SSR-based genetic mapping of QTLs determining chilling requirements for time of initial vegetative budbreak in domesticated apple (Malus x domestica Borkh.) cultivar ‘Anna’ x ‘Austin’. by Paidashe Hove A thesis submitted in partial fulfilment of the requirements for the degree of MAGISTER SCIENTIAE (M.Sc.) Department of Biotechnology University of the Western Cape Bellville Supervisor: Dr. D. J. G. Rees Co-supervisor: Prof. B. Ndimba June 2012 ABSTRACT The Rosaceae family contains major temperate crops such as the domesticated apple (Malus x domestica Borkh.), peach (Prunus persica L. Batsch) and European pear (Pyrus communis L.). However, despite its evident economic importance, it is generally poorly studied in genomic terms, relative to the other major crop groups. Microsatellite and Diversity Array Technology (DArT) genetic markers have been exploited in this work and are essential tools in genetic map construction and marker-assisted selection (MAS) of high quality apples and other rosaceous crops. Microsatellites are advantageous in that they are co-dominant, highly polymorphic, abundant, transferable and reliably reproducible; hence their use in this study. In order for budbreak to take place in a timely and homogenous fashion, apple trees need a period of exposure to low temperatures. Within orchards the application of chemicals that induce budbreak in unsuitable environments is required to produce apples from cultivars that require high chilling levels. However, this and other practices using chemicals in orchards tend to pollute the environment. One of the solutions to this problem is to breed low chill apples such as ‘Anna’ cultivar, which was used as one of the parents in this study. This work was aimed at understanding the underlying genetic factors that determine chilling requirements for the time of initial vegetative budbreak trait in the apple cross ‘Anna’ x ‘Austin’. This was achieved through linkage map construction using SSR and DArT molecular markers followed by QTL analysis. This thesis has therefore exploited the large number of Expressed Sequence Tags (ESTs) and genome sequence data for the apple, using Tandem Repeats Finder, to design a total of 98 new SSR primers pairs. The i other 369 SSR markers used in this work were from published work. JoinMap! 4.1 software was used to create an integrated genetic map with 17 linkage groups, for the domesticated apple cultivar, ‘Austin’ x ‘Anna’ mapping population with 80 individuals. The result of this process was a genetic map 1 212cM in length, and a total of 429 markers (314 DArT and 115 SSR), at an average density of a marker every 4 cM. This map was used identify the Quantitative Trait Loci (QTLs) determining chilling requirements for time of vegetative budbreak (IVB). In this process, putative IVB QTLs were identified in the ‘Anna’ x ‘Austin’ mapping population using the rMQM analysis function of MapQTL! 6.0, for both adult and seedling data collected over 3 growing seasons from 1996 to 1998. These QTLs were detected on linkage groups 2, 9 and 14, and explained 0.3 to 12.8 % of the observed phenotypic variation for the adult population, and 5.3 - 21 % for the seedling population. Seedling (LG 14) and adult (LGs 5, 7, 10) specific QTLs were also detected for the ‘Anna’ x ‘Austin’ cross. These QTLs will provide the basis for marker validation on related mapping populations in the apple breeding programme, and for the future identification of candidate genes controlling the process of budbreak. ii LIST OF ACRONYMS AND ABBREVIATIONS ABI Applied Biosystems AFLP Amplified Fragment Length Polymorphism AgNO3 Silver nitrate An Anna APS Ammonium PeroxidiSulphate ARC Agricultural Research Council Au Austin BLAST Basic Local Alignment Search Tool bp base pair CIA Chloroform Isoamyl Alcohol cm centimetre cM centiMorgan CR Chilling Requirement CTAB N-acetyl-N-N-N trimethyl ammonium bromide CU Chilling Units/ Cold Units DArT Diversity Array Technology DFPT Deciduous Fruit Producers Trust dNTPs DeoxyriboNucleic-5"-TriPhosphate DNA DeoxyriboNucleic Acid DNOC DiNitro Ortho Cresol mineral oil °C degrees Celcius iii EtOH ethanol EDTA Ethylene Diamine Tetraacetic Acid (disodium salt) EST Expressed Sequence Tag FAOSTAT Food and Agricultural Organization Statistical Database (United Nations) F1 First filial generation g gram GD ‘Golden Delicious’ gDNA genomic DNA x g centrifugal force ha hectare IRB time of Initial Vegetative Budbreak IVB time of Initial Reproductive Budbreak kb kilo basepairs kV kilo Volt LG Linkage Group l litre LOD Logarithm (base 10) of ODds M Molar MAB Marker Assisted Breeding MAS Marker Assisted Selection Mbp Mega basepairs/ Million basepairs ml milli litre iv min minute mM milli Molar MT Metric Tons NaBH4 Sodium Borohydride NaCl sodium chloride NaOH sodium hydroxide ng nano gram NH4Ac Ammonium Acetate PAGE PolyAcrylamide Gel Electrophoresis PCR Polymerase Chain Reaction PDS Prolonged Dormancy Symptoms PIC Polymorphism Information Content PPECB Perishable Product Export Control Board QTL Quantitative Trait Locus RAPD Random Amplified Polymorphic DNA RFLP Restriction Fragment Length Polymorphism RNA RiboNucleic Acid s second SNP Single Nucleotide Polymorphism SSR Simple Sequence Repeat TBE Tris Borate EDTA TE Tris EDTA TEMED N. N. N". N"-Tetra Ethyl Methyl-Ethylene Diamine v Tm melting temperature Tris (base) Tris hydroxymethyl amino methane µg microgram µl microlitre UV Ultra Violet V Volts v/v volume per volume w/v weight per volume x g centrifugal force vi ACKNOWLEDGEMENTS Firstly, I would like to express my most sincere gratitude to my supervisor Dr. Jasper Rees and co-supervisor Prof. Bongani Ndimba for their guidance and mentorship throughout the duration of this research. Special thanks also goes to Dr. Iwan Labuschagné and Mr. Trevor Koopman for the work they did at the ARC in generating and maintaining the plant material used in this study. I would also like to thank Dr. Daleen van Dyk and Dr. Khashief Soeker; many thanks are in order for your insight and help with linkage and QTL mapping. Members of the ARC Biotechnology Platform and the Department of Biotechnology at the University of the Western Cape deserve special mention for their camaraderie and support. Financial support for this work was supplied by THRIP and DFPT and was most appreciated. To my parents, family and friends, thank you for your continued great patience and encouragement thus far. Above all, I would like to thank God for affording me the life and good health to be able to apply myself in this academic endeavour. vii DECLARATION I herewith declare that the work presented in this thesis is my own work and has not been submitted for any degree or examination in any other university, and that all the sources I have used or quoted have been indicated and acknowledged by complete references. Paidashe Hove June 2012 Signed……………………… viii LIST OF FIGURES Figure 1: A diagrammatic representation of the signals and the typical seasons corresponding to the three different types of dormancy. .................................................. 17! Figure 2: The annual cycle of a Populus tree growing at Umea University, Umea Sweden.. ............................................................................................................................ 19! Figure 3: A simplified view of the four pathways that control flowering time in Arabidopsis, showing the major genes involved. ............................................................. 30! Figure 4: ‘Anna’ x ‘Austin’ adult tree IVB frequency distribution data for the years 1996 to 1998. ............................................................................................................................. 74! Figure 5: ‘Anna’ x ‘Austin’ seedling IVB frequency distribution data for the years 1996 to 1998. ............................................................................................................................. 75! Figure 6: A graphical representation of ‘Anna’ x ‘Austin’ adult tree year-to-year IVB data compared as a data trend, over the years 1996, 1997 and 1998. ............................... 76! Figure 7: A graphical representation of ‘Anna’ x ‘Austin’ seedling year-to-year IVB data compared as a trend, over the years 1996, 1997 and 1998. .............................................. 76! Figure 8: A 1 % Agarose gel run of the ‘Austin’ x ‘Anna’ genomic DNA. .................... 78! Figure 9: The user interface of the Tandem Repeats Database ....................................... 80! Figure 10: A graphical display of a typical sequence shown in the tandem repeats database. ............................................................................................................................ 81! Figure 11: A nucleotide level or fasta format view of the dinucleotide repeat sequence array. ................................................................................................................................. 82! Figure 12: A 6 % silver stained polyacrylamide gel of PCR amplicons derived form parental cultivar DNA and marker CH04e03. .................................................................. 83! ix Figure 13: A 6 % silver stained PAGE of a four-primer PCR multiplex run for DNA from four apple cultivars.. ................................ ................................................................. 84! Figure 14: Electropherograms obtained after amplification of the ‘Anna’ parental DNA using megaplex 17 ...........................................................................................................