Molecular Mapping and Characterization of Phenylpropanoid Pathway Genes in Common Bean (Phaseolus vulgaris L.) By Zeinab Yadegari A Thesis Presented to The University of Guelph In partial fulfillment of requirements for the degree of Doctor of Philosophy in Plant Agriculture Guelph, Ontario, Canada © Zeinab Yadegari, June 2013 ABSTRACT MOLECULAR MAPPING AND CHARACTERIZATION OF PHENYLPROPANOID PATHWAY GENES IN COMMON BEAN (PHASEOLUS VULGARIS L.) Zeinab Yadegari Advisor: University of Guelph, 2013 Professor K. P. Pauls Common bean is a nutritionally and economically important food crop and a major source of dietary protein in many developing countries throughout the world. Seed coat colour and size in this crop are the main factors determining its marketability in different parts of the world. Flavonoid compounds that are responsible for seed coat colour in beans have been shown to have anti-oxidant, anti-proliferative, anti-tumor, anti-inflammatory, and pro- apoptotic activities. They also may enhance the resistance of beans to pest and disease. A better understanding of the relationships between seed coat colour and flavonoid metabolism in the seed coat may help breeders to select for more nutritionally-beneficial bean varieties. The objective of this research was to test the hypothesis that the genes determining colour in beans are structural and regulatory genes of the phenylpropanoid pathway. The map positions of phenylpropanoid genes were determined in two recombinant inbred populations. Segregation patterns of 18 phenylpropanoid pathway genes in the BAT93 × Jalo EEP 558 RIL population and five phenylpropanoid pathway genes in OAC Rex × SVM Taylor were used to place them on the linkage maps for these populations. Five out of 18 genes were mapped within 2-17 cM of colour gene loci in the BAT93 × Jalo EEP 558 RIL population. The sequences of central genes of the phenylpropanoid pathway were determined by sequencing 6 BAC clones selected with probes for two PAL genes, two CHS genes, DFR, and Myb. The functional annotations of the BAC clones were determined and the similarities between bean phenylpropanoid genes and their corresponding orthologs in other plant species were investigated. A recently developed approach of whole genome sequence comparison was utilized to compare the microsynteny of the sequenced BAC clones with regions of the soybean genome. The physical locations of BAC clones were verified on the bean genome and their counterpart locations on the soybean genome were confirmed. The results agreed with previous studies that indicated that bean genome segments have two homologous segments in soybean and confirmed the high degree of microsynteny that is shared between bean and soybean. ACKNOWLEDGEMENTS I would like to express my deep and sincere gratitude to my advisor Dr. Peter Pauls for his excellent support and guidance throughout this project. I am thankful for having the opportunity to work within his group. Special thanks to my advisory committee, Dr. Judith Strommer, Dr. Istvan Rajcan and Dr. Annette Nassuth for their valuable input during the course of this research, its writing and defense. I am deeply grateful to Dr. Matthew Blair the external examiner of this thesis for his thorough review and constructive comments. My special thanks extend to Dr. Alireza Navabi for his great comments on this work. I would like to acknowledge the help of members of Peter’s lab specially Jan Brazolot, Dr. Yarmilla Reinprecht and Dr. Loo-Sar Chia. Special thanks go to Tom Smith for his help with greenhouse work. I am grateful for the love and support of my family specially my husband Ali who helped me a lot during bioinformatics analysis of the data. Also my sincere thanks go to my parents for their encouragement and support throughout my life. iv TABLE OF CONTENTS I. INTRODUCTION ......................................................................................................................... 1 The bean crop in the world and Ontario ................................................................................. 1 Centres of origin for P. vulgaris ............................................................................................. 1 II. LITERATURE REVIEW ............................................................................................................. 3 Nutritional importance of dry bean ........................................................................................ 3 The genetics of seed coat colour in bean ................................................................................ 4 Association between colour genes and the phenylpropanoid pathway in common bean ...... 6 Phenylpropanoid pathway and colour .................................................................................. 10 Phenylalanine ammonia lyase (PAL) .......................................................................... 13 Chalcone synthase (CHS) ............................................................................................ 18 Dihydroflavonol 4-reductase (DFR) ........................................................................... 24 Myb Transcription factor ............................................................................................. 29 Phenylpropanoid compounds and disease resistance in common bean ............................... 32 Linkage mapping in common bean ...................................................................................... 33 Physical map in common bean ............................................................................................. 34 Genomics and bioinformatics in Legumes ........................................................................... 34 Genomic features of common bean ...................................................................................... 38 Available Common Bean Genomic Resources .................................................................... 38 Thesis hypothesis and objectives ......................................................................................... 38 III. MAPPING OF PHENYLPROPANOID PATHWAY GENES IN COMMON BEAN (PHASEOLUS VULGARIS L.) ............................................................................................. 40 Abstract ................................................................................................................................ 40 Introduction .......................................................................................................................... 42 Materials and methods .......................................................................................................... 44 Plant materials ............................................................................................................. 44 DNA extraction, digestion, and electrophoresis .......................................................... 45 Southern blotting, hybridization and autoradiography ................................................ 47 SSR and SNP genotyping in the OAC population ...................................................... 47 Statistical and linkage analysis .................................................................................... 48 Results .................................................................................................................................. 49 Marker polymorphism and segregation ....................................................................... 49 Core population ................................................................................................. 49 OAC Rex × SVM Taylor RI population ........................................................... 54 Construction of the linkage map ................................................................................. 57 Core population ................................................................................................. 57 OAC Rex × SVM Taylor RI population ........................................................... 58 Integrated genetic map and genome sequence ............................................................ 65 Discussion ............................................................................................................................ 71 Phenylpropanoid genes in core population ................................................................. 71 Cosegregation of DFR1 and flower colour in bean ..................................................... 74 v IV. MOLECULAR CHARACTERIZATION OF PHENYLPROPANOID GENES IN COMMON BEAN (PHASEOLUS VULGARIS L.) .............................................................. 76 Abstract ................................................................................................................................ 76 Introduction .......................................................................................................................... 77 Materials and Methods ......................................................................................................... 79 Extraction of total RNA .............................................................................................. 79 Amplification of partial genomic sequence ................................................................. 79 PCR products detection and sequence analysis ........................................................... 80 RT–PCR characterization of PvCHS-A, PvCHS-B and PvDFR expression in seed coat of P.vulgaris .......................................................................................................