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Genomic Studies on Floral and Vegetative Development in the Genus Streptocarpus (Gesneriaceae) Yun-Yu Chen Doctor of Philosophy The University of Edinburgh Royal Botanic Garden Edinburgh 2019 Declaration I declare that this thesis has been composed solely by myself and that it has not been submitted, in whole or in part, in any previous application for a degree. Except where stated otherwised by reference or acknowledgement, the work presented is entirely my own. _________________ Yun-Yu Chen Date 15/07/2019 i Acknowledgements I would like to express my utmost gratitude to the wonderful people who joined and contributed to this project and helped me throughout the PhD. First are my supervisors, Michael Möller, Catherine Kidner, and Kanae Nishii – thank you for giving me the opportunity, guidance, joy, intellectual support, and sometimes physical hard work! - throughout my study. Thank you all very much for your time and effort in discussing and commenting the works we produced together, and in helping me to practice and improve myself through numerous posters, presentations, and written pieces of work. This thesis is a result of all of our dedications. I would also like to thank our collaborators, Christine Hackett from the BioSS for the course and discussion on linkage mapping and QTL mapping, and Atsushi Nagano from Ryukoku University for the RAD-Seq experiments. I feel extremely lucky and grateful to have worked with you all, and the memory will forever stay dearly in my heart. My sincerest thanks also goes to my examiners, Michelle Hart and Minsung Kim, on reviewing this brick of papers and for stimulating the questions and discussions during the viva. You have helped me improve the work and myself even more. My gratitude also goes to the supporting teams at the University of Edinburgh and the Royal Botanic Garden Edinburgh. To my thesis committee member, Prof Andrew Hudson, who joined all my PhD milestones, providing invaluable discussions about the project. To all members in the RBGE horticultural team, particularly Sadie Barber, Nate Kelso and Andy Ensoll for taking care of the plant materials – without you the project would not have happened. Thanks to the RBGE STS team, Laura Forrest and Ruth Holland for the support in molecular lab, and to Frieda Christie for the training and discussion in microscopy and SEM. Thanks to the ICT team members for fulfilling my endless software and server requests. A big thank you to all the friends in Edinburgh - Julieth, Roosevelt, Javier, Karina, Flávia, Kristine, Pakkapol, Andrés, Mauricio, Lucía, Surabhi, Nadia, Thibauld and so many more - I will always remember the happiness and time we shared together in the BO8 office as well as in the pubs. To people in the tropical science group: Axel, Peter, Mark, Tiina, Toby and others, for the fun times and intellectual discussions. Special thanks to my former landlady Susan and her family for their hospitality. Also to Moth, their little granny cat for the cuddling service provided. Finally, my sincerest gratitude goes to my family - for my parents’ love and endless supports, for waiting for me at the other side of the world patiently. To my wife Andrea, who appeared at the most surprising moment, and accompanied me to complete this journey. Thank you for your patience and understanding, going through all the hard work and stressful moments with me. Wherever I am, I know I can look to you and feel the calmness and supports you reserved for me. I owe you everything. ii Abbreviations BAM Binary sequence alignment map (file format) BLAST Basic local alignment search tool BLAT BLAST-like alignment tool BM Basal meristem Bp Base pairs BTL Binary trait loci BUSCO Benchmarking universal single-copy orthologs cM Centimorgan CTAB Cetyl trimethylammonium bromide DBG De Bruijn graph DNA Deoxyribonucleic acid EDTA Ethylenediaminetetraacetic acid Gbp Giga base pairs gDNA Genomic DNA GM Groove meristem GWAS Genome wide association study KEGG Kyoto Encyclopedia of Genes and Genomes Mbp Mega base pairs NGS Next generation sequencing ORF Open reading frames PCR Polymerase chain reaction QTL Quantitative trait loci RAD-Seq Restriction-site associated DNA sequencing RBGE Royal Botanic Garden Edinburgh RNA Ribonucleic acid RNA-Seq RNA sequencing Rpm Revolution per minute SAM Sequence alignment map (file format) SEM Scanning electron microscope iii Abstract The genus Streptocarpus consists of around 180 species with diverse morphologies. At least three main types of vegetative growth forms can be distinguished: caulescent, rosulate (acaulescents with multiple leaves), and unifoliate (acaulescents with one leaf). Floral size, shape, and pigmentation pattern are also highly variable between species. Previous studies have suggested that some of the morphological characters are inherited as Mendelian traits. For instance, the rosulate growth form is dominant over the unifoliate, and the rosulate / unifoliate growth form was hypothesised to be determined by two genetic loci, based on the Mendelian segregation ratios recorded in backcross and F2 populations. However, the identity of the loci and the underlying molecular mechanisms remain unknown. In this study, Streptocarpus rexii (rosulate) and Streptocarpus grandis (unifoliate) were used to study the genetic basis of morphological variation in Streptocarpus . The aim is to use modern next generation sequencing (NGS) technologies to build draft genomes, transcriptomes, and genetic maps for the non-model Streptocarpus plants, and carry out quantitative trait loci (QTL) mapping to locate the causative loci. First, suitable DNA and RNA extraction methods for obtaining NGS-quality nucleic acids from Streptocarpus were established. For DNA extraction this was a modified protocol of the ChargeSwitch gDNA Plant Kit, and for RNA extraction a TRIzol reagent plus phenol:chloroform:isoamyl alcohol wash protocol was devised. The nucleic acid samples extracted were subsequently used for library preparation and NGS sequencing experiments. Whole genome shotgun sequencing was performed for S. rexii and S. grandis using Illumina HiSeq 4000 and HiSeq X. De novo assembly of the sequence data produced a S. rexii draft genome of 596,583,869 bp, with 95,845 scaffolds and an N50 value of 35,609 bp. The S. grandis draft genome had a total span of 843,329,708 bp, with 127,951 scaffolds and an N50 value of 31,638 bp. The genome assemblies served as references for subsequent NGS data analysis. The RNA samples derived from various vegetative and floral tissues of S. rexii and S. grandis were sequenced on MiSeq and HiSeq 4000 platforms. The transcriptome assembly was carried out using de novo and reference-based methods (i.e. mapped to the obtained draft genomes), followed by putative protein-coding open reading frame identification and annotation. For S. rexii , 60,500 and 53,322 transcripts were constructed in the de novo and reference-based assemblies respectively. For S. grandis , 51,267 and 46,429 transcripts were constructed respectively. A Streptocarpus genetic map was constructed using restriction-site associated DNA sequencing (RAD-Seq) genotyping of a backcross population (( S. grandis × S. rexii ) × S. grandis ). The RAD-Seq data were analysed using a de novo approach and reference-based approaches with two different aligners, and the RAD-markers recovered from the three iv approaches were combined to maximise the genetic map density. Different marker-filtering strategies with varying stringencies were also tested and compared. The results showed that the most stringently filtered map had 377 mapped markers in 17 linkage groups, and a total distance of 1,144.2 cM. On the other hand, the densest map consisted of 853 markers in 16 linkage groups (matching the basic haploid chromosome number of the Streptocarpus species used here), and a total distance of 1,389.9 cM. The maps constructed were used for QTL mapping of growth form variation, identifying up to 5 effective loci for the rosulate / unifoliate phenotypes, with two of the loci on LG2 and LG14 consistently found in all mapping attempts. The results suggest that the variation in growth form may be regulated by two major loci, but a few additional minor loci might also be associated with the trait. Several QTLs for floral dimension, flowering time, and floral pigmentation patterns were also found, and the genetic regions associated with the floral traits of Streptocarpus were revealed for the first time. During this study valuable genomic resources were generated for future research to identify the genes underlying different morphologies in the genus Streptocarpus . The reported QTLs narrow down the genetic region for fine-mapping studies, and the genome and transcriptome resources will aid the isolation of candidate gene sequences. Identifying the genetic loci and their crosstalk behind the variable morphologies in future work will greatly add to our knowledge on how the highly diverse genus Streptocarpus has evolved and on how fundamental developmental processes of plants are regulated. v Lay summary An important question in biology is how differences in shape and form between species have evolved. To answer this question, a key step is to investigate how shape and form develop and understand the genetic mechanisms regulating these processes.