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Research Project: Management of Temperate-Adapted Fruit, Nut, and Specialty Crop Genetic Resources and Associated Information

Location: National Clonal Germplasm Repository

Title: Increased phylogenetic resolution using target enrichment in Rubus

Author
item Carter, Katie - Oregon State University
item Alice, Lawrence - Western Kentucky University
item Bryant, D - Danforth Plant Science Center
item Bushakra, Jill
item Liston, Aaron - Oregon State University
item Mockler, Todd - Danforth Plant Science Center
item Bassil, Nahla
item Hummer, Kim

Submitted to: HortScience
Publication Type: Abstract Only
Publication Acceptance Date: 6/28/2017
Publication Date: 9/18/2017
Citation: Carter, K., Alice, L., Bryant, D., Bushakra, J., Liston, A., Mockler, T., Bassil, N.V., Hummer, K.E. 2017. Increased phylogenetic resolution using target enrichment in Rubus. HortScience. Available: https://ashs.confex.com/ashs/2017/meetingapp.cgi/Paper/26717.

Interpretive Summary: Understanding the evolution of blackberries, raspberries, and their wild relatives (Rubus) has been challenging because the group exhibits many complicated biologically features. Species can breed with each other, have multiple copies of each chromosome set, and produce seed without pollen. The availability of DNA sequence data has clarified evolutionary relationships in other plants, but these characteristics complicate analyses within Rubus. In the past, this group has been studies using only a few genes or only focusing on a few species. A large amount of sequence data over a wide species sampling is needed to untangle the evolutionary history of these berries. The objective of our study was to use an innovative targeted DNA sequencing approach to provide clarity concerning relationships within Rubus. This procedure involved sequencing specific regions of DNA instead of the entire set of genes. This allowed more samples to be sequenced at one time. We sequenced 4,445 genes from the black raspberry genome and 247 genes that were found in the apples, peachs, and strawberries. These genes exist as a single copy, and are particularly useful for evolutionary studies like this one. This represents a significant increase in the amount of data compared to other recent studies. When initially described in the early 1900’s, this genus was divided into 12 groups to better categorize similar species. Groups at this level of classification, within a genus, are called subgenera. We sequenced 96 samples, including representatives from each of the 12 groups and five known hybrids or economically important cultivars. Representative samples were studied from every continent on which caneberries grow. Preliminary analyses resulted in a branching family tree indicating the evolutionary relationships between species. A taxonomic group name is useful if all members come from a common ancestor. Then they form one group. The members of subgenus Orobatus, caneberries from South America, form a unique group. Nearly all species of subgenus Rubus, commonly called blackberries, form a separate group. Members of some groups were divided throughout the tree, indicating that traditional taxonomic names should be redefined. The groupings we obtained were similar to those seen from a previous study. However, relationships between the major groups were somewhat different in our tree, likely due to the increased number of genes studied. In particular, some wild crop relatives currently classified in two subgenera, Anoplobatus and Dalibardastrum, form a unique group separated from all other species. Subgenus Idaeobatus, containing raspberries, was surprisingly split between two groups. This robust dataset will produce further insights into the evolutionary history of caneberry species. This research will be informative for breeders who select blackberries and raspberries for cultivar development. It also demonstrates how this gene targeting technique can clarify relationships in complex groups such as the blackberry/raspberry crop wild relatives.

Technical Abstract: Phylogenetic analyses in Rubus L. have been challenging due to polyploidy, hybridization, and apomixis within the genus. Wide morphological diversity occurs within and between species, contributing to challenges at lower and higher systematic levels. Phylogenetic inferences to date have been based on limited nuclear and chloroplast loci or have focused on one subgenus or section. To clarify complex infrageneric relationships in Rubus, a thorough sampling of each subgenus at multiple loci is needed. Target enrichment is an innovative phylogenomic approach that could provide clarity. The objective of our study was to use target enrichment to determine phylogenetic relationships within Rubus. Our target enrichment probe synthesis encompassed loci from the Rubus occidentalis L. genome and a comparison of the apple, peach and strawberry genomes. Specifically, we chose probe sequences from transcripts of 4,445 single copy R. occidentalis nuclear genes. In addition, we included probes for 247 single copy nuclear genes, as identified from the apple, peach and strawberry genome sequences. Illumina sequencing was performed for 96 enriched libraries, including representatives from each of 12 subgenera and five known hybrids or economically important cultivars. The final sequencing library contained a high proportion of the captured sequences, enabling high levels of sample multiplexing while retaining good depth of coverage at each locus. More than three species were sampled for each subgenus except for Comaropsis and the monotypic subgenus Chamaemorus. Representative samples from every continent on which Rubus is known were studied. Preliminary analysis differentiated certain members of subgenera Anoplobatus and Dalibardastrum into a unique clade relative to the remainder of the genus. Observed clades were similar to those seen in a previous nuclear ribosomal internal transcribed spacer (ITS) tree, however, relationships among clades were different. Idaeobatus was polyphyletic in the ITS and the target enrichment phylogeny, but the trees place alternate Idaeobatus clades sister to the clade containing subgenus Rubus. The target enrichment phylogeny provided additional resolution within clades, likely due to the increased number of loci. Orobatus was monophyletic and all species of subgenus Rubus except R. ursinus and R. caesius grouped together. Other subgenera were either para- or polyphyletic, suggesting that traditional subgeneric divisions of Rubus should be redefined. This robust dataset will produce further insights into the role of polyploidy and interspecific hybridization in the evolutionary history of Rubus.