2017 ASHS Annual Conference

Penstemon is a large genera native to the United States and contains nearly 300 species. Speciation is thought to have occurred during the latter part of the Pleistocene epoch. The majority of species are untapped for commercial use and are not used in breeding programs, largely, because of species incompatibility during interspecific hybridization. We are interested in a gene that is necessary for proper chromosomal segregation during cell division: Centromeric Histone 3 (CENH3). This histone protein differs from others in that it 1) localizes only to the centromeric region of a chromosome and 2) evolves rapidly particularly at the 5ʹ end and the DNA-binding region in Loop 1. Mutations in this gene lead to sterility and uniparental chromosome elimination when crossed with wild types. We hypothesized that this gene was also rapidly evolving in Penstemon and may aid or be involved in speciation. We were able to utilize sequence data from Genbank to design degenerate primers to conserved regions within the coding sequence of CENH3 from unrelated angiosperm species. From those primers we successfully amplified two copies of CENH3, termed CENH3a and CENH3b. Next generation sequencing of Penstemon DNA revealed that a gene duplication event took place prior to speciation within the genus. The coding sequence of CENH3b is approximately 78 bp shorter than CENH3a because of a deletion in the hypervariable 5ʹ region, and it is approximately 1,800 bp shorter in gDNA because of the absence of two introns. Alternative splice variants were cloned from both copies of CENH3, but they occurred nearly 10 times more frequently in CENH3b than in CENH3a. Most of the splice variants caused frameshift mutations that would lead to the creation of early stop codons. It is unclear if these splice variants are functional. A phylogeny was built to compare the taxonomic classifications of Penstemon with the sequence divergence of CENH3 among species. The phylogenetic data was in many ways similar to the taxonomic data. However the data suggest that subgenus Habroanthus evolved from the subgenus Penstemon. The data also suggest that Saccanthera and Dasanthera shared a common evolutionary ancestor that is different from the subgenera Habroanthus and Penstemon. More work will be needed to determine the translational profile of CENH3 mRNAs and their role in cell division and speciation within Penstemon.