Study of Molecular phylogenetic and matk protein structure in "Susan -e Chelcheragh" (Lilium ledebourii [Baker] Boiss)

Chloroplastic matk gene is one of the best genes for a plant barcode and study of phylogenic/evolutionary relationships between plant species and families, possesses a high evolutionary rate at a nucleotide/amino acid level. Lilium, with ~100 species, is the most important genus in the Liliaceae that have been classified. The classification of Liliaceae is continuously studied morphologically, cariologically, and phylogenetically. New molecular phylogenic approaches have lead in fundamental changes in this classification. Lilium ledebourii is a valuable species endemic to Iran, but it is seriously endangered. Notwithstanding its valuable characteristics, Lilium ledebourii has never been phylogenetically studied together with the other members of the Liliaceae. Using matk sequence, we studied the taxonomic position and evolutionary relationships of Lilium ledebourii and 41 other Lilium species.

First, DNA was extracted by using Qiagen DNEasy. After verifying DNA quality and quantity, real-time sequencing (SMRT) was performed using the next generation PacBio platform and a complete matK sequence was obtained using bioinformatics tools. Finally, it was registered in NCBI (ID: MN557236 - Pending publishing). For the study of the phylogenetic relationships, matk sequences of the 41 species were downloaded from NCBI. The sequences were aligned by Mega7 software, following the ClustalW method. Phylogenetic tree was drawn according to the maximum likelihood method. Genetic distance was calculated by the K2P method. The maximum composite likelihood of the nucleotide substitution pattern was estimated using the substitution matrix. By using FFPred, InterProScan, TargetP, and Phyre2, secondary structures such as Alpha helix, Beta turn, and random coil, as well as the tertiary structure of the protein, were studied.

According to the phylogenetic tree, the Lilium species were classified into 4 clusters: A, B, C, and D. The Iranian species was placed in cluster B. According to phylogenic topology as well as to the study of genetic distance, Lilium ledebourii had the highest similarities to L. pyrenaicum, L. ciliatum, and L. candidum with 97% bootstrap. In bioinformatic analysis, a high level of conservation was observed in the alignment of this protein. Lilium ledebourii contained arginine at position #271, which was similar only to L. pyrenaicum, L. ciliatum, and L. candidum. The other species had lysine at this position. The species displayed many differences in the positions of amino acids, such as at #317, #346, #363, and #417. The study of the similarities of the secondary structure of matk in L. ledebourii showed this protein to have 233 amino acids at α helix (45.51%), 24 amino acids at β turn (4.69%), and 156 amino acids at random coil (30.74%).

The present study analyzed matk in Lilium ledebourii for the first time, revealing its secondary structure, the position of α- helix and β- turn. Additionally, the Tertiary structure of this protein was proposed for the first time. According to phylogenic topology as well as to the study of genetic distance, Lilium ledebourii had the highest similarities to L. pyrenaicum, L. ciliatum, and L. candidum. Overall, the molecular relationship (phylogenetics and protein structure) of Lilium ledebourii with other Lilium species was studied.