Discipline: Ecology Environmental and Earth Sciences
Subcategory: Plant Research
Nia Worth - Harris-Stowe State University
Co-Author(s): David Bogler, Harris Stowe State University, Missouri, Saint Louis.
Agave is a genus of 200 species in dry habitats of Mexico, Southwestern U.S., and the Caribbean, classified in Asparagaceae, subfamily Agavoideae. Karyotypes are distinctively bimodal in this group, commonly n=30 with 25 small chromosomes and 5 large. However, some basal members have n=15 and possibly represent the oldest lineages. We hypothesize that the basal taxa in the phylogeny will have the lowest chromosome numbers and that major clades of the phylogeny will be defined by doubling of the n=15 base chromosome number. The objective of our study was to assess the current status of Agave phylogeny using sequences downloaded from GenBank. Also, we sequenced the entire chloroplast genome of Agave arizonica using the Oxford Nanopore MinION sequencer to assess its potential for future studies using whole chloroplast genomes, or regions with a known variation. The MinION is a ‘Third Generation’ sequencer that plugs into a laptop. Large fragments of DNA are passed through pores embedded in a membrane, with changes in electrical current identifying the bases. The MinION is relatively inexpensive and holds great promise for low-cost generation of large amounts of sequence data. The literature was reviewed to assemble the chromosome number and ploidy level for Agavoideae taxa. The sequences rbcL, matK, trnL-F spacer, trnL intron, and ITS2 from 25 Agavoideae were downloaded and aligned, the matrices concatenated into a single matrix with 2,700 characters, and analyzed using MrBayes. The resulting tree was well-resolved, and compared favorably with recent whole chloroplast genome studies, although resolution within Agave is still weak. The n=15 taxa of Agavoideae are basal on this tree and this suggests the group originated in western North America. The MinION experiment resulted in 1,103 cpDNA sequence fragments that were aligned to a reference chloroplast genome of Agave americana from GenBank using Geneious software. The average coverage was 4.9 and the average read length was about 800 bp. The next step in our experiment is to locate variable regions of the cpDNA genome of Agave species and use the MinION to sequence large fragments (8-10,000 base length) in multiple species to obtain a further resolution of the species groups. Funder Acknowledgements: Funding was provided by HBCU-UP Implementation grant, HRD-0811219, increasing STEM Degree Production through Undergraduate Research and Collaborations, administered by Dr. Tommie Turner. NSF provided support in the form of a Research Experiences for undergraduates award to Nia Worth. We thank HSSU for granting access to laboratory space for use of the MinION, and Missouri Botanical Garden for access to the library, herbarium, and garden. Would also like to thank Faculty Advisor/Mentor: David J. Bogler.
Funder Acknowledgement(s): Funding was provided by HBCU-UP Implementation grant, HRD-0811219, increasing STEM Degree Production through Undergraduate Research and Collaborations, administered by Dr. Tommie Turner. NSF also provided support in the form of a Research Experiences for undergraduates award.
Faculty Advisor: David Bogler, email@example.com
Role: Arranged lists of agave species to be included in the experiment, focusing on karyotypes specific to what the objective of our experiment was. Help with troubleshooting the use of the software which was involved in work with our experiment. Purified sample for use with MinION with the assistance of a mentor and loaded sample. Researched and looked up general information already known about agave genus and its taxonomic whereabouts.