DIVERSITY OF TALL FESCUE AND RELATIONSHIPS WITHIN FESTUCA SUBGENUS SCHEDONOURUS BASED ON NUCLEAR AND CHLOROPLAST SIMPLE SEQUENCE REPEAT MARKERS By VINCENZO AVERELLO IV A Thesis Submitted to the School of Graduate Studies Rutgers, The State University of New Jersey In partial fulfillment of the requirements For the Degree of Master of Science Graduate Program in Plant Biology Written Under the Direction of Stacy Bonos and William Meyer And Approved By _________________________________________ _________________________________________ _________________________________________ _________________________________________ New Brunswick, NJ October, 2017 ABSTRACT OF THE THESIS Diversity of tall fescue and relationships within Festuca subgenus Schedonourus based on nuclear and chloroplast simple sequence repeat markers By VINCENZO AVERELLO IV Thesis Director: Dr. Stacy A. Bonos and Dr. William A. Meyer Tall fescue (Festuca arundinacea Schreb. syn. Lolium arundinaceaum [Schreb.] Darbysh. syn. Schedonourus arundinaceus [Schreb.] Dumort.) is an allohexaploid grass species that is found throughout Europe, much of Asia, and North Africa. As it is currently understood, there are two gene pools within Festuca subgenus, the Continental morphotype, which can be found in Europe and Asia, and the Mediterranean morphotype, which can be found in Northern Africa. The aims of this thesis were to investigate the level of diversity present in a tall fescue germplasm collection from the center of origin for the species and recent cultivars and to determine the relationships between the collections and cultivars, as well as between the species and subspecies within the subgenus Schedonorus. Sixteen individuals from ninety-eight collections, cultivars, and accessions of Festuca and Lolium were genotyped using two tall fescue nuclear EST-SSRs, eleven tall fescue nuclear genomic SSRs, and eighteen tall fescue chloroplast SSRs. One chloroplast marker was used to assign each cultivars, collection, or accession to a morphotype. The nuclear SSR markers found that the turf-type cultivars of tall fescue were closely related ii but still genetically distinct from each other, agreeing with known pedigree information. The collections generally clustered by geographic origin. Bayesian cluster analysis showed that the cultivars and collections exhibit a high level of admixture. Chloroplast microsatellites were not capable of discriminating between cultivars and collections, as the nuclear satellites were. Both marker systems, separated the collections from North Africa from the other collections, as well as other species and subspecies from that region, suggesting they are not closely related. This work showed that nuclear SSRs were capable of distinguishing between cultivars and collections of tall fescue, while chloroplast SSRs were only capable of distinguishing between species and subspecies. iii Acknowledgements I cannot express enough gratitude for my amazing advisors, Stacy Bonos and William Meyer, who have always pushed me to make sure that I am doing the best possible work I can. Their comments on this work and everything else I have done with them has been incredibly helpful, supportive, and motivating. Their dedication to me, their students, and all their work will serve to inspire me for years to come. I want to thank for Josh Honig. He trained me on all the lab skills that I needed to complete this project. He was always available and helpful when problems arose with material and analyses. Working with him has allowed me to grow into a better person and a better scientist. I also want to thank Ning Zhang for serving on the thesis committee and her constructive comments on my work. Jennifer Vaiciunnas and Christine Kubik were invaluable in the laboratory, assisting with everything from collecting and maintaining plant material to troubleshooting and maintaining an incredible work space for all. Everyone who helped collecting material or with field and greenhouse, Dirk Smith, Austin Grimshaw, Eric Weibel, Trent Tate, and many others. To Megan Muehlbauer, Stephanie Fong, Ariane Vasilatis, Robert Mattera, Philip Vines, and many others, thank you for being helpful and supportive friends from my time here in Plant Biology. I want to thank my family for their many years of support through all my academic endeavors. Their unwavering support has encouraged me through any rough times I have add during my studies. iv Table of Contents Abstract of the thesis ........................................................................................................................ ii Acknowledgements ......................................................................................................................... iv Table of Figures ............................................................................................................................. vii Table of Tables ............................................................................................................................. viii Literature Review............................................................................................................................. 1 Tall Fescue Biology, Use, and Breeding...................................................................................... 1 Phylogeny and Systematics of Festuca and Lolium ..................................................................... 3 Tall Fescue Center of Origin and Within Species Diversity ........................................................ 9 Use and Development of Nuclear Microsatellite (nuSSR) Markers in Other Species ............... 15 Use of Chloroplast Microsatellites (cpSSR) Markers for Diversity Research ........................... 22 Tall Fescue Genomic Resources and Other Applications .......................................................... 27 References .................................................................................................................................. 33 Diversity of Tall Fescue and Relationships within Festuca subgenus Schedonorus ..................... 39 Introduction ................................................................................................................................ 39 Materials and Methods ............................................................................................................... 42 Plant Material ........................................................................................................................ 42 Nuclear and Chloroplast SSR Markers .................................................................................. 54 SSR genotyping ...................................................................................................................... 57 Allele scoring and SSR summary statistics ............................................................................ 58 Analysis of molecular variation (AMOVA) ............................................................................ 59 Genetic dissimilarity and neighbor joining dendrogram construction .................................. 60 Bayesian model based clustering ........................................................................................... 61 Results and Discussion .............................................................................................................. 61 nuSSR summary statistics ...................................................................................................... 61 cpSSR and haplotype summary statistics ............................................................................... 62 Festuca and Lolium species and sub-species relationships based on nuSSR AMOVA, Morphotype Assignment, Pairwise ΦPT Values, interpopulation pairwise genetic distance and neighbor joining tree analysis......................................................................................... 65 Festuca and Lolium species and sub-species relationships based on cpSSR AMOVA, Morphotype Assignment, Pairwise ΦPT Values, interpopulation pairwise genetic distance and neighbor joining tree analysis....................................................................................... 102 Continental Festuca arundinacea germplasm relationships ............................................... 138 Comparison of relationships based on nuSSRs and relationships based on cpSSRs ........... 139 v Genetic diversity based on nuSSR markers, model based clustering analysis..................... 140 Conclusion ............................................................................................................................... 157 References ................................................................................................................................ 159 vi Table of Figures Figure 1- Neighbor-Joining Tree based on nuclear SSR Markers. The shape to the left of the node name is the result of Chl045 marker to determine morphotype. An open circle represents that all 16 individuals have the continental allele, a filled circle represents that all individuals have the Mediterranean allele. An open square represents that 15 individuals has the continental allele while 1 individual has the Mediterranean allele. A filled square represents that that 15 individuals have the Mediterranean allele while 1 individual has the Continental allele. A filled triangle represents that there is mixture of the two alleles. Numbers at nodes are support values, only values great than 70 are shown. ..................