Discipline: Biological Sciences
Nia Worth - Harris Stowe State University
Co-Author(s): Dr. David Bogler, Harris Stowe State University, Saint Louis, MO and Missouri Botanical Garden, Saint Louis, MO
Agave is a genus of 200 species in dry habitats of Mexico, Southwestern U.S., and Caribbean, classified in Asparagaceae, subfamily Agavoideae. Very little is known about subgeneric relationships in Agave due to low sequence variation. The objective of our study was to assess the current status of Agave phylogenomics using a combination of karyotype information and sequences downloaded from GenBank. In addition 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. 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 generation large amounts of sequence data. Bud material from living Agavoideae was collected and chromosomes imaged using the acetocarmine squash technique. The literature was reviewed to assemble chromosome number and ploidy level for Agave and 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. Higher polyploidy occurs in some species of Agave. The downloaded sequences (rbcL, matK, trnL-F spacer, trnL intron, and ITS2) from 25 Agavoideae were aligned, the matrices concatenated into a single matrix with 2,700 characters, and analyzed using MrBayes. The resulting tree was well-resolved, although resolution within Agave is weak. The n=15 taxa of Agavoideae are basal and this suggests the group originated in the New World. 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 average read length was about 800 bp. With more careful purification and quantification of DNA extracts we hope to barcode 12 or more species samples and run these in a single MinION run.
Funder Acknowledgement(s): HBCU-UP Implementation Grant
Faculty Advisor: David Bogler, Ph.D., BoglerD@hssu.edu
Role: I performed chromosome counts and gathered background information about the genus in general. I gathered information on how many types of ploidy were in the genus, the different species pollination biology, and the economic importance of species in the genus. I learned how to use both the Geneious and MEGA software for use with the Minion. I also performed gel electrophoresis on several purified agave DNA samples to find out which ones were fragmented enough to be used for sequencing. Finally I carried out sequencing using the Minion sequencer with the purified DNA samples.