| Summary: | Terpenoids are a wide variety of natural products and terpene synthase (TPS) plays a key role in the biosynthesis of terpenoids. <i>Mentha</i> plants are rich in essential oils, whose main components are terpenoids, and their biosynthetic pathways have been basically elucidated. However, there is a lack of systematic identification and study of TPS in <i>Mentha</i> plants. In this work, we genome-widely identified and analyzed the <i>TPS</i> gene family in <i>Mentha longifolia</i>, a model plant for functional genomic research in the genus <i>Mentha</i>. A total of 63 <i>TPS</i> genes were identified in the <i>M. longifolia</i> genome sequence assembly, which could be divided into six subfamilies. The <i>TPS-b</i> subfamily had the largest number of genes, which might be related to the abundant monoterpenoids in <i>Mentha</i> plants. The <i>TPS-e</i> subfamily had 18 members and showed a significant species-specific expansion compared with other sequenced Lamiaceae plant species. The 63 <i>TPS</i> genes could be mapped to nine scaffolds of the <i>M. longifolia</i> genome sequence assembly and the distribution of these genes is uneven. Tandem duplicates and fragment duplicates contributed greatly to the increase in the number of <i>TPS</i> genes in <i>M. longifolia</i>. The conserved motifs (RR(X)8W, NSE/DTE, RXR, and DDXXD) were analyzed in <i>M. longifolia</i> TPSs, and significant differentiation was found between different subfamilies. Adaptive evolution analysis showed that <i>M. longifolia TPSs</i> were subjected to purifying selection after the species-specific expansion, and some amino acid residues under positive selection were identified. Furthermore, we also cloned and analyzed the catalytic activity of a single terpene synthase, <i>MlongTPS29</i>, which belongs to the <i>TPS-b</i> subfamily. <i>MlongTPS29</i> could encode a limonene synthase and catalyze the biosynthesis of limonene, an important precursor of essential oils from the genus <i>Mentha</i>. This study provides useful information for the biosynthesis of terpenoids in the genus <i>Mentha</i>.
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