Investigating the North American Crepis (Asteraceae) agamic complex for evidence of homoploid hybridization using next-generation sequencing techniques by Kathleen McGrath B.Sc.FSc. Trent University, 2007 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Botany) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) September 2014 © Kathleen McGrath, 2014 Abstract Despite the classic place that the North American Crepis (Asteraceae) agamic complex holds in evolutionary literature, few of the hypotheses about the group presented by Babcock and Stebbins 1938 monograph have been tested. In particular, they hypothesized that the seven sexual diploids had strong interfertility barriers that prevented the formation of diploid hybrids. Here I present an analysis of two previously unrecognized diploid morphotypes, which belong to an unresolved clade with Crepis pleurocarpa and Crepis occidentalis based on plastid DNA variation. Morphological traits suggest that both morphotypes may be the product of diploid x diploid hybridization. I gathered nuclear SNP markers using genotyping by sequencing to assess the origins of these two lineages. I constructed a de novo assembly of the nuclear genome of Crepis monticola to serve as a reference for SNP discovery. Analysis of contig length,number, and coverage indicate that the nuclear genome of Crepis is highly repetitive and shares features in common with other plant genomes that make angiosperm genomes challenging to work with. This complexity, as well as technical challenges likely due to partial enzymatic digestion of genomic DNA during GBS library preparation, resulted in only 19 SNPs passing the quality filters. Nonetheless, these 19 markers were used to provide a preliminary assessment of the origins of novel morphotypes. A signal of mixed ancestry was found for one of these morphotypes with the majority of their genome being distinct from both C. occidentalis subsp. occidentalis and C. pleurocarpa. The second morphotype is of non-mixed ancestry most closely resembling Crepis occidentalis. In a separate study, I provide a draft assembly of the Crepis monticola chloroplast genome. I show that gene order and content are unchanged from other members of Asteraceae with the exception of the rpl16 gene, which retains an intron that is reported as lost multiple times in Asteraceae. Results of a data analysis detailing the presence or absence of the first exon of rpl16 in published Asteraceae plastome sequences indicates that most of these supposed losses are errors, pointing to the need for careful examination of plastome assemblies gathered from databases. ii Preface This thesis is original, unpublished, independent work by the author, K. McGrath. All plant material used in this thesis was collected by Chris Sears, with Crepis runcinata samples obtained by Jeannette Whitton. The genotyping-by-sequencing protocol used was modified from protocols created by Greg Baute, Greg Owens, Kristen Nurkowski, David Toews, Miguel Alcaide, Sampath Seneviratne, and Haley Kenyon. The reference based UNIX pipeline used in data analysis was graciously provided by Greg Baute and Greg Owens. Additional scripts for data parsing were provided by David Tack. iii Table of contents Abstract .......................................................................................................................... ii Preface .......................................................................................................................... iii Table of contents.......................................................................................................... iv List of tables ................................................................................................................ vii List of figures ............................................................................................................. viii Acknowledgments........................................................................................................ ix Dedication ...................................................................................................................... x 1. Introduction ............................................................................................................. 1 1.1. DNA sequencing: then and now ...................................................................... 1 1.2. Next-generation sequencing: limitations and challenges ............................. 2 1.3. Genotyping-by-sequencing ............................................................................. 3 1.4. Angiosperm nuclear genomes ........................................................................ 3 1.5. Thesis chapters summary ............................................................................... 4 1.5.1. Chapter two ................................................................................................. 4 1.5.2. Chapter three ............................................................................................... 5 2. Investigating the North American Crepis (Asteraceae) agamic complex for evidence of homoploid hybridization .......................................................................... 6 2.1. Introduction ........................................................................................................ 6 2.1.1. Historical background ................................................................................ 6 2.1.2. Current taxonomic circumscriptions and putative hybrids ..................... 7 2.1.3. Homoploid hybridization ............................................................................ 8 2.1.4. Goals and objectives .................................................................................. 9 2.2. Materials and methods ..................................................................................... 10 2.2.1. Plant materials and DNA extractions ........................................................ 10 2.2.2. Isolation of Crepis monticola DNA for genomic paired-end Illumina sequencing ........................................................................................................... 11 iv 2.2.3. Genotyping-by-sequencing protocol ....................................................... 12 2.2.4. Raw sequence analysis and quality assessment ................................... 14 2.2.4.1. Crepis monticola assembly ............................................................. 14 2.2.4.2. .. Genotyping-by-sequencing data within reference based pipeline .................................................................................................................... 15 2.2.4.3. Stacks .............................................................................................. 16 2.2.4.4. UNEAK ............................................................................................. 17 2.2.5. Crepis monticola De Novo assembly ...................................................... 18 2.2.6. Alignment of genotyping-by-sequencing data to Crepis monticola contigs .................................................................................................................. 20 2.2.7. De Novo assembly in STACKS ................................................................ 20 2.2.7.1. Ustacks ............................................................................................ 21 2.2.7.2. Cstacks ............................................................................................ 21 2.2.7.3. Sstacks............................................................................................. 22 2.2.7.4 Populations ....................................................................................... 22 2.2.8. De Novo assembly in UNEAK .................................................................. 23 2.2.9.1. Reference based SNP calling ......................................................... 24 2.2.9.2. De Novo SNP calling ....................................................................... 26 2.2.10. Analysis of SNP data .................................................................... 27 2.3. Results .............................................................................................................. 33 2.3.1. Crepis monticola sequencing and contig assembly .............................. 33 2.3.2. Genotyping-by-sequencing read analysis and assembly ...................... 35 2.3.2.1. Sequence analysis .......................................................................... 35 2.3.2.2. De-multiplexing ............................................................................... 35 2.3.3. SNP identification...................................................................................... 37 2.3.4. Data analysis ............................................................................................. 39 2.3.4.1. Pair-wise Fsts .................................................................................. 41 2.3.4.2. STRUCTURE analysis ..................................................................... 41 2.3.4.3. Principal component analysis ........................................................ 43 2.4. Discussion ........................................................................................................ 44 v 2.4.1. ... Crepis monticola genome composition and reference based alignment of GBS data ..........................................................................................................