Phylogenetic relationships, marker analysis, and investigation of genes mediating high and low methyl salicylate biosynthesis in different birch species (Betula L., Betulaceae)
The perennial woody plant birch (Betula L.) is crucial for the economy and to maintain the biodiversity of the boreal forests. Besides their conventional benefits, some birches constitutively synthesize the anti-inflammatory methyl salicylate (MeSA). It is used in many drugs as an ointment for relief against rheumatic complaints, muscles, and joint pains. The molecules involved in the biosynthesis of MeSA following the evolutionary relationships of high and low- MeSA producing birch and markers associated with high MeSA content were never identified. In the first examination, eight chloroplast and five nuclear genomic regions were analyzed to determine the phylogenetic relationships and genetic variations present in high and low MeSA producing birch species. Betula lenta, always clustered within a clade with other high MeSA-producing birches and also formed an ancestral node in the network analysis. The phylogenetic and network analysis suggests, B. lenta is one of the ancestors and can produce high MeSA. Further, candidate genes, salicylic acid methyltransferase (SAMT), and salicylic acid-binding protein 2 (SABP2) mediate the MeSA biosynthesis were studied from the eight high (B. lenta, B. alleghaniensis, B. medwediewii, and B. grossa) and low (B. pendula, B. utilis, B. alnoides, and B. nana) MeSA producing birches. Additionally, six distinguishable single nucleotide substitution markers were determined and validated with 38 additional individuals originated from different botanical gardens to acknowledge high and low MeSA-producing birches. The tissue-specific expression and bioinformatics analysis suggested that SAMT and SABP2 have a significant effect on MeSA biosynthesis in birches. Furthermore, the detailed examination of the SAMT and SABP2 genes lead to the identification of their affiliated gene and enzyme families, SABATH (SAMT, benzoic acid carboxyl methyltransferase, and theobromine synthase) and MES (methylesterases), respectively in the Betula pendula (BpSABATH and BpMES). Bioinformatics and phylogenetic analysis revealed the putative substrate specificities of BpSABATH and BpMES enzymes. The valuable information of the candidates, BpSABATH and BpMES could be exploited for genetic modification or targeted mutagenesis to decide only the function of a gene followed by enzymatic substrate specificity analysis. Identified nuclear markers will be used in the birch breeding for mass production of organic MeSA. This thesis encourages the commercial production of birches for the timber in short-rotation coppices (SRC) along with pharmacological benefits through extracting MeSA from the bark. The SRC and extracted MeSA would be highly advantageous for forest farming as well as for bio-economy.