Comparative Mitogenomic Analysis of the D. buzzatii cluster

MENSCH, J; HURTADO, J; MOREYRA, N; HASSON, E

ALAMOS, MEXICO

Simposio; Simposio Evolution and Ecology: The Drosophila-Cactus model system; 2018

LANGEBIO-University of California San Diego

Comparative Mitogenomic Analysis of the D. buzzatii clusterMtDNA has became a popular marker in evolutionary biology and populationgenetics given its high mutation rate respect to nuclear genetic markers,which allow to infer phylogenetics relationships between species andpopulations. Also, the lack of recombination, linear ancestry (inheritance) andhigh copy number frequently makes mitochondrial DNA the molecular markerof choice (Ladoukakis et al. 2017)How we have done itWhy we chose mitochondrial DNA! What we have doneFor this project, it were employed Paired Ends Illumina reads from bothgenomic and transcriptomic sequences. All the process can be summarizedby the next six steps:(1)Mapping genomic and transcriptomic reads to D. mojavensismitochondrial genome with bowtie2.(2)Extracting only confident mapped reads with Samtools and join to createone dataset per each species.(3)Performing an assembly with Mira assembler (B. Chevreux et al. 2014),using D. mojavensis as reference. At this stage, it is important to trydifferent genome coverage and set the Mira parameters.(4)Running MITObim (Hahn et al. 2013) with the complete set of reads oneach species to re-assembly the uncompleted regions of the resultingassembly in step 3. This software manage the genome coverage and set theoptimal value for each dataset.(5)Carrying out the genome annotation using the Mitos WebServer (Bernt etal. 2013) and the gene annotations of D. mojavensis.The resulting assemblies allow us to perform a phylogenetic analysis withthe complete mitochondrial genomes of D. buzzatii, D. koepferae, D.antonietae and D. borborema, using D. mojavensis as outgroup. In addition,in order to validate the annotated genes, we compared the mitochondrialgenes previously reported by Oliveira et al. (2012) (nad2, cox1, cox2 andlsRNA) with the respective assemblies obtained in this project.What we have achievedWhat is there to improve and what to highlight?The statistics for the assemblies are shown in Table 1. At least the 90% the total sequence of each mitogenome was assembled and all annotated genes on eachcorresponding assembly were in the correct order and sense. Phylogenetic tree using the complete mitochondrial genomes are similar with those obtained bymorphological characters and nuclear sequences, being D. buzzatii the most divergent species of the cluster (Figure 1). Importantly, our annotated genesperfectly matched with those obtained by Oliveira et al. (2012) and formed groups in each species (Figure 2) although we need to employ the wholemitogenomes to reconstruct a high-confidence phylogeny (Figure 1).Table 1. Assembly statistics of the five mitochondrial genomes included in thisstudy.Figure 1. Maximum Likelihood Phylogenetic Three using the completeassemblies of the five Drosophila species.Figure 2. Maximum Likelihood Phylogenetic Three using 4 previously reported generegions with the corresponding sequences of our assemblies.The unknown regions (N´s) must be analyzed by other methods given the variability in the total length. It should be noted that the less performance assembly(D. buzzatii) was only made with transcriptomic sequencing data, highlighting the relevance of having genomic information, probably because of its greaterenrichment in mitochondrial sequencing reads. As a future perspective, it is planned to sequence the unrevealed regions. This action will allow to evaluateconsistency of the assemblies and filling the unknown bases.The aim of the project is to design a workflow to reconstruct and obtain, for thefirst time, the mitochondrial complete genomes assemblies of four species of theDrosophila buzzatii cluster directly from genomic and transcriptomic sequencingdata. Subsequently, it were studied the phylogenetic relationships among speciesand compared the results with the small set of mitochondrial markers previouslyreported (Oliveira et al. 2012).ReferencesChevreux et al. Using the miraEST Assembler for Reliable and Automated mRNA Transcript Assembly and SNP Detection in Sequenced ESTs. Genome Research 2004. 14:1147-1159.Oliveira et al. Monophyly, divergence times, and evolution of host plant use inferred from a revised phylogeny of the Drosophila repleta species group. Molecular Phylogenetics and Evolution Hahn64 (2012) 533?544.Hahn et al.Reconstructing mitochondrial genomes directly from genomic next-generation sequencing reads?a baiting and iterative mapping approach, Nucleic Acids Research, Volume 41, Issue 13, 1 July 2013, Pages e129.Ladoukakis et al. Evolution and inheritance of animal mitochondrial DNA: rules and exceptions. Journal of Biological Research-Thessaloniki, 2017, vol. 24, no 1, p. 2.Species D. borborema D. antonietae D. buzzatii D. koepferae D. mojavensisTotal length(bp) 14938 14938 14926 14933 14904Unknownsequencepercent (N?s%)3,7% 2,2% 9,55% 0,21% 0%N° ofannotatedgenes37 36 30 38 38Correctedgenes order � � � � �