*2.2. Chromosomal Distributions and Synteny Analysis of Rice Trihelix Genes*

The extraction of the chromosomal information of the *OsMSL*s identified their chromosomal locations. As shown in Figure 1, all *OsMSL*s had precise positions in the chromosomes. Each rice chromosome contains ≥1 *OsMSL*. The *OsMSL*s are unevenly and non-randomly distributed on 12 chromosomes. Chr2 (chromosomal 2) contains the largest number of *OsMSL*s (eight) whereas Chr6 contains only one. The first four chromosomes contain 23 trihelix genes while chromosomes 5–12 have on average only 2–3 genes per chromosome. Therefore, *OsMSL*s are distributed mainly on the first four rice chromosomes. Although Chr2 is relatively short, it contains the most *OsMSL*s. Chr1 is the longest in rice and also contains numerous *OsMSL*s. Chr10, the shortest chromosome, contains two *OsMSL*s. In contrast, Chr6 is longer than Chr10 but contains only one *OsMSL*. There is no apparent correlation between the chromosome length and *OsMSL* gene distribution. Moreover, only *OsMSL12* and *OsMSL13* form gene clusters on Chr2.

*Int. J. Mol. Sci.* **2018**, *19*, x FOR PEER REVIEW 7 of 29

Synteny was also used to analyze rice trihelix gene duplication. Chromosomal region within 200 kb containing two or more genes is defined as a tandem duplication event [16]. As shown in Figure 1, four rice trihelix genes (*OsMSL09/10* and *OsMSL12/13*) were clustered into two tandem duplication event regions on rice chromosomal 2. Besides the tandem duplication events, segmental duplications were also investigated by BLASTP and MCScanX methods [17]. Four segmental duplication events with eight rice trihelix genes were also identified, which are located on duplicated segments on chromosomes 1, 2, 4, 5, and 11 (Figure 2). This finding is consistent with the highly divergent, non-conservative evolution of *Int. J. Mol. Sci.* **2018**, *19*, x FOR PEER REVIEW *OsMSL*s. 8 of 29

**Figure 2.** Schematic representations of segmental duplications of rice trihelix genes. Different color lines indicate all synteny blocks in rice genome between each chromosome, and the thick red lines indicate duplicated trihelix gene pairs. The chromosome number is indicated at the bottom of each chromosome. Scale bar marked on the chromosome indicating chromosome lengths (Mb). **Figure 2.** Schematic representations of segmental duplications of rice trihelix genes. Different color lines indicate all synteny blocks in rice genome between each chromosome, and the thick red lines indicate duplicated trihelix gene pairs. The chromosome number is indicated at the bottom of each chromosome. Scale bar marked on the chromosome indicating chromosome lengths (Mb).

To further understand the gene duplication mechanisms of the rice trihelix family, we constructed four comparative syntenic maps of rice associated with four representative species, including one dicots (*Arabidopsis*) (Figure 3A) and three monocots (*Brachypodium distachyon*, wheat and maize) (Figure 3B). A total of 23 rice trihelix genes showed a syntenic relationship with those in maize, followed by wheat (21), *Brachypodium distachyon* (19) and *Arabidopsis* (2), indicating that in comparison with monocotyledonous plants, rice trihelix genes show a high evolution divergence with dicotyledonous plants. Congruously, previous research reported that 14 pairs of orthologous trihelix genes were found between tomato and *Arabidopsis* [8]. Some *OsMSL*s were found to be associated with at least three syntenic gene pairs, such as *OsMSL14*, *OsMSL17*, and *OsMSL21*. These genes may have played a crucial role in the trihelix gene family during evolution. To better understand the evolutionary constraints acting on the trihelix gene family, the Ka/Ks ratios of the trihelix gene pairs were calculated (Tables S1–S5). All segmental and tandem duplicated *OsMSL* gene pairs, and the

majority of orthologous trihelix gene pairs had Ka/Ks < 1, suggesting that the rice trihelix gene family might have experienced a strong purifying selective pressure during evolution. *Int. J. Mol. Sci.* **2018**, *19*, x FOR PEER REVIEW 9 of 29

**Figure 3.** Synteny analysis of trihelix genes between rice and (**A**) dicotyledonous plant *Arabidopsi thaliana*, (**B**) monocotyledonous plant *Brachypodium distachyon*, wheat and maize. Gray lines in the background indicate the collinear blocks within rice and other plant genomes, while the red lines highlight the syntenic trihelix gene pairs. The species names with the prefixes '*A. thaliana*', '*B. distachyon*', '*T. aestivum*', '*Z. mays*' and '*O. sativa*' indicate *Arabidopsi thaliana*, *Brachypodium distachyon*, *Triticum aestivum*, *Zea mays* and *Oryza sativa*, respectively. Red or green bars represent the chromosomes. The chromosome number is labeled at the top or bottom of each chromosome. **Figure 3.** Synteny analysis of trihelix genes between rice and (**A**) dicotyledonous plant *Arabidopsi thaliana*, (**B**) monocotyledonous plant *Brachypodium distachyon*, wheat and maize. Gray lines in the background indicate the collinear blocks within rice and other plant genomes, while the red lines highlight the syntenic trihelix gene pairs. The species names with the prefixes '*A. thaliana*', '*B. distachyon*', '*T. aestivum*', '*Z. mays*' and '*O. sativa*' indicate *Arabidopsi thaliana*, *Brachypodium distachyon*, *Triticum aestivum*, *Zea mays* and *Oryza sativa*, respectively. Red or green bars represent the chromosomes. The chromosome number is labeled at the top or bottom of each chromosome.
