University of New Hampshire University of New Hampshire Scholars' Repository Master's Theses and Capstones Student Scholarship Winter 2017 Developing a molecular pipeline to identify Chenopodium species in New England Erin Neff University of New Hampshire, Durham Follow this and additional works at: https://scholars.unh.edu/thesis Recommended Citation Neff, Erin, "Developing a molecular pipeline to identify Chenopodium species in New England" (2017). Master's Theses and Capstones. 1156. https://scholars.unh.edu/thesis/1156 This Thesis is brought to you for free and open access by the Student Scholarship at University of New Hampshire Scholars' Repository. It has been accepted for inclusion in Master's Theses and Capstones by an authorized administrator of University of New Hampshire Scholars' Repository. For more information, please contact [email protected]. DEVELOPING A MOLECULAR PIPELINE TO IDENTIFY CHENOPODIUM SPECIES IN NEW ENGLAND BY ERIN NEFF B.S. Biochemistry, Grove City College, 2015 THESIS Submitted to the University of New Hampshire in Partial Fulfillment of the Requirements for the Degree of Master of Science in Integrative and Organismal Biology December, 2017 This thesis has been examined and approved in partial fulfillment of the requirements for the degree of Master of Science in Integrative and Organismal Biology by: Thesis Director, Thomas M. Davis, Ph.D., Biological Sciences Janet R. Sullivan, Ph.D., Biological Sciences Cheryl A. Smith, Ph.D., Agriculture, Nutrition, and Food Systems Richard G. Smith, Ph.D., Natural Resources & the Environment On November 27, 2017 Original approval signatures are on file with the University of New Hampshire Graduate School. ii TABLE OF CONTENTS DEDICATION LIST OF TABLES LIST OF FIGURES ABSTRACT CHAPTER PAGE INTRODUCTION ………………………………………………………………………..……. 1 THE GENUS CHENOPODIUM ………………………...…………….………...........… 1 CHENOPODIUM SPECIES IN NORTHERN NEW ENGLAND ……………………. 2 COMPLICATED TAXONOMY: CHENOPODIUM ALBUM AS A SHORT CASE STUDY …………… …………………………………………………………………... 10 CURRENT STATE OF GENETIC RESOURCES AND GERMPLASM AVAILABILITY FOR THE CHENOPODIUM GENUS ………………………….….. 13 GERMPLASM RESOURCES ………………………………………………………… 14 AIMS OF THIS PROJECT …………………………………………………………...... 15 I. METHODS……………………………………………………………………......... 17 a. DETERMINATION OF COLLECTION SITES ……………………………… 17 b. PLANT COLLECTION PROTOCOL ……………………………….………... 18 iii c. STANDARD COMPARATORS USED IN THIS STUDY ……………............ 19 d. ANALYSIS OF COLLECTED PLANT MATERIAL: DETERMINATION OF PLOIDY VIA FLOW CYTOMETRY …………………………………………. 20 e. SPECIES IDENTIFICATION AND ASSESSMENT OF WITHIN-SPECIES DIVERSITY VIA RAPD PCR ………………………………………………… 23 f. CONFIRMATION OF IDENTITY FOR USDA STANDARDS VIA DNA SEQUENCING ………………………………………………………..….......... 27 i. CHLOROPLAST DNA SEQUENCING ………………………..……... 27 ii. SALT OVERLY-SENSITIVE 1 (SOS1) DNA SEQUENCING ……………………………………………………………………........... 28 g. DEVELOPMENT OF DIPLOID MODEL CHENOPOD FOR FUTURE GENETIC STUDY VIA CROSSES ………………………………...….……… 32 II. RESULTS ………………………………………………………………………….. 36 a. PLANT COLLECTIONS ……………………………………………………… 36 b. FLOW CYTOMETRY ………………………………………………………… 38 c. RAPD PCR ……………………………………………………………..……… 42 d. CONFIRMATION OF USDA REFERENCE STANDARDS VIA DNA SEQUENCING ………………………………………………………….……... 45 i. CHLOROPLAST DNA SEQUENCING AND ALIGNMENT ….......... 45 ii. SOS1 DNA SEQUENCING AND ALIGNMENT …………………….. 47 e. DIPLOID SPECIES CROSSES ...……………………………………………… 49 III. DISCUSSION ……………………………………………………………………… 52 a. TAXONOMY ………………………………………………………………….. 52 iv b. DISCUSSION OF USEFULNESS OF MOLECULAR IDENTIFICATION TESTS i. FLOW CYTOMETRY ………………………………………………… 54 ii. RAPD PCR …………………………………………………….............. 55 iii. DNA SEQUENCING ………………………………………………….. 56 1. CPDNA SEQUENCING …………………………………...….. 57 2. SOS1 DNA SEQUENCING …………………………………… 58 c. CHENOPODIUM BERLANDIERI VAR. MACROCALYCIUM …................. 59 d. CHENOPODIUM BERLANDIERI VAR. BUSHIANUM ……………………. 60 e. CHENOPODIUM BERLANDIERI VAR. ZSCHACKEI ………...….……….. 61 f. CHENOPODIUM STRICTUM ………………………………………………... 62 g. CHENOPODIUM ALBUM ……………………………………....…………… 62 h. CHENOPODIUM FICIFOLIUM ……………………………………..……….. 63 i. CHENOPODIUM STANDLEYANUM ……………………………..………… 64 j. CHENOPODIUM FOGGII ....…………………………………...…….............. 64 k. DIPLOID HYBRID ASSESSMENT ……………………………….….............. 66 IV. CONCLUSIONS ………………………………………………………….……….. 67 V. LIST OF REFERENCES …………………………………………………………... 68 VI. APPENDICIES a. APPENDIX 1: PREPARATION OF DE LAAT’S BUFFER FOR FLOW CYTOMETRY …………………………………………………………………. 74 b. APPENDIX 2: PREPARATION OF PROPIDIUM IODINE STAIN FOR FLOW CYTOMETRY …………………………………………………………………. 75 v c. APPENDIX 3: QUANTIFICATION OF DNA USING A QUBIT FLUORMETER ………………………………………………………………... 76 d. APPENDIX 4: PREPARATION OF 2% ELECTROPHORESIS GEL FOR RAPD PCR …………………………………………………………………….. 77 e. APPENDIX 5: PREPARATION OF 1X TBE BUFFER FOR ELECTROPHORESIS GEL ……………………………………………..…….. 78 f. APPENDIX 6: CHLOROPLAST REFERENCE SEQUENCE DATA USED IN THIS STUDY …………………………………...………………………….….. 79 g. APPENDIX 7: CHLOROPLAST PHYLOGENETIC TREE GENERATED FOR THIS STUDY ………………………………………………………………..… 80 h. APPENDIX 8: COLLECTION SITES, 2016 – 2017.……………….….…........ 81 i. APPENDIX 9: SOS1 INTRON 16 REFERENCE SEQUENCE DATA ……………………………………………………………………..…………..... 85 j. APPENDIX 10: SOS1 INTRON 16 PHYLOGENETIC TREE GENERATED FOR THIS STUDY ………………………………………………….………… 86 k. APPENDIX 11: COLLECTION DATA, SUMMER 2016 AND 2017………... 87 l. APPENDIX 12: FLOW CYTOMETRY DATA, SUMMER 2016 AND 2017 ……………………………………………………………………………….….. 89 m. APPENDIX 13: CHLOROPLAST DNA SEQUENCE IDENTITY MATRIX ………………………………………………………………………………....... 93 n. APPENDIX 14: SOS1 INTRON 16 SEQUENCE IDENTITY MATRIX …………………………………………………………………………….......… 94 vi DEDICATION This work is dedicated to my best friend and husband, Joseph Neff, whose patient support and kind encouragement has made the completion of this research and the writing of this thesis possible. vii LIST OF TABLES TABLE 1: CHENOPOD SPECIES IN THE NORTHERN NEW ENGLAND REGION ……… 3 TABLE 2: PUBLISHED 1C VALUES FOR NNE CHENOPODS AND QUINOA ………….. 14 TABLE 3: USDA COMPARATORS USED IN THIS STUDY ………………………………. 20 TABLE 4: RAPD PRIMERS USED IN THIS STUDY ……………………………………….. 26 TABLE 5: COLLECTION CODES FOR SPECIMENS SUBMITTED TO UNH HODGDON HERBARIUM AND USDA NORTH CENTRAL REGIONAL PLANT INTRODUCTION STATION ……………………………………………………………………………………… 37 TABLE 6: SPECIES SEQUENCED USING TRNL-F CHLOROPLAST PRIMERS ………... 46 TABLE 7: SPECIES SEQUENCED USING SOS1 INTRON 16 PRIMERS ………………… 49 TABLE 8: DIPLOID HYBRIDS OBTAINED FROM CROSSES …………………………… 52 viii LIST OF FIGURES FIGURE 1: CHENOPODIUM PHYLOGENETIC TREE FROM WALSH ET AL. (2015) …… 6 FIGURE 2: CHROMOSOME COUNTS OF CHENOPODIUM SPECIES FROM MANDAK ET AL. (2012) ……………………………………………………………………………………….. 9 FIGURE 3A: EMASCULATION OF C. FICIFOLIUM INFLORESCENCES …………...….. 34 FIGURE 3B: COVERED C. FICIFOLIUM INFLORESCENCES FOLLOWING POLLINATION ………………………………………………………………………………... 34 FIGURE 4A: CO-CHOP FLOW CYTOMETRY OUTPUT FROM C. FICIFOLIUM ……….. 39 FIGURE 4B: SINGLE-CHOP FLOW CYTOMETRY OUTPUT FROM REFERENCE STANDARD …………………………………………………………………………………… 39 FIGURE 4C: SINGLE-CHOP FLOW CYTOMETRY OUTPUT FROM C. FICIFOLIUM ..... 39 FIGURE 5: CALCULATED 1C FLOW CYTOMETRY VALUES OF CHENOPODS AND USDA STANDARDS ………………………………………………………………………….. 41 FIGURE 6: RAPD PCR OUTPUT FOR IDENTIFYING C. BERLANDIERI SPECIMENS TO THE SUBSPECIES-LEVEL …………………………………………………………………... 43 FIGURE 7: RAPD PCR OUTPUT FOR ASSESSING WITHIN-SPECIES DIVERSITY OF C. BERLANDIERI VAR. MACROCALYCIUM SPECIMENS ………………………………… 44 FIGURE 8: RAPD PCR OUTPUT COMPARING C. STANDLEYANUM AND C. FOGGII …………………………………………………………………………………………………... 44 FIGURE 9: CHOOSING DIPLOID PARENTS VIA RAPD PCR ………………………...….. 45 ix FIGURES 10 – 12: ASSESSMENT OF DIPLOID HYBRIDS VIA PCR WITH FTL PRIMERS ………………………………………………………………………………………………50 – 51 x ABSTRACT DEVELOPING A MOLECULAR PIPELINE TO IDENTIFY CHENOPODIUM SPECIES (AMARANTHACEAE) IN NEW ENGLAND By Erin Neff University of New Hampshire, December, 2017 Weedy species from the genus Chenopodium may provide useful genetic resources for improving quinoa (C. quinoa), a highly nutritious and economically-important crop. Before this can be accomplished, the weedy species in the Northern New England (NNE) region must be enumerated, characterized, and represented in germplasm collections and herbaria. In this study, Chenopodium germplasm was collected from the NNE region and identified via a pipeline consisting of morphological identification, flow cytometric C-value determination, gel-visualization of RAPD and gene-specific PCR products, and comparative DNA sequencing. The collected specimens were compared to plants obtained from the USDA National Plant Germplasm System. In total, five different species, including the rare C. foggii, were collected and examined, and three of the nine studied USDA comparator accessions were found to be incorrectly identified. Representative specimens were submitted to the Hodgdon Herbarium at the University of New Hampshire and the USDA National Plant Germplasm System. As a step toward developing germplasm needed for constructing a linkage map as a genomic resource for Chenopodium, crosses were performed between genetically distinct representatives of a wild diploid species, C. ficifolium, and four confirmed F1 hybrids were obtained. xi xii INTRODUCTION THE GENUS CHENOPODIUM The number of plant species belonging to the genus Chenopodium (Amaranthaceae) has been reported to be between 100 and 150 (Cole