## 2 Results
### Sequence Alignment and Phylogenetic Analysis of McJAZ Genes  
To analyze the similarity of the homologous sequences among the candidate McJAZ proteins, multiple sequence alignment analysis of the seven McJAZ proteins or the pairwise McJAZ proteins was carried out. The results showed that except for McJAZ6 and McJAZ7, which shared 80.80% identity at the amino acid sequence level (Supplemental Table S1), the rest of pairwise McJAZ proteins contained differential amino acid identity ranging from 6.94% to 27.80% shared identity, analyzed by local DNAMAN 6.0 software (Figure 2A and Supplemental Table S1). In contrast to the sequences overall, the typical TIFY and Jas domains were conserved in all McJAZ proteins in _M. canadensis_ (Figure 2A). Similarly, the yield sequence logos drawn using the MEME tool also showed highly conserved amino acid sequence within the TIFY and Jas domains (Figure 2B). In addition, we also found that McJAZ6 and McJAZ7 possessed the typical EAR (LELRL) motif in the N-terminal region that is widely present in JAZ proteins across different plants (Figure 2A). Interestingly, evidence suggests that the TIFY or Jas motif is responsible for mediating protein-protein interactions for itself or other partners [19; 34]. Overall, these results showed the possibility that the function of the seven McJAZ proteins may be conserved within _M. canadensis_.  
Figure 1: _McJAZ genes and domain architectures of McJAZ proteins in M. canadensis._ Shown above are the coding sequences (CDs) of the seven focal _McJAZ_ genes, and the domain architectures of these McJAZ proteins. The McJAZ proteins share a conserved TIFY domain (indicated by a pink box), and Jas domain (indicated by a blue box) with other JAZ homologous proteins in other plant species.  
To understand the evolutionary and phylogenetic relationships of the _JAZ_ family genes between _M. canadensis_ and other known species including _A. thaliana_, _O. sativa_, and _A. annuua_, a phylogenetic tree was constructed based on an unrooted neighbor-joining (NJ) method using twelve AtJAZ proteins from _A. thaliana_, fifteen OsJAZ proteins from _O. sativa_, nine AaJAZ proteins from _A. annuua_, and seven McJAZ proteins from _M. canadensis_. The results showed that all the JAZ proteins were clustered into four groups (Groups A-D) and further classified into A1, A2, B1-B3, C, and D subgroups according to the reported phylogenetic analysis of AtJAZ proteins in _Arabidopsis_[34]. Among them, all McJAZ proteins were distributed into 5 subgroups, such that subgroup A1 contains McJAZ5, subgroup A2 contains McJAZ3 and McJAZ4, subgroups B1 and B2 contain McJAZ1 and McJAZ2 respectively, and McJAZ6, McJAZ7 belongs to subgroup D (Figure 3).  
Figure 2: **Multiple sequence alignment of McJAZ proteins from _M. canadensis_. (A) Alignment of the amino acid sequences of McJAZ proteins from _M. canadensis_. The EAR motif is indicated by a red rectangle. The conserved TIFY and Jas domains are outlined with a saffron yellow straight line. (B) The sequence logos of the TIFY and Jas motif are highly conserved across all McJAZ proteins. The height of each stack indicates conservation of the sequence at the labeled positions, and the height of each letter within each stack indicates the relative frequency of the corresponding amino acid.