Testing Alternatic Breeding Methods in White Clover
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Lincoln University Digital Thesis Copyright Statement The digital copy of this thesis is protected by the Copyright Act 1994 (New Zealand). This thesis may be consulted by you, provided you comply with the provisions of the Act and the following conditions of use: you will use the copy only for the purposes of research or private study you will recognise the author's right to be identified as the author of the thesis and due acknowledgement will be made to the author where appropriate you will obtain the author's permission before publishing any material from the thesis. Testing alternative breeding methods in white clover A thesis submitted in partial fulfilment of the requirements for the Degree of Doctor of Philosophy at Lincoln University by Richard Mark George Lincoln University 2014 Abstract of a thesis submitted in partial fulfilment of the requirements for the Degree of Doctor of Philosophy. Testing alternative breeding methods in white clover by Richard Mark George A series of experiments were conducted to estimate the genetic parameters for an elite breeding population of white clover (Trifolium repens L.) in both an irrigated and dryland environment. Data collected from two environments using spaced-planted and mini-plot trials were used to demonstrate the rationale for pursing various traditional and modern marker- assisted selection breeding methods in white clover. In addition, and under the over-arching theme of investigating breeding strategies, supplementary findings are presented regarding pollination patterns within a white clover population in isolation cages. In an experiment designed to generate half-sib and full-sib families for progeny evaluation, pollen dispersal within isolation cages using wild bumble bees (Bombus sp.) was monitored using simple sequence repeat molecular markers (SSR markers). Within bi-parental crosses, no detectable levels of self-fertilised germinated seedlings were detected, nor were there any detectable levels of foreign pollen sources. Within polycross isolations, no detectable levels of foreign pollen sources were detected, and self-fertilised rates were negligible (<1%). Outcross paternal progeny counts deviated significantly from random mating in two 20 parent polycrosses. Siring success of paternal genotypes decayed as the distance between them and recipient maternal parents increased. Low levels of paternal pollen dispersal at increasing distances from maternal recipients within isolation cages demonstrated the requirement for randomised multi-clonal replication in white clover polycrosses, especially when being used for the generation of half-sib families for progeny evaluation. Development and application of a novel paternity testing method in white clover was successful at assigning paternity to known maternal half-sib progeny from a 20 parent polycross. A panel of seven pre-determined SSR markers successfully assigned paternity to 92% of the half-sib progeny. Data collected from known maternal and molecular maker determined paternal half-sib progeny demonstrated that their respective additive genetic variances were similar across a range of morphological traits and two experimental sites. Combining both known maternal and molecular marker determined paternal half-sib ii selection, the rate of expected genetic gain was doubled compared to traditional half-sib family selection alone. Supplementary phenotypic selection within molecular marker determined full-sib families further improved expected genetic gain. Discrepancies in heritabilities calculated on a half-sib family means basis and that on a single plant basis support the rationale for breeders to pursue half-sib family selection methods for low heritable traits such as clover herbage yield, whereas traits with moderate heritabilities on a single plant basis, such as leaf size, support the use of phenotypic selection methods. In a multi-site mini-plot trial conducted in two Canterbury soils which differed in soil moisture content and irrigation application, significant genetic variation for both autumn vegetative persistence and herbage yield was observed. Utilising a North Carolina I mating design, second year autumn vegetative persistence (plot coverage) ranged among full-sib families from 15 to 100% at the irrigated site and from 10 to 70% at the dryland site, clover herbage yield from 496 to 1382 kgDM/ha at the irrigated site and 93 to 326 kgDM/ha at the dryland site, and growing point density from 356 to 3111 and 115 to 867 growing points m-2, at the irrigated at dryland sites, respectively. Physiological measurements indicated significant soil moisture stress at the dryland site, with water potentials up to 3.5 fold lower than the irrigated site. The extent of genetic variation observed for autumn recovery supports the notion for the use of intra-population selection for the development of improved cultivars that can persist through moderate drought conditions and demonstrate improved recovery rates following rain. Similar to the spaced-planted trials, large discrepancies in heritabilities calculated on a family means basis and that on a single plant basis support the rationale for breeders to pursue family selection methods for clover herbage yield and most vegetative stolon attributes. Heritabilities for traits such as leaf size and stolon thickness support the use of phenotypic selection methods. Partitioning of genetic variation into both additive and non-additive genetic variation was hindered by large standard errors. Inconclusive evidence for a significant proportion of non- additive genetic variation for clover herbage yield warrants further enquiry. Significant family × replicate, family × year, family × environment and family × year × environment variance components demonstrated the requirement for breeders to include multi-site and multi-year trials with adequate plot size to identify families with broad adaptation for field application. Keywords: Trifolium repens, white clover, breeding methods, genetic variances, paternity testing, half-sib families, full-sib families, spaced-planted, mini-plots, clover herbage yield, persistence, moisture stress, pollination. iii Acknowledgements The completion of this PhD research project and thesis was made possible by the support and advice given to me by a number of people for whom I am immensely grateful for. I was fortunate to have an outstanding advisory and supervisory team whose wealth of both academic and practical knowledge contributed to not only this thesis but also my professional development. I would like to thank my principal supervisors, Dr Rainer Hofmann (Lincoln University) and Dr Zulfi Jahufer (AgResearch) for their guidance and support throughout my project. Their humility was inspiring. I would also like to extend this gratitude to my advisors at AgResearch, Brent Barrett and Keith Widdup. I am exceedingly grateful for the mentoring role Dr Derek Woodfield from PGG Wrightson Seeds played throughout the course of my project, his vision, and the significant personal development opportunities he has provided me with. Likewise, I would like to thank both Dr Alan Stewart and Michael Norriss from PGG Wrightson Seeds for their practical advice and discussions regarding commercial breeding programs. I also extend this gratitude to the wider PGG Wrightson Seeds team at the Kimihia Research Centre. I thank AgResearch staff Ivan Baird, John Ford and Greig Cousins for not only their help in the field but also their invaluable wealth of practical breeding knowledge that they have shared with me. Thanks to Dr Andrew Griffiths, Craig Anderson and Dr Benjamin Franzmayr for their assistance and guidance in the molecular biology lab and Dr Alasdair Noble and Dr John Koolard for statistical advice. A special thanks to Anouck De Bonth and Michelle Ebbett of AgResearch for the accommodation and bike loans on my visits to Palmerston North. I thank Stephen Stilwell, Jenny Zhao, Rosy Tung, Daniel Dash, Keith Pollock and Dave Jack for their technical assistance at Lincoln University. I thank Emma Byrne for her support and encouragement during the project. Finally, I wish to thank my close friends, parents, brothers and partner Colette for their love and support. I would like to acknowledge the financial support from PGG Wrightson Seeds, MBIE (PGGS1001) and AgResearch. iv Table of Contents Acknowledgements ............................................................................................................................... iv Table of Contents .................................................................................................................................. v List of Tables ........................................................................................................................................ ix List of Figures ........................................................................................................................................ x List of Plates .......................................................................................................................................... xi Abbreviations ....................................................................................................................................... xii Chapter 1 Introduction .......................................................................................................................... 1 1.1 Background ...................................................................................................................................