Taxonomy …………………………………………………… 28 1.2.2 Origin ……………………………………………………… 28 1.2.3 Botany ……………………………………………………… 31 1.2.4 Importance …………………………………………………… 31 1.2.5 Brief synopsis of Vasconcellea species …………………….. 33 1.3 Papaya ringspot virus (PRSV-P)…………………………. 42 1.3.1 Overview ………………………………………………………. 42 1.3.2 Distribution …………………………………………………… 43 1.3.3 Symptoms and effects ……………………………………….. .. 44 1.3.4 Transmission …………………………………………………. 45 1.3.5 Control ………………………………………………………. .. 46 1.4 Breeding ………………………………………………………. 47 1.4.1 Disease resistance …………………………………………….. 47 1.4.2 Biotechnology………………………………………………….. 50 1.4.3 Intergeneric hybridisation…………………………………….. 51 1.4.4 Genetic transformation………………………………………… 54 1.4.5 DNA analysis ………………………………………………...... 56 1.4.6 SCAR marker for PRSV-P resistance…………………………. 60 3 1.5 Research plan ……………………………………………….. 62 Project objective …………………………………………… 62 Project aims ………………………………………………… 62 Flow chart …………………………………………………... 63 Chapter 2 Intrageneric and intergeneric hybridisation between Vasconcellea species and Carica papaya L. …. 65 2.1 Introduction …………………………………………………… 67 2.2 Materials and methods ……………………………………….. 70 2.2.1 Plant material ……………………………………………….. …. 70 2.2.2 Cross pollination ……………………………………………. …. 71 2.2.3 Progeny produced by seed …………………………………...…. 73 2.2.4 Progeny produced by embryo rescue …………………………… 73 2.2.5 Morphological characters ………………………………………. 75 2.2.6 Pollen fertility……………………………………………………. 75 2.2.7 Screening for PRSV-P resistance ………………………………. 76 2.3 Results ………………………………………………………. ….. 77 2.3.1 V. pubescens x V. parviflora [F1]……………………………….. 77 2.3.2 V. pubescens x V. parviflora [F2]………………………………… 80 2.3.3 V. pubescens x V. parviflora [F3] RR …………………………… 81 2.3.4 (V. pubescens x V. parviflora F2 RR) to V. parviflora [BC1]…. 84 2.3.5 {(V. pubescens x V. parviflora F2 RR) to V. parviflora} BC1 to V. parviflora [BC2] ………………………………………….. 85 2.3.6 C. papaya x V. parviflora [F1]……………………………………. 87 2.3.7 C. papaya x V. pubescens [F1]…………………………………… 89 2.3.8 C. papaya x (V. pubescens x V. parviflora F2 RR) ……………… 91 2.4 Discussion ………………………………………………………… 95 Chapter 3 The use of a SCAR and CAPS marker to assist hybridisation between C. papaya L. and Vasconcellea species ……….……………………. …………… 101 3.1 Introduction ……………………………………………………… 102 3.2 Materials and methods ………………………………………… 104 3.2.1 Plant material …………………………………………………… 104 4 3.2.2 DNA extraction and marker analysis …………………………….. 105 3.3 Results ………………………………………………………….. … 112 3.3.1 V. pubescens x V. parviflora [F1] ………………………………………………… …. 112 3.3.2 V. pubescens x V. parviflora [F3]……………………………………………………. 114 3.3.3 (V. pubescens x V. parviflora F2 RR) to V. parviflora [BC1]………. 116 3.3.4 {(V. pubescens x V. parviflora F2 RR) to V. parviflora BC1} to V. parviflora [BC2]……………………………………………… 118 3.3.5 Vasconcellea species and hybrids (V. quercifolia, V. stipulata, V. cauliflora, V. goudotiana and C. papaya x V. quercifolia [F1])… 118 3.3.6 C. papaya genotypes tested with Opk4_1r marker …………………. 121 3.3.7 C. papaya x V. pubescens [F1] and C. papaya x V. parviflora [F1] 123 3.3.8 C. papaya x V. parviflora [F1] ………………………………………………………… 125 3.3.9 C. papaya x (V. pubescens x V. parviflora [F2] containing RR allele for PRSV-P resistance) ………………………………… 125 3.4 Discussion ………………………………………………………… 130 Appendix A ……………………………………………………………. 133 Drew and Smith media ……. ………………………………… 133 Brewbaker and Kwack medium…. .………………………… 135 References …………………………………………………………… 136 5 List of Figures Figure 1.1.3.1 Morphological characters of C. papaya ……………… 21 Figure 1.1.3.2 Female C. papaya plant ……………………………….. 23 Figure 1.1.3.3 Male C. papaya plant ………………………………….. 24 Figure 1.2.5.1 Morphological characters of V. pubescens ……………. 33 Figure 1.2.5.2 Morphological characters of V. parviflora ……………. 35 Figure 1.2.5.3 Morphological characters of V. quercifolia ……………… 36 Figure 1.3.3.1 PRSV-P symptoms on papaya leaves, fruit and petioles........................................................................... 44 Figure 2.2.4.1 Immature seeds 90 days after an intergeneric cross……. 73 Figure 2.2.4.2 In vitro V. pubescens x V. parviflora [F3] hybrid seedling 74 Figure 2.2.4.3 Humidity cabinet used for acclimatisation…………….. 74 Figure 2.2.7.1 PRSV-P infection on a C. papaya leaf …........................ 76 Figure 2.3.1.1 Flowers of C. papaya, V. pubescens, V. parviflora, C. papaya x (V. pubescens x V. parviflora) and V. pubescens x V. parviflora [F1] ………………………………. 77 Figure 2.3.1.2 Leaf shape of V. parviflora, V. pubescens and V. pubescens x V. parviflora [F1]……………………………… 78 Figure 2.3.2.1 Variation observed in colour and morphology of flowers resulting from crosses of V. pubescens x V. parviflora [F1], [F2] generations……………………………… 80 Figure 2.3.3.1 Variation observed in leaves of F1, F2 and F3 generations of V. pubescens x V. parviflora crosses ……… 82 Figure 2.3.4.1 Variation observed in colour and morphology of flowers resulting from crosses of V. pubescens x V. parviflora [F1], [F2] [BC1] generations …………………………………………….… 84 Figure 2.3.5.1 Group of BC2 plants {(V. pubescens x V. parviflora F2 RR) to V. parviflora BC1} to V. parviflora……… 85 Figure 2.3.5.2 Female flowers of BC2 {(V. pubescens x V. parviflora F2 RR) to V. parviflora BC1} to V. parviflora………. 86 6 Figure 2.3.5.3 Male flowers of BC2 {(V. pubescens x V. parviflora F2 RR) to V. parviflora BC1} to V. parviflora…… 86 Figure 2.3.6.1 Flowers of C. papaya; V. parviflora and C. papaya x V. parviflora [F1]……………………………………………… 87 Figure 2.3.6.2 C. papaya x V. parviflora [F1] leaves showing varying degrees of mottling…………………………………… 88 Figure 2.3.6.3 Leaves of C. papaya, V. parviflora and C. papaya x V. parviflora [F1]………………………………… 89 Figure 2.3.7.1 Flowers of C. papaya, V. pubescens and C. papaya x V. pubescens [F1]………………………………… 90 Figure 2.3.7.2 Leaves of C. papaya, V. pubescens and C. papaya x V. pubescens [F1]………………………………… 91 Figure 2.3.8.1 Variations in flower colour on plants of C. papaya x (V. pubescens x V. parviflora F2 RR) in comparison to flower colour of V. parviflora and C. papaya female and male plants………………….. ……………………………. 93 Figure 2.3.8.2 Variations in leaf shape on plants of C. papaya x (V. pubescens x V. parviflora F2 RR) in comparison to leaves of parent plants ………………………………………… 94 Figure 3.3.1.1 Amplified bands of the Opk4_1r SCAR marker in V. pubescens x V. parviflora [F1]……………………………… 112 Figure 3.3.1.2 Application of CAPS Psi1k4 marker to V. pubescens x V. parviflora [F1]……………………………… 113 Figure 3.3.2.1 Amplified bands of the Opk4_1r SCAR marker in V. pubescens x V. parviflora [F3]…………………………………… 114 Figure 3.3.2.2 Application of CAPS Psi1k4 marker to V. pubescens x V. parviflora [F3]………………………………… 115 Figure 3.3.3.1 Amplified bands of the Opk4_1r SCAR marker in backcross population (V. pubescens x V. parviflora F2 RR) to V. parviflora [BC1] and [BC2] populations…………………… 116 Figure 3.3.3.2 Application of CAPS Psi1k4 marker to (V. pubescens x V. parviflora F2 RR) to V. parviflora [BC1]………..117 7 Figure 3.3.5.1 Amplified bands of the Opk4_1r SCAR marker in Vasconcellea spp. (V. quercifolia, V. stipulata, V. cauliflora, V. goudotiana and C. papaya x V. quercifolia [F1]) ……………… 119 Figure 3.3.5.2 Application of CAPS Psi1k4 marker to Vasconcellea spp. (V. quercifolia, V. stipulata, V. cauliflora, V. goudotiana and C. papaya x V. quercifolia [F1])……………... 120 Figure 3.3.6.1 Amplified bands of the Opk4_1r SCAR marker in C. papaya……………………………………………………….……… 121 Figure 3.3.6.2 Application of CAPS Psi1k4 marker to C. papaya………..122 Figure 3.3.7.1 Amplified bands of the Opk4_1r SCAR marker in C. papaya x V. pubescens [F1] and C. papaya x V. parviflora [F1]……………………………………... 123 Figure 3.3.7.2 Application of CAPS Psi1k4 marker to C. papaya x V. pubescens [F1] and C. papaya x V. parviflora [F1]…………….. 124 Figure 3.3.9.1 Amplified bands of the Opk4_1r SCAR marker in C. papaya x (V. pubescens x V. parviflora F2)………………… 126 Figure 3.3.9.2 Application of CAPS Psi1k4 marker to C. papaya x (V. pubescens x V. parviflora F2)………………………………. 127 Figure 3.3.9.3 Application of CAPS Psi1k4 marker to C. papaya x (V. pubescens x V. parviflora F2)……………………………. 128 8 List of Tables Table 1.1.3.1 Characteristics of the major flower types in papaya ………… 22 Table 1.1.4.1 Top 10 papaya-producing countries in 2005 ………………… 26 Table 1.2.2.1 Vasconcellea species ................................................................ 29 Table 2.3.1.1 A summary of morphological characters for C. papaya, V. pubescens, V. parviflora and their F1 and F2 hybrids. ……………. 79 Table 2.3.3.1 Summary of hybridisation of V. pubescens x V. parviflora [F3] RR…………………………………………………… 81 Table 2.3.3.2 A summary of morphological characters for C.
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