We utilized the full-length Fel d 1 chain 1 (NCBI accession X62477) and chain 2 (NCBI accession X62478) DNA sequences to perform a basic local alignment search (BLAST) to identify Fel d 1 orthologous sequences.We constructed maximum likelihood (ML) trees for DNA sequences using MEGAX [1].
For chain1, 11 felid species were analyzed, including Prionailurus bengalensis, Prionailurus viverrinus, Leopardus geoffroyi, Neofelis nebulosa, Panthera leo, Panthera uncia, Felis catus, Acinonyx jubatus, Lynx rufus, Lynx canadensis, Puma yagouaroundi. Additionally,Suricata suricatta was chosen as an outgroup for constructing the phylogenetic tree.
![](https://static.igem.wiki/teams/4645/wiki/model/molecular-phylogenetic-analysis/ch1-tree1.png)
For chain2, 11 felid species were analyzed, including Prionailurus bengalensis, Prionailurus viverrinus, Leopardus geoffroyi, Neofelis nebulosa, Panthera leo, Panthera uncia, Felis catus, Acinonyx jubatus, Lynx rufus, Puma yagouaroundi, Puma concolor (Due to the lack of sequencing data, we did not find orthologous sequences of chain 2 in Lynx candensis. Puma concolor were chosen as a supplement). Additionally, Canis lupus familiars was chosen as an outgroup for constructing the phylogenetic tree.
![](https://static.igem.wiki/teams/4645/wiki/model/molecular-phylogenetic-analysis/ch2-tree2.png)
Our selection of species spans several lineages of family Felidae in order to more fully reveal the evolutionary history of Fel d 1 orthologs across different species.Chain 1 was found to roughly conform to the correct phylogenetic tree for felid species, but chain 2 deviated considerably due to greater differences between orthologs.
In order to better understand the evolutionary conservation of Fel d1, we used Sequence Demarcation Tool Version 1.2 (SDTv1.2) [2] to produce pheatmaps for sequence identities of Fel d 1 chains 1 (left) and 2 (right) orthologs in feline species.
![](https://static.igem.wiki/teams/4645/wiki/model/molecular-phylogenetic-analysis/indentitynew.png)
Multiple sequence alignments indicate that the Fel d 1 gene sequence is not conserved, and its relatively low sequence identity (94.8% for CH1 and 94.6% for CH2, respectively) is roughly mirroring to the genome-wide noncoding sequences between species [3]. The lack of evolutionary conservation implies that the Fel d 1 gene may be nonessential for cats, thus suggesting to some extent that our neutralization of Fel d 1 in domestic cats is low-risk.
[1] Kumar S, Stecher G, Li M, Knyaz C, Tamura K. MEGA X: Molecular Evolutionary Genetics Analysis across Computing Platforms. Mol Biol Evol. 2018 Jun 1;35(6):1547-1549.
[2] Muhire BM, Varsani A, Martin DP. SDT: a virus classification tool based on pairwise sequence alignment and identity calculation. PLoS One. 2014 Sep 26;9(9):e108277.
[3] Cho YS, Hu L, Hou H, Lee H, Xu J, Kwon S, Oh S, Kim HM, Jho S, Kim S, Shin YA, Kim BC, Kim H, Kim CU, Luo SJ, Johnson WE, Koepfli KP, Schmidt-Küntzel A, Turner JA, Marker L, Harper C, Miller SM, Jacobs W, Bertola LD, Kim TH, Lee S, Zhou Q, Jung HJ, Xu X, Gadhvi P, Xu P, Xiong Y, Luo Y, Pan S, Gou C, Chu X, Zhang J, Liu S, He J, Chen Y, Yang L, Yang Y, He J, Liu S, Wang J, Kim CH, Kwak H, Kim JS, Hwang S, Ko J, Kim CB, Kim S, Bayarlkhagva D, Paek WK, Kim SJ, O'Brien SJ, Wang J, Bhak J. The tiger genome and comparative analysis with lion and snow leopard genomes. Nat Commun. 2013;4:2433.