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ISOLATION OF SIRENIAN WHOLE MITOCHONDRIAL GENOME USING LONG-RANGE PCR FOR HIGH THROUGHPUT SEQUENCING
Sirenians are herbivorous aquatic mammals that inhabit shallow waters of Indian, Pacific, and Atlantic oceans, rivers and estuaries zones. Few genomic studies that focused on understanding the genetic diversity of Sirenians have been done, however, focusing on one coding region or few nuclear loci. Nowadays, new sequencing technologies are able to generate entire genomes in one sequencing run, reducing wet lab time and overall cost. Mitogenomes are suitable for different genetic studies due to its main features, such as no recombination and high substitution rate. In this context, we aimed using a new approach to isolate the whole mitochondrial genome of Sirenians from genomic DNA (gDNA), through long-range Polymerase Chain Reaction (LR-PCR). Mitogenome sequences of Elephant (Elephas maximus), Dugong (Dugong dugon) and Florida West Indian Manatee (Trichechus manatus latirostris) available on GenBank (NCBI) were aligned using MEGA and a consensus sequence was used for designing two pairs of putative primers within conserved regions of the Tethytheria clade, using Primer 3 Plus tool (GC content > 40%, target fragment range 7000-10000). These two primer pairs split the 16 kb circular mitogenome into two amplification reactions (reaction 1: amplicon length 10878 bp; reaction 2: amplicon length 6346 bp). Quality of primers was checked in-silico using the OligoAnalyzer IDT DNA tool to avoid hairpins and self/heterodimers, while specificity to Sirenians was checked using BLAST and UCSC In-Silico PCR tool. Two reactions were carried out using Platinum Taq Polymerase High Fidelity (Invitrogen) in 25 µl reaction mix (0.2 mM each dNTP, 1U Polymerase, 0.2 mM PrimerF, 0.2 mM PrimerR, 1x Buffer, 2.0 mM Mg2SO4, 20.7 µl H2O) with 1.5 µl of gDNA (50 ng/µl). For the 10.8 kb fragment amplicon, cycling conditions were: 30 seconds at 94℃, 35 cycles of 15 seconds denaturation at 94℃, 30 seconds of annealing at 58℃, and 13 minutes extension at 68℃, with a final 10 minutes extension at 68℃. For the 6.3 kb amplicon, cycling conditions were the same, except for annealing temperature at 55℃ and 7 minutes of extension time. PCR products were checked using agarose gel 0.8% electrophoresis stained with ethidium bromide. Partial sequencing was done using Sanger technology, and generated sequences were blasted on Genebank, with a mean of 98% of Florida manatee mitogenomes identity. We tested this LR-PCR protocol in 28 samples of Marine manatees from Brazil (T. manatus manatus) and 4 samples of Amazon Manatees (Trichechus inunguis) with a success rate of 81% (26 of 32). Samples with no PCR product showed a fragmented gDNA, which may be the reason for no amplification. Overall, LR-PCR seems to be an easy and accessible way to isolate the entire mitochondrial genomes of non-fragmented Sirenians DNA samples. Together with massive throughput sequencing, which we are currently testing, it might work as a fast and promising protocol to access the mitochondrial genetic diversity of Sirenians and other mammals.
Sirenia, Mitogenome, Long-range PCR, NGS
This work is part of the Marine Manatee Mitogenome Project, funded by the National Geographic Society and CAPES.
Fabricio Rauan Garcia Furni, Bruna Palma Matta, Cibele Bonvicino, Pedro Cordeiro-Estrela