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Maryum Bhatti1, Aaron Lee1, Hiba Rahman-Vyas1, Dianella G

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Maryum Bhatti1, Aaron Lee1, Hiba Rahman-Vyas1, Dianella G NAC SUBFAMILY 1A GENE TREE Combining benchwork and bioinformatics to reconstruct the evolutionary history of CUP- SHAPED COTYLEDON in honeysuckles and relatives Maryum Bhatti1, Aaron Lee1, Hiba Rahman-Vyas1, Dianella G. Howarth2, Michael J. Donoghue3, Wendy L. Clement1 1Department of Biology, The College of New Jersey, Ewing, NJ; 2Department of Biological Sciences, St. John’s University, Jamaica, NY; 3Department of Ecology and Evolution, Yale University, New Haven, CT RECOVERING CUC FROM INTRODUCTION DIPSACALES Context Fusion among adjacent parts is a phenomena that occurs Sampling and Isolating CUC throughout flowering plants. CUP-SHAPED COTYLEDON, CUC, • 38 species were selected across the Dipsacales for a member of the NAC Subfamily 1a transcription factors, and has been shown to affect organ boundary formation.1,2 Honeysuckles, species that had no available genomic resources or Lonicera (Caprifoliaceae, Dipsacales), are known for fusing • Degenerate primers were designed to isolate exons petals into long tubes and also exhibit fusion among ovaries, bracts 1 and 2 of CUC1/2 and CUC3 based on reference and leaves. Variation in fusion across 160 species of Lonicera genomes (e.g., Arabidopsis, Petunia, Snapdragon) make them an excellent system to investigate the evolution of • CUC specific primers were also created including fusion. all introns and exons of CUC Goals • Successful reactions near 500 bp were isolated and • Recover CUC from the phylogenetic diversity of Dipsacales, cloned using PCR and cloning; sample target species in which no • Multiple clones per species were sequenced at the genomic data is available. Yale Sequencing on the Hill Facility • Reconstruct a gene tree for NAC Subfamily 1a and CUC using • Sequences were assembled and manually edited both direct sequences gained in this study and data extracted 3 from available genomic resources. using Geneious v.9 • Assembled sequences were subject to a BLASTn4 against the Arabidopsis thaliana genome to verify DIPSACALES SPECIES SAMPLED and/or determine which NAC gene was recovered Alignment and Gene Tree Figure 3. Maximum clade Reconstruction credibility tree from the Bayesian analysis of exons 1 • Sequences were combined and 2 of NAC Subfamily 1a. All with a larger dataset of NAC NAC and CUC clades are Subfamily 1a (Lee et al. highlighted and labeled. ML unpublished) recovered bootstrap values are displayed from available genomic at the nodes and colored circles Dipelta floribunda resources. From here, two Lonicera ferdinandii indicate posterior probabilities datasets were formed; (1) following the key on the left NAC dataset with exons and side of the figure. Recovery of (2) CUC dataset that two copies of CUC1/2, namely included introns (not shown CUC2A and CUC2B from the in this poster) were aligned same accession of Diervilla in MUSCLE5 and MEGA8 lonicera, Abelia chinensis, and • Phylogenetic analyses were Dipelta floribunda help conducted using MrBayes6,7 confirm a duplication of CUC2 Weigela japonica Diervilla sessilifolia under a single partition with prior to the origin of Figure 1. Representatives of four clades of Dipsacales sampled in this study. the GTR+I+G model for a Caprifoliaceae. No Collections and observations of these species were made from the living million generations Caprifoliaceae sequences have collections at the Arnold Arboretum of Harvard University been recovered for CUC3. KEY FINDINGS Annotations: We recovered 15 CUC and one NAC100 sequence from a total of 38 species sampled across the Dipsacales. CUC3 • Black circle = nodes at which duplications have was not successfully amplified or sequenced. occurred • Red circle =Directly Focusing on the evolution of CUC across Dipsacales, we recovered a duplication of CUC1/2 and one loss of CUC3 sequenced species that specific to Caprifoliaceae (Fig. 2): are duplicated • Our inclusion of Diervilla and Weigela, which together form a clade sister to the rest of Caprifoliaceae, confirms • Adoxaceae clade = the duplication of CUC1/2 occurred prior to the diversification of Caprifoliaceae rather than within Caprifoliaceae. indicated at the CUC3 • Our inability to amplify CUC3 from Caprifoliaceae continues to support the loss of this gene that occurs clade throughout angiosperms. • Caprifoliaceae clade = indicated by the black Directly isolating and sequencing CUC was an important approach to include species for which no genomic data lines and includes were available (e.g., Diervilla, Dipelta, and Weigela). CUC2A and CUC2B References: 1Aida, M. et al. 1997. The Plant Cell 23:54-68. 2Zhong, J. et al. 2016. 3Geneious 9. Biomatters Ltd., New Zealand. 4Camacho, C. et al. 2008. BLAST+: architecture and Acknowledgements: We thank TCNJ School of Science and Department of Biology as well as the Mentored Undergraduate Summer Experience applications. BMC Bioinformatics 10:421. 5Edgar, R. C. 2004. Nucleic Acids Research 32:1792-1797. 6Huelsenbeck JP and F Ronquist. 2001. Bioinformatics 17: 754-755. 7Ronquist F and for funding and members of Dr. Clement’s lab at TCNJ. This work is also supported by NSF grants NSF DEB-1929670 and NSF OAC-1828163 JP Huelsenbeck. 2003. Bioinformatics 19: 1572-1574. 8Kumar S,, Stecher G, Li M, Knyaz C, and Tamura K. 2018. MEGA X. Molecular Evolutionary Genetics Analysis across computing platforms. Molecular Biology and Evolution 35: 1547-1549.
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