The nuclear genome of Rhazya stricta and the evolution of alkaloid diversity in a medically relevant clade of Apocynaceae

Abstract

Alkaloid accumulation in plants is activated in response to stress, is limited in distribution and specific alkaloid repertoires are variable across taxa. Rauvolfioideae (Apocynaceae, Gentianales) represents a major center of structural expansion in the monoterpenoid indole alkaloids (MIAs) yielding thousands of unique molecules including highly valuable chemotherapeutics. The paucity of genome-level data for Apocynaceae precludes a deeper understanding of MIA pathway evolution hindering the elucidation of remaining pathway enzymes and the improvement of MIA availability in planta or in vitro. We sequenced the nuclear genome of Rhazya stricta (Apocynaceae, Rauvolfioideae) and present this high quality assembly in comparison with that of coffee (Rubiaceae, Coffea canephora, Gentianales) and others to investigate the evolution of genome-scale features. The annotated Rhazya genome was used to develop the community resource, RhaCyc, a metabolic pathway database. Gene family trees were constructed to identify homologs of MIA pathway genes and to examine their evolutionary history. We found that, unlike Coffea, the Rhazya lineage has experienced many structural rearrangements. Gene tree analyses suggest recent, lineage-specific expansion and diversification among homologs encoding MIA pathway genes in Gentianales and provide candidate sequences with the potential to close gaps in characterized pathways and support prospecting for new MIA production avenues.

Figure 1. Divergent structural evolution in Rhazya stricta.

Genome level rearrangements leading from the ancestral core eudicot, represented by Vitis (upper right hand corner) to Rhazya and Coffea. Each linear arrangement represents successive steps from the eudicot ancestor toward the Gentianales ancestor. An arrow leads from the last of these stages to the relevant portion of the Gentianales ancestor. From this ancestor, two arrows indicate the divergence of Coffea and Rhazya, and successive rearrangements within each lineage are again indicated by linear arrangements of colored blocks. Inferred rearrangements between lineages are given at the arrows. Extant genomes carry the species name. The circular diagrams at the lower left and right represent the current orthologies between Rhazya superscaffolds 6 and 15, and Coffea and Vitis, respectively. Syntenic blocks are colored according to the 21 ancestral core eudicot chromosomes and do not represent biologically significant units.

Figure 2. Expansion, loss and duplication of selected monoterpenoid indole alkaloid (MIA) sequences across Gentianales.

The cladogram (top, center) depicts relationships among six eudicots based on the Angiosperm Phylogeny Group (http://www.mobot.org/MOBOT/research/APweb/). Histograms below each species indicate the number of sequences returned in BLAST searches to identify homologs for each of the eight gene families. The maximum likelihood trees along the left and right show subclades of reconciled gene phylogenies that include known homologs in either Catharanthus (cra.Genbank) or Rauvolfia (rsa.Genbank) for seven of the eight MIA genes (THAS is shown in Fig. 3). Complete reconciled phylogenies for each of the eight sequences are shown in Supplementary Fig. S3. cra, Catharanthus roseus; cof, Coffea canephora; rha, Rhazya stricta; tom, Solanum lycopersicum; rsa, Rauvolfia serpentina. Blue font indicates GenBank sequences and red font indicates Rhazya stricta sequences. Red square = gene duplication; green circle = speciation event; gray circles = gene loss. Values above branches indicate bootstrap support for the clade, numbers below nodes give bootstrap support for the event (duplication or speciation) at the node. Bootstrap values less than 50 are not shown; inferred loss events are indicated with an asterisk and do not have associated bootstrap values.

Figure 3. Duplication and loss of Tetrahydroalstonine synthase (THAS) across six angiosperms based on gene tree/species tree reconciliation.

Internal and terminal branches in black reflect the amount of change along branch; dashed gray branch extensions are present for branches whose lengths were too short to enable uniform representation with branch support and length information. cra, Catharanthus roseus; cof, Coffea canephora; rha, Rhazya stricta; rsa, Rauvolfia serpentina; tom, Solanum lycopersicum; the, Theobroma cacao. Species indicated in blue font were downloaded from GenBank and those in red are from Rhazya stricta. Red square = gene duplication; green circle = speciation event; wide gray dashed line ending with a circle = gene loss. Values above branches indicate bootstrap support for the clade, numbers below nodes give bootstrap support for the event (duplication or speciation) at the node. Bootstrap values less than 50 are not shown; inferred loss events are indicated with an asterisk and do not have associated bootstrap values. Scale bar represents number of amino acid substitutions per site. The maximum likelihood (ML) tree shown is the tree with the best ML score.