*2.3. The Chromosomal Distribution of EXO70s in Triticeae Species*

The identified 174 *EXO70s* from four *Triticeae* species (*T. aestivum*, *Ae. tauschii*, *T. dicoccoides* and *H. vulgare*) were assigned to corresponding chromosomes (including three on unknown chromosome). With the exception of wheat 6D, all chromosomes have at least one *EXO70* gene (Figure 3). The *EXO70s* were not evenly distributed on difference chromosomes or in different homologous groups. In total, there were 9, 44, 27, 14, 19, 7 and 51 *EXO70s* in the homologous groups 1 to 7, with group 7 having the most *EXO70s* (29.31%), followed by group 2 (25.29%) (Table 2). The *EXO70s* in group 7 was dispersely distributed along the chromosome, while those in group 2 were clutered at the dital region of the long arm (Figure 3). We found that nine groups *EXO70s* were conversely present on the same homoeologous groups of the four species, such as the B1 in group1, F2/3 in group 2, E1 and F3/2 in group 3, C2/C1 in group 4, CI and D1 in group 5, F1 and F4 in group 7 (Table S2, Figure 3).

For homologous chromosomes from different genomes of wheat and its ancestral/related species, most of the corresponding *EXO70* orthologs were present at the syntenic genome regions. For example, *EXO70B1s* were on the long chromosome arm of homoeologous group 1 and *EXO70E1* on group 3 chromosomes (Figure 3). There are some exceptions. The *EXO70G1* is present on all group 6 chromosomes except for sub-genome 6D of the hexaploidy wheat. The *EXO70H1* is located on chromosomes 2A, 2B of common wheat and *T. dicoccoides*, 2D of *Ae. tauschii* and 2H of barley and on 1D of common wheat (Figure 3). We found that the *EXO70C2* is on the 4BS of common wheat and *T. dicoccoides* (Figure 3a,b), on the 4DS of common wheat and *Ae. tauschii* (Figure 3a,d), but on the 4AL of common wheat and *T. dicoccoides* (Figure 3a,b). This also supports the presence of an inter-arm translocation of 4A during the evolution from diploid to tetraploid wheat [48,49].

*Int. J. Mol. Sci.* **2018**, *19*, x 7 of 22

**Figure 3.** The chromosomal distribution of the *EXO70* gene family in four *Triticeae* species*.* (**a**‒ **d**) represent *T. aestivum*, *T. dicoccoides*, *A. tauschii* and *H. vulgare*, respectively. Chromosome numbers are indicated at the top of each bar and the number in parentheses corresponds to the number of *EXO70* genes present on that chromosome. The name of each gene is to the left of each chromosome. Gene names labeled with red, blue, or black indicate that they are conserved in four species, missing in one due to incomplete data, or missing in more than two, **Figure 3.** The chromosomal distribution of the *EXO70* gene family in four *Triticeae* species. (**a**–**d**) represent *T. aestivum*, *T. dicoccoides*, *A. tauschii* and *H. vulgare*, respectively. Chromosome numbers are indicated at the top of each bar and the number in parentheses corresponds to the number of *EXO70* genes present on that chromosome. The name of each gene is to the left of each chromosome. Gene names labeled with red, blue, or black indicate that they are conserved in four species, missing in one due to incomplete data, or missing in more than two, respectively.

#### respectively. *2.4. The Diversification of Gene Structure of Triticeae EXO70s*

biological role is different from that of other subgroups.

*2.4. The Diversification of Gene Structure of Triticeae EXO70s*  The C-terminal PFam03081 domain, which may determine the function or structure of the proteins, is a specific characteristic of the *EXO70* superfamily [47]. All the predicted 200 and 15 homologous cloned EXO70 proteins possessed such a domain; however, their amino acid The C-terminal PFam03081 domain, which may determine the function or structure of the proteins, is a specific characteristic of the *EXO70* superfamily [47]. All the predicted 200 and 15 homologous cloned EXO70 proteins possessed such a domain; however, their amino acid sequence length is different for different *EXO70s*, varying from 103aa to 669aa and with an average length of 345aa (Table S1).

sequence length is different for different *EXO70s*, varying from 103aa to 669aa and with an average length of 345aa (Table S1). The exon‒intron structure of the 200 *Triticeae EXO70s* was visualized by using the online Gene Structure Display Server and compared among different subgroups or within each individual subgroup. The exon-intron structures for most of the *EXO70s* within the same subgroups were relatively conserved among *Triticeae* species, was similar to in *Arabidopsis* or rice [7,36]. Compared with other subgroups, the subgroup *EXO70A* with 30 genes has the most introns on average, e.g., *TuEXO70A3* has the most introns (20) and *HvEXO70A4-2H* has the fewest introns (five) (Figure 4a). The 170 *EXO70* genes in the remaining eight subgroups only have about one intron on average. Among them, 83 genes (41.50%) are intronless, including eight *EXO70Bs*, seven *EXO70Cs*, nine *EXO70Ds*, one *EXO70Es*, 18 *EXO70sF*, 12 *EXO70Gs*, five *EXO70Hs* and 23 *EXO70sI*; 60 genes (30.00%) have one intron (e.g., *AetEXO70B1* and *TaEXO70C1-5BL*), 19 genes (9.50%) have two introns (e.g., *TuEXO70C2* and *TaEXO70G2-6BS*) and eight genes (4.00%) have three to six introns (e.g., *AetEXO70I3* and *TaEXO70D1-5AL*). We also observed that genes that have a closer phylogenetic relationship in the same subgroup have a similar gene structure; however, within the same subgroup, some genes showed quite different gene structure. For instance, *TaEXO70D1-5AL* has six introns in *EXO70D* (Figure 4d) The exon–intron structure of the 200 *Triticeae EXO70s* was visualized by using the online Gene Structure Display Server and compared among different subgroups or within each individual subgroup. The exon-intron structures for most of the *EXO70s* within the same subgroups were relatively conserved among *Triticeae* species, was similar to in *Arabidopsis* or rice [7,36]. Compared with other subgroups, the subgroup *EXO70A* with 30 genes has the most introns on average, e.g., *TuEXO70A3* has the most introns (20) and *HvEXO70A4-2H* has the fewest introns (five) (Figure 4a). The 170 *EXO70* genes in the remaining eight subgroups only have about one intron on average. Among them, 83 genes (41.50%) are intronless, including eight *EXO70Bs*, seven *EXO70Cs*, nine *EXO70Ds*, one *EXO70Es*, 18 *EXO70sF*, 12 *EXO70Gs*, five *EXO70Hs* and 23 *EXO70sI*; 60 genes (30.00%) have one intron (e.g., *AetEXO70B1* and *TaEXO70C1-5BL*), 19 genes (9.50%) have two introns (e.g., *TuEXO70C2* and *TaEXO70G2-6BS*) and eight genes (4.00%) have three to six introns (e.g., *AetEXO70I3* and *TaEXO70D1-5AL*). We also observed that genes that have a closer phylogenetic relationship in the same subgroup have a similar gene structure; however, within the same subgroup, some genes showed quite different gene structure. For instance, *TaEXO70D1-5AL* has six introns in *EXO70D* (Figure 4d) and the three copies of *TaEXO70I5* and *AetEXO70I3* have five introns, while the other genes in the same subgroup (*EXO70D* or *EXO70I*) only have one or two introns (Figure 4i). The most diversified gene structure of the *EXO70A* subgroup may be a clue that their diversified biological role is different from that of other subgroups.

and the three copies of *TaEXO70I5* and *AetEXO70I3* have five introns, while the other genes in the same subgroup (*EXO70D* or *EXO70I*) only have one or two introns (Figure 4i). The most diversified gene structure of the *EXO70A* subgroup may be a clue that their diversified

*Int. J. Mol. Sci.* **2018**, *19*, x 8 of 22

**Figure 4.** Phylogenetic analysis and exon‒intron structures of *EXO70* gene family in common wheat and related *Triticeae* species. The phylogenetic analysis was performed using the sequences of the conserved domain of *EXO70*; proteins were aligned by ClustalW, constructed by MEGA6 using the N-J method, with 1000 bootstrap replicates; the branch length scale bar indicates the evolutionary distance. The left column identifies subgroups and is marked with different alternating background tones to make subgroup identification easier. Introns and exons are represented by black lines and colored boxes, respectively. **Figure 4.** Phylogenetic analysis and exon–intron structures of *EXO70* gene family in common wheat and related *Triticeae* species. The phylogenetic analysis was performed using the sequences of the conserved domain of *EXO70*; proteins were aligned by ClustalW, constructed by MEGA6 using the N-J method, with 1000 bootstrap replicates; the branch length scale bar indicates the evolutionary distance. The left column identifies subgroups and is marked with different alternating background tones to make subgroup identification easier. Introns and exons are represented by black lines and colored boxes, respectively.

#### *2.5. The Expression Pattern of TaEXO70 Genes 2.5. The Expression Pattern of TaEXO70 Genes*

I (Figure 5a).

The expression patterns of different members in a gene family will help us predict their potential biological roles. To elucidate the potential roles of the identified *EXO70s*, their expression in different tissues or in responses to various biotic and abiotic stresses was investigated by *in silico* expression profiling or qRT-PCR analysis. The expression patterns of wheat *TaEXO70s* in different tissues (root, stem and leaf of seeding stage), under two biotic stresses (stripe rust pathogen *CYR31* and powdery mildew pathogen *E09*) and two abiotic stresses (drought and heat) [50,51] were first investigated using the wheat RNA-seq data from the publicly available databases. The expression level was measured as tags per million (TPM). To facilitate the portraits of transcript abundance, we assume the expression was high if TPM ≥ 2.5; moderate if 2.5 > TPM ≥ 1.5; low if 1.5 >TPM > 0; and undetectable if TPM = 0. All 75 *TaEXO70* genes exhibited significantly diverse expression patterns (Figure 5). Their expression can be classified into five groups. The group "e" includes 23 *TaEXO70s* (30.67%). Their transcript abundance was undetectable; among them 11 were from subgroup I and six were from subgroup C (Figure 5e). The group "d" includes 20 genes (26.67%). They had detectable but weak transcript abundance; among them six were from subgroup A and six were from subgroup I (Figure 5d). The group "c" has six genes (8.00%), whose expression is high in roots and stems, whereas it does not respond to the four stresses (Figure 5c). The group "b" has 15 genes (20.00%). These genes were found to be negligibly to moderately expressed in the three tissue types and in response to one or two stresses. Among them, nine were from the subgroup F (Figure 5b). The group "a" has ten genes (13.33%). Their expression was generally high, either The expression patterns of different members in a gene family will help us predict their potential biological roles. To elucidate the potential roles of the identified *EXO70s*, their expression in different tissues or in responses to various biotic and abiotic stresses was investigated by in silico expression profiling or qRT-PCR analysis. The expression patterns of wheat *TaEXO70s* in different tissues (root, stem and leaf of seeding stage), under two biotic stresses (stripe rust pathogen *CYR31* and powdery mildew pathogen *E09*) and two abiotic stresses (drought and heat) [50,51] were first investigated using the wheat RNA-seq data from the publicly available databases. The expression level was measured as tags per million (TPM). To facilitate the portraits of transcript abundance, we assume the expression was high if TPM ≥ 2.5; moderate if 2.5 > TPM ≥ 1.5; low if 1.5 >TPM > 0; and undetectable if TPM = 0. All 75 *TaEXO70* genes exhibited significantly diverse expression patterns (Figure 5). Their expression can be classified into five groups. The group "e" includes 23 *TaEXO70s* (30.67%). Their transcript abundance was undetectable; among them 11 were from subgroup I and six were from subgroup C (Figure 5e). The group "d" includes 20 genes (26.67%). They had detectable but weak transcript abundance; among them six were from subgroup A and six were from subgroup I (Figure 5d). The group "c" has six genes (8.00%), whose expression is high in roots and stems, whereas it does not respond to the four stresses (Figure 5c). The group "b" has 15 genes (20.00%). These genes were found to be negligibly to moderately expressed in the three tissue types and in response to one or two stresses. Among them, nine were from the subgroup F (Figure 5b). The group "a" has ten genes (13.33%). Their expression was generally high, either in different tissues or in response to the four stresses and none of them were from the subgroup I (Figure 5a).

in different tissues or in response to the four stresses and none of them were from the subgroup

*Int. J. Mol. Sci.* **2018**, *19*, x 9 of 22

**Figure 5.** Heat map of the expression profiling of wheat *EXO70* genes in different tissues and under various stresses. The color scale bar represents the expression values of the genes. (**a**–**e**): Genes with different expression types. Abbreviations: *Bgt*, powdery mildew; *Pst*: Stripe rust. **Figure 5.** Heat map of the expression profiling of wheat *EXO70* genes in different tissues and under various stresses. The color scale bar represents the expression values of the genes. (**a**–**e**): Genes with different expression types. Abbreviations: *Bgt*, powdery mildew; *Pst*: Stripe rust.

Six genes were upregulated in response to both biotic and abiotic stresses. *TaEXO70E1-3B* was the only gene that was highly expressed in three tissues and upregulated in response to all four stresses. Its homologs on 3AL *TaEXO70E1-3AL* displayed moderate expression in three organs and were only responsive to *Bgt* inoculation and drought stress. The *TaEXO70D1-5DL* and *TaEXO70F3-3B* also displayed moderate expression in three organs and stresses. The *TaEXO70H1-1DL* expression was higher in root/leaf tissue and showed upregulation under both *Pst* infection and heat treatment. The *TaEXO70B1-1BL/1DL* expression was higher in root/stem tissue, and it was downregulated in response to *Bgt* and *Pst* inoculation but upregulated by drought treatment. *TaEXO70A3-2AL/2BL/2DL* had a similar expression pattern, being expressed in three tissues (stems > roots > leaves) and downregulated by *Pst* inoculation but upregulated by both drought and heat treatment. *TaEXO70D2-7BS* and *TaEXO70G1-7DL*  were both only responsive to biotic stresses; however, they showed opposite patterns, with *TaEXO70D2-7BS* upregulated and *TaEXO70G1-7DL* downregulated. Twelve genes were only responsive to one of the four stresses; five were only upregulated in response to *Bgt* or *Pst* inoculation; and seven were only upregulated in response to drought or heat stress. Conservation roles were observed for all three homolog genes from different genomes, such as Six genes were upregulated in response to both biotic and abiotic stresses. *TaEXO70E1-3B* was the only gene that was highly expressed in three tissues and upregulated in response to all four stresses. Its homologs on 3AL *TaEXO70E1-3AL* displayed moderate expression in three organs and were only responsive to *Bgt* inoculation and drought stress. The *TaEXO70D1-5DL* and *TaEXO70F3-3B* also displayed moderate expression in three organs and stresses. The *TaEXO70H1-1DL* expression was higher in root/leaf tissue and showed upregulation under both *Pst* infection and heat treatment. The *TaEXO70B1-1BL/1DL* expression was higher in root/stem tissue, and it was downregulated in response to *Bgt* and *Pst* inoculation but upregulated by drought treatment. *TaEXO70A3-2AL/2BL/2DL* had a similar expression pattern, being expressed in three tissues (stems > roots > leaves) and downregulated by *Pst* inoculation but upregulated by both drought and heat treatment. *TaEXO70D2-7BS* and *TaEXO70G1-7DL* were both only responsive to biotic stresses; however, they showed opposite patterns, with *TaEXO70D2-7BS* upregulated and *TaEXO70G1-7DL* downregulated. Twelve genes were only responsive to one of the four stresses; five were only upregulated in response to *Bgt* or *Pst* inoculation; and seven were only upregulated in response to drought or heat stress. Conservation roles were observed for all three homolog genes from different genomes, such as *TaEXO70A3(2AL/2BL/2DL)* and *TaEXO70F2(2AL/2BL/2DL)*.

#### *TaEXO70A3(2AL/2BL/2DL)* and *TaEXO70F2(2AL/2BL/2DL)*. *2.6. Differential Expression of 15 EXO70 Genes from H. villosa*

*2.6. Differential Expression of 15 EXO70 Genes from H. villosa* The expression patterns of 15 *EXO70*-Vs in different tissues and in response to different stresses or treatments in *H. villosa* were investigated by qRT-PCR. Different expression patterns were observed for the analyzed genes (Figure 6). Six of the genes (*EXO70D1-V*, *EXO70A2-V*, The expression patterns of 15 *EXO70*-Vs in different tissues and in response to different stresses or treatments in *H. villosa* were investigated by qRT-PCR. Different expression patterns were observed for the analyzed genes (Figure 6). Six of the genes (*EXO70D1-V*, *EXO70A2-V*, *EXO70F3-V*, *EXO70H1*-V, *EXO70I1*-V and *EXO70G2*-V) were not differentially expressed in all three tissues. Four genes

*EXO70F3-V*, *EXO70H1*-V, *EXO70I1*-V and *EXO70G2*-V) were not differentially expressed in all

(*EXO70B1*-V, *EXO70G1*-V, *EXO70A1*-V, *EXO70C1*-V) showed more abundant transcript in stem; seven genes (*EXO70E1*-V, *EXO70A3*-V, *EXO70B1*-V, *EXO70G1*-V, *EXO70F1*-V, *EXO70D2*-V and *EXO70F2*-V) showed higher expression level in leaves (Figure 6a). *EXO70F1*-V, *EXO70D2*-V and *EXO70F2*-V) showed higher expression level in leaves (Figure 6a). *EXO70A2-V* showed a similar expression level in all the three tissues; however, its

abundant transcript in stem; seven genes (*EXO70E1*-V, *EXO70A3*-V, *EXO70B1*-V, *EXO70G1*-V,

*Int. J. Mol. Sci.* **2018**, *19*, x 10 of 22

*EXO70A2-V* showed a similar expression level in all the three tissues; however, its expression was significantly increased in response to *Bgt* inoculation and treatments by chitin, flg22, heat stress, phytohormones, or H2O2. *EXO70G1-V* showed a similar expression level in the stems and leaves; its expression was also significantly increased by *Bgt* inoculation and treatment by chitin, flg22, four phytohormones, or H2O2. The expression of *EXO70H1-V* was strongly upregulated by *Bgt* inoculation and cold stress, and moderately upregulated by chitin, Flg22, ET and heat stress. *EXO70I1-V* only responded to abiotic stresses (drought, salt and cold) and ABA treatment. *EXOG2-V* was responsive to flg22 treatment and drought stress, and *EXO70A3-V* was only responsive to heat stress (Figure 6b,c). The divergence of expression patterns of different gene members indicated their clear-cut roles in the adaptation of *H. villosa* to various environments or stresses. expression was significantly increased in response to *Bgt* inoculation and treatments by chitin, flg22, heat stress, phytohormones, or H2O2. *EXO70G1-V* showed a similar expression level in the stems and leaves; its expression was also significantly increased by *Bgt* inoculation and treatment by chitin, flg22, four phytohormones, or H2O2. The expression of *EXO70H1-V* was strongly upregulated by *Bgt* inoculation and cold stress, and moderately upregulated by chitin, Flg22, ET and heat stress. *EXO70I1-V* only responded to abiotic stresses (drought, salt and cold) and ABA treatment. *EXOG2-V* was responsive to flg22 treatment and drought stress, and *EXO70A3-V* was only responsive to heat stress (Figure 6b,c). The divergence of expression patterns of different gene members indicated their clear-cut roles in the adaptation of *H. villosa* to various environments or stresses.

**Figure 6.** Heat map of the expression profiling of wheat and *H. villosa EXO70* genes in tissues and in response to biotic/abiotic stress, phytohormones and H2O2 treatments. (**a**) Tissue-specific expression pattern of 15 *EXO70-V* genes in *H. villosa*. (**b**) Expression levels of *EXO70-V* genes in biotic/abiotic stresses of *H. villosa*. (**c**) Expression profiling of *EXO70-V* genes in response to phytohormones and H2O2 treatments. The scale bar showing expression level of the genes. Abbreviations: *Bgt*, powdery mildew; SA, salicylic acid; MeJA, methyl jasmonate; ET, **Figure 6.** Heat map of the expression profiling of wheat and *H. villosa EXO70* genes in tissues and in response to biotic/abiotic stress, phytohormones and H2O<sup>2</sup> treatments. (**a**) Tissue-specific expression pattern of 15 *EXO70-V* genes in *H. villosa*. (**b**) Expression levels of *EXO70-V* genes in biotic/abiotic stresses of *H. villosa*. (**c**) Expression profiling of *EXO70-V* genes in response to phytohormones and H2O<sup>2</sup> treatments. The scale bar showing expression level of the genes. Abbreviations: *Bgt*, powdery mildew; SA, salicylic acid; MeJA, methyl jasmonate; ET, ethephon; ABA, abscisic acid; H2O<sup>2</sup> , hydrogen peroxide.

#### ethephon; ABA, abscisic acid; H2O2, hydrogen peroxide. *2.7. The Subcellular Localization of EXO70s from H. villosa*

*2.7. The Subcellular Localization of EXO70s from H. villosa*  Knowledge of the subcellular localization of a plant protein can help us predict its potential role in the biological process. The subcellular localization of EXO70-Vs was investigated by transiently expressing the construct into leaves of *Nicotiana tabacum* via *Agrobacterium* method. Eleven EXO70-Vs generated fluorescence signals. Compared with the relatively even distribution of GFP signals in the cell (Figure 7a), the 11 confusion proteins had distinct localization patterns. EXO70A1, A3 and F1-V displayed weak signals on the plasma membrane (PM) (Figure 7b‒d), while the PM signals for EXO70C1-V and EXO70D2-V were more intensive (Figure 7e,f). EXO70B1, E1 and F3-V displayed signals both in the PM and the Knowledge of the subcellular localization of a plant protein can help us predict its potential role in the biological process. The subcellular localization of EXO70-Vs was investigated by transiently expressing the construct into leaves of *Nicotiana tabacum* via *Agrobacterium* method. Eleven EXO70-Vs generated fluorescence signals. Compared with the relatively even distribution of GFP signals in the cell (Figure 7a), the 11 confusion proteins had distinct localization patterns. EXO70A1, A3 and F1-V displayed weak signals on the plasma membrane (PM) (Figure 7b–d), while the PM signals for EXO70C1-V and EXO70D2-V were more intensive (Figure 7e,f). EXO70B1, E1 and F3-V displayed signals both in the PM and the nucleus (Figure 7g–i). EXO70D1-V and EXO70F2-V also produced signals in the PM; in addition, they also had small and discrete spot signals in the PM (Figure 7j,k). EXO70I1-V was the only one with no continuous PM localized signal; however, we observed discrete punctate signals along the PM (Figure 7l).

signals along the PM (Figure 7l).

nucleus (Figure 7g‒i). EXO70D1-V and EXO70F2-V also produced signals in the PM; in addition, they also had small and discrete spot signals in the PM (Figure 7j,k). EXO70I1-V was

**Figure 7.** Subcellular localization of *H. villosa* EXO70-GFP-Vs proteins. *H. villosa* EXO70-GFP-Vs proteins were transiently expressed in *N. benthamiana* leaves carried out with injection of *Agrobacterium* and examined by a confocal microscope. Green fluorescence was observed 48 h after infection. Bar = 20 μm. (**a**) The empty GFP vector was used as the control. The green channel shows that GFP signals were localized in the nucleus and cytoplasmic and plasma membranes. (**b**‒**l**) The subcellular localization pattern of EXO70A1-V to EXO70I1-V, **Figure 7.** Subcellular localization of *H. villosa* EXO70-GFP-Vs proteins. *H. villosa* EXO70-GFP-Vs proteins were transiently expressed in *N. benthamiana* leaves carried out with injection of *Agrobacterium* and examined by a confocal microscope. Green fluorescence was observed 48 h after infection. Bar = 20 µm. (**a**) The empty GFP vector was used as the control. The green channel shows that GFP signals were localized in the nucleus and cytoplasmic and plasma membranes. (**b**–**l**) The subcellular localization pattern of EXO70A1-V to EXO70I1-V, respectively.

#### respectively. **3. Discussion**

#### **3. Discussion**  *3.1. Evolutionary Relationship of the EXO70 Gene Family in Wheat and Its Relatives*

*3.1. Evolutionary Relationship of the EXO70 Gene Family in Wheat and Its Relatives*  The evolutionary relationships of the *EXO70* gene family (between wheat, *T. urartu, Ae. tauschii, T. dicoccoides, H. vulgare* and *H. villosa*) have been speculated about based on the total number, classification, chromosomal distribution and structure. The surveyed diploid species of seven chromosome pairs except for *H. villosa* all possessed 26 *EXO70* genes, which suggested this gene family appeared before the divergence among Triticum species [52]. Allohexaploid wheat originated from two hybridizations between three diploid progenitors approximately 2.5‒4.5 million years ago [53]. The number of *EXO70* genes in tetraploid and common wheat (a total of 47 and 75, respectively) is approximately twice and three times as many as diploids, implying they have undergone one and two rounds of polyploidization events [54]. Although the polyploidization event induced rapid and extensive genetic and epigenetic changes in the genome which were related to a large range of molecular and physiological adjustment [55] as well as a significant loss of gene family members upon domestication [53], by comparing with diploid species, the *EXO70* gene family did not go through a wide range of expansion or The evolutionary relationships of the *EXO70* gene family (between wheat, *T. urartu, Ae. tauschii, T. dicoccoides, H. vulgare* and *H. villosa*) have been speculated about based on the total number, classification, chromosomal distribution and structure. The surveyed diploid species of seven chromosome pairs except for *H. villosa* all possessed 26 *EXO70* genes, which suggested this gene family appeared before the divergence among Triticum species [52]. Allohexaploid wheat originated from two hybridizations between three diploid progenitors approximately 2.5–4.5 million years ago [53]. The number of *EXO70* genes in tetraploid and common wheat (a total of 47 and 75, respectively) is approximately twice and three times as many as diploids, implying they have undergone one and two rounds of polyploidization events [54]. Although the polyploidization event induced rapid and extensive genetic and epigenetic changes in the genome which were related to a large range of molecular and physiological adjustment [55] as well as a significant loss of gene family members upon domestication [53], by comparing with diploid species, the *EXO70* gene family did not go through a wide range of expansion or diminution in tetraploid wheat and allohexaploid wheat. This deduction is also supported by their chromosomal location analysis, which showed that 73.1% of genes have good collinearity among wheat, *Ae. tauschii, T. dicoccoides* and *H. vulgare*, and most of the same type of orthologous genes maintain the relative order of their ancestral genes (Figures 3 and 4). As for the small difference in the number of genes on individual subgroups among wheat and its relatives, this may be because of the quality of the genome assembling or the gene duplication of subgroup *EXO70I*.

Phylogenetic analysis showed that all three groups (*EXO70.1*, *EXO70.2* and *EXO70.3*) and nine subgroups (*EXO70A* to *EXO70I*) are represented in each of the six *Triticeae* species. A similar gene structure was found in the same subgroup; subgroup *EXO70A* consists of multiple introns, while the other eight subgroups had fewer or were intronless. The variable intron numbers confirmed the classifications of the *EXO70* genes. Additionally, *EXO70* subgroups diversified before the divergence within polyploid wheat and related species during the evolutionary process of the *EXO70* gene family; however, no new groups/subgroups have emerged.

*EXO70I* members were most represented in wheat and its five relatives (57), as well as in rice (16), but not in *Arabidopsis* [6,36]. *EXO70I* belongs to *EXO70.2*, not *EXO70.3*, which is different from what was found in previous studies [1,7,36]. This suggests that the *EXO70I* subgroup arose before the evolutionary divergence of rice from other *Triticeae* crops and disappeared during the evolution of *Arabidopsis*. The *EXO70I* subgroup underwent rapid divergence, producing a large number of members; this event can probably be explained by unequal cross-over or segmental chromosomal duplication [56,57]. During long-term natural selection, numerous *EXO70* genes diverged and evolved in order to respond to various conditions. The study of *Arabidopsis* showed that the duplicated gene loss process is non-random; those involved in DNA repair are more likely to be lost, while genes involved in signal transduction and transcription have been preferentially retained [56]. Therefore, the function of *EXO70I* subgroup in *Arabidopsis* was inclined to responses to DNA repair, and in grass species may participate in signal transduction. Research on a larger range of species is needed to figure out whether the *EXO70I* branch is unique to monocotyledons.
