## 4 Discussion  
The genus _Mentha_ has important economic value for its abundance of essential oils. The major constituents of mint essential oils are monoterepenes and sesquiterpenes [18; 19]. _Mentha_ plants (especially peppermint and spearmint) have been employed as model systems for the study of monoterepene biosynthesis [20; 21]. However, the complex polyploidy and lack of genomic information limited further study. Horse mint (_M. longifolia_) is a diploid ancestor species of the genus _Mentha_, which has been developed as a model species for mint genomics [22]. The completion of _M. longifolia_ genome sequencing provides opportunity to perform functional genomic studies of _Mentha_ plants [23]. In this study, the _TPS_ gene family, which is positioned at the branch point and is a key enzyme for terpenoid biosynthesis, was genome-widely identified and analyzed in _M. longifolia_ genome sequence assembly. A total of 63 complete _TPS_ genes were identified in the _M. longifolia_ genome sequence assembly according to the conserved N-terminal and C-terminal domains of TPS. _TPS_ belongs to a medium-sized gene family, with various gene numbers (approximately 20-150) among different plants [12]. The number of _TPS_ genes in _M. longifolia_ genome sequence assembly is moderate when compared to that of other reported plants. Figure 6: GC-MS analysis of the products formed by recombinant MlongTPS29 proteins via in vitro assays. **(A,B)** Total ion current of products yielded by pET28a and pET28a-MlongTPS29, respectively. **(C)** Mass spectrum of the indicated peak. According to the phylogenetic analysis, _TPSs_ of _M. longifolia_ fall into six known Angiosperm _TPS_ subfamilies (_TPS-a_, _TPS-b_, _TPS-c_, _TPS-e_, _TPS-f_, and _TPS-g_). No gymnosperm-specific _TPS-d_ subfamily or _S. moellendorffii_-specific _TPS-h_ subfamily genes were identified. However, recent studies indicated that the _TPS-d_ subfamily is not gymnosperm-specific, it was also found in _Anunas comosus_ and _Marchantia polymorpha_[13]. _TPS-b_ is the largest subfamily in _M. longifolia_ genome sequence, and it has more members than the _TPS-a_ subfamily (34.9%_TPS-b_ genes and 20.6% _TPS-a_ genes). This is in contrast to most other plants, such as _A. thaliana_ (18.8% _TPS-b_ genes and 68.8% _TPS-a_ genes) [50], _Vitis vinifera_ (29.0% _TPS-b_ genes and 43.5% _TPS-a_ genes) [46], and _Oryza sativa_ (5.0% _TPS-b_ genes and 62.5% _TPS-a_ genes) [13]. The genomic distribution analysis showed that there were some tandem duplicates and segment duplicates in _TPS-b_ genes, which might be the cause of the increase in the number of TPS-b subfamily genes in _M. longifolia_ genome sequence [13]. The _TPS-b_ subfamily is mainly responsible for catalyzing the biosynthesis of monoterpenoids, and monoterpenoids are the main components of the essential oils of _Mentha_ plants [1,18]. Therefore, we speculate that the expansion of the _TPS-b_ subfamily of _Mentha_ may be related to the rich monoterenoid content. Another interesting phenomenon is that there are 18 _TPS-e_ subfamily genes in _M. longifolia_ genome sequence, which is much higher than that of most other plants. It is worth noting that most _TPS-e_ genes (15 of 18) are distributed on scaffold9, and tandem duplicates also exist in this subfamily. Whether the species-specific expansion of _TPS-e_ in _M. longifolia_ causes functional differentiation remains unclear. The integrated chemical-genomic-phylogenetic approach in Lamiaceae revealed that gene family expansion rather than increasing the enzyme promiscuity of terpene synthase is correlated with mono- and sesquiterpene diversity [51]. GC-MS analysis showed that the diversity of mono- and sesquiterpene in the genus _Mentha_ was more abundant than that in other genera of Lamiaceae [51]. The catalytic function of the expanded _TPS-e_ subfamily needs further investigation.