Differential Evolution of Antiretroviral Restriction Factors in Pteropid Bats as Revealed by APOBEC3 Gene Complexity

Hayward, Joshua A; Tachedjian, Mary; Cui, Jie; Cheng, Adam Z; Johnson, Adam; Baker, Michelle L; Harris, Reuben S; Wang, Lin-Fa; Tachedjian, Gilda

The generation of the pteropid A3 loci was accomplished in a step-wise manner outlined in supplementary fig. S2 , Supplementary Material online. The A3 gene locus in Eutherian mammals is located between the CBX6 and CBX7 genes (LaRue et al. 2008) . BLASTn searches using the P. alecto A3 cDNA products as queries revealed that publicly accessible bat genomes of P. alecto and P. vampyrus did not contain assembled A3 gene loci located within the boundaries of CBX6 and CBX7. However, the bat A3 cDNA could be mapped, albeit across numerous discontiguous scaffolds, to the P. vampyrus genome accessible through the Ensembl database. The A3 locus contains many large, repetitive sequence elements that result in the failure of automated assembly. This problem was overcome by matching exons derived from sequenced cDNA, allowing the informed pairing of discontiguous scaffolds. This was not possible using the P. alecto genome as it did not contain sufficient assembled scaffolds to which A3 cDNA products could be mapped. It was reasonable to map P. alecto cDNA products against the P. vampyrus genome as these species are very closely related, with a recent divergence of $4.4 Ma and 97-99% genomic nucleotide sequence identity (Almeida et al. 2014 ). The P. vampyrus A3 locus was then generated through a step-wise extension of preexisting P. vampyrus gene scaffolds to join the gaps between exon-paired scaffolds, followed by remapping of the sequence read archives (SRA) to validate and generate the 190 kB P. vampyrus A3 locus. The P. alecto A3 locus was generated by mapping P. alecto reads against the P. vampyrus A3 locus and was found to be slightly shorter at approximately 188 kB as a result of multiple short 3-20 nt insertions and deletions. These indels occurred in the A3 loci since the divergence of P. alecto and P. vampyrus, likely as a result of nonhomologous recombination events, the frequency of which is known to be increased in regions of the genome that contain sections of repetitive DNA (Metzenberg et al. 1991) .