*2.5. Chromosome Distribution and Covariance Analysis of HSF Genes in M. sativa*

*M. sativa HSF* genes were unevenly distributed on the six chromosomes (Figure 5). This included chromosomes 1, 3, 4, 5, 6, and 7, with the largest number of genes found on chromosome 6 with 5 genes, followed by 3 genes found on chromosome 4. All the remaining chromosomes contained only 2 *MsHSF* genes each.

**Figure 4.** Phylogenetic relationship tree (**A**), gene structure (**B**) and conserved patterns (**C**) of HSF in *M. sativa*.

**Figure 5.** Distribution of *MsHSF* genes on the chromosomal scaffolds of *M. sativa*.

Gene duplication events are common in all species; they can generate new functional genes and drive species evolution. Therefore, we used MCScanX genomic homozygosity analysis to explore duplications in the *M. sativa* HSF gene family (Figure 6). Three (*MsHSF10*, *MsHSF11*, and *MsHSF12*) tandem duplicated genes and two pairs (*MsHSF01*, *MsHSF02*, *MsHSF15*, and *MsHSF16*) of codominant genes were detected in the MsHSF family.

**Figure 6.** Chromosome distribution and interchromosomal relationships of *MsHSF* genes. Gray lines indicate synthetic blocks within the *M. sativa* genome, and red lines indicate duplicated *MsHSF* gene pairs.

Additionally, the proportion of *HSF* genes shared by *M. sativa* and other species may reflect the evolutionary relationships of the HSF gene family among *M. sativa*, dicotyledons, and monocotyledons. Therefore, in this study, we constructed a comparative homozygous map of three *HSF* genes from *M. sativa* and three dicots (*A. thaliana*, *G. max*, and *Medicago truncatula*) and one monocot (*Z. mays*) (Figure 7). A total of 44 direct homologous pairs (including one *M. sativa* gene corresponding to more than one *G. max* gene) were studied between *M. sativa* and *G. max HSF* genes. 20, 11, and 7 pairs of direct homologous genes were presented in *M. truncatula*, *A. thaliana*, and *Z. mays*, respectively. During evolution, most of the *HSF* genes of *M. sativa* have more than two direct homologs in *A. thaliana*, which further suggests that *M. sativa* has experienced more whole gene duplication events.

**Figure 7.** Synthetic analysis of the *M. sativa* genome with the genomes of one monocotyledon and three dicotyledon plants. Gray lines represent alignment blocks between paired genomes, and blue lines indicate synthetic *HSF* gene pairs.
