# Identifying Terpenoid Biosynthesis Genes in _Euphoria maculata_ via Full-Length cDNA Sequencing
## 4 Discussion  
NGS technologies have revolutionized many areas of genetics. Transcriptomics captures a snapshot of the total transcripts in a cell at a specific time and is used to quantify gene expression profiles during development [19; 35]. High-throughput short RNA-Seq analysis was used to identify the genes involved in the biosynthesis of phytochemicals in medicinal plants [25; 27; 36]. Here, we used transcriptome profiling to analyze the genes involved in terpenoid biosynthesis in the medicinal plant _E. maculata_ L., which is used in folk medicine in oriental countries [4]. Terpenoids are major secondary metabolites in _E. maculata_ that have pharmacological benefits including anti-inflammation, antioxidant, antitumor, hepatoprotection, and anti-HIV protease activity [4; 5; 7; 37]. The _E. maculata_ genome has not been sequenced; therefore, we obtained transcriptome sequences from PacBio SMRT full-length cDNA sequencing. We obtained 20,172 full-length unigenes, which is similar to that obtained in _Berberis koreanu_ (23,246) by PacBio SMRT sequencing [27]. Although full-length unigenes may not accurately represent the number of genes in a species, the number of genes in _E. maculata_ may be low compared to other plant species. Gene numbers in plants range from 20,000 to 124,000. The small genome of _Arabidopsis thaliana_ encodes 26,000 genes [38]. We previously reported an Illumina NovaSeq-derived transcriptome of _Euphorbia jolkini_ having 123,215 assembled transcripts [27]. In our functional annotation of _E. maculata_ genes, 19,190 (92.6%) and 19,407 (93.65%) matched with the Nt and Nr databases in NCBI, respectively, indicating that the function of most of the transcripts is known and only about 7% of the transcripts have not been annotated. The top three species BLAST-matching with _E. maculata_ transcripts were the Para rubber tree (_Heven brasiliensis_), castor bean (_Ricinus communis_), and cassava (_Manibot esculent_), all in Euphorbiaceae. These plants produce a milky latex containing terpenes [18; 39; 40]. The high match to these species may be because they have well-characterized transcriptome data due to their economic importance, as reported in the Para rubber tree [41; 42], castor bean [43], and cassava [44; 45]. GO allows the comparison and functional classification of genes and their products across species ([[http://www.geneontology.org/](http://www.geneontology.org/)]([http://www.geneontology.org/](http://www.geneontology.org/)); accessed on 1 April 2022) and covers three domains: cellular components, molecular functions, and biological processes. In our _E. maculata_ transcriptomes, the distribution of genes in the different functional categories was similar to that of other medicinal plants [26; 27; 46]. PacBio SMRT sequencing is a third-generation sequencing system that allows the identification of isoforms [20; 47]. Paralogs are homologous genes in a species that arise from the duplication of a single ancestral gene [48]. We identified isoforms and paralogs in our PacBio SMRT sequencing data. In humans, approximately 70% of protein-coding genes have at least one paralog [49]. _Arabidopsis_ has at least 21,843 paralogs, which account for approximately 84% of its protein-coding genes [50]. However, 97.8% of the _E. maculata_ unigenes were single copy, which is unexpectedly high because most eukaryotes underwent several whole-genome duplication events that resulted in the duplication of ancestral genes. Thus, it will be interesting to determine the number of paralogs in other _Euphorbia_ species to verify our findings. Currently, only one _Euphorbia_ transcriptome has been reported, but it was generated by Illumina NovaSeq, which does not permit the analysis of paralogs of full-length transcripts [26]. Transcript isoforms are derived from alternative splicing of the introns and the differential initiation or termination of translation from primary transcripts, which allows a single gene to code for multiple forms of a protein [51].