*2.1. Identification, Classification and Annotation of Chitin Genes in Pepper*

To comprehensively investigate and analyze the chitin-genes in pepper, the Hidden Markov Model (HMM) profile of the chitin-binding protein (Accession no. PF00187.17) was blast-searched in the pepper genome. As a result, 21 and 17 chitin genes were retrieved form CM334 and Zunla-1 databases, respectively. The gene sequences were aligned to avoid repetition and alternative splicing, and the longest sequences among them were chosen for further analysis. Among those 21 and 17 genes mined from the CM334 and Zunla databases, the genes having similar sequences with each other, were considered as a single gene. Consequently, we designed primer pairs (Table S1) for the amplification and confirmation of the doubtful gene sequences through cloning and sequencing. Finally, 16 predicted gene sequences were confirmed and then blast searched in NCBI. Nomenclature for the 16 CaChi was assigned based on their domains and chromosomal locations (Table 1 and Figure 1). The SMART results show that Chitin Binding Domain (CBD) was found in all 16 members while additional functional domains such as glycoside hydrolase\_19\_super family (*CaChiI1*, *CaChiI2*, and *CaChiI3*), chitinase glycoside\_hydrolase\_19 (*CaChiIV1* and *CaChiIV2*) and Barwin (*CaChiVI2* and *CaChi3*) were also found in this gene family (Figure 1 and Table S2). In addition, the characteristics of gene structure and protein size were quite different in CaChi gene family. The CDS of CaChi genes ranged from 258 bp (*CaChiVI1*) to 996 bp (*CaChiI1*), whereas the deduced proteins had 85–331 amino acids. The predicted *p*I values ranged from 4.71 (*CaChiIII7*) to 9.19 (*CaChiI2*), MW ranged from 9.06 (*CaChiVI1*) to 35.49 (*CaChiI1*) kDa and the instability index varied from 18.45 (*CaChiIV1*) to 68.89 (*CaChiIII4*) (Table 1 and Figure 1). The molecular formula shows that *CaChiIII3* contains the most (36) sulfur elements while *CaChiVI1* has the fewest (9) sulfur elements. All deduced proteins are shown in Table S2.

**Table 1.** List of Chitin-binding protein family genes identified in pepper and their sequence characteristics. Chr: chromosome; CDS: codding sequence; MW: molecular weight (kDa). the proteomic information was obtained from ExPASy (Available online: http://web.expasy.org/protparam/).


**Figure 1.** Domain architecture of CaChi classes I–VII in pepper and other plant species. The logos of domain organization were generated by Pfam database (Available online: http://pfam.xfam.org/search#tabview=tab0), and then further amendments were made with PhotoScape X. the aa: the number of amino acids; *p*I: isoelectric point; green : chitin binding domain (CBD); blue : glycoside hydrolase 19 super family; orange : chitinase glycoside **Figure 1.** Domain architecture of CaChi classes I–VII in pepper and other plant species. The logos of domain organization were generated by Pfam database (Available online: http://pfam.xfam.org/ search#tabview=tab0), and then further amendments were made with PhotoScape X. the aa: the number of amino acids; *p*I: isoelectric point; green **Figure 1.** Domain architecture of CaChi classes I–VII in pepper and other plant species. The logos of domain organization were generated by Pfam database (Available online: http://pfam.xfam.org/search#tabview=tab0), and then further amendments were made with PhotoScape X. the aa: the number of amino acids; *p*I: isoelectric point; green : chitin binding domain (CBD); blue : glycoside hydrolase 19 super family; orange : chitinase glycoside : chitin binding domain (CBD); blue **Figure 1.** Domain architecture of CaChi classes I–VII in pepper and other plant species. The logos of domain organization were generated by Pfam database (Available online: http://pfam.xfam.org/search#tabview=tab0), and then further amendments were made with PhotoScape X. the aa: the number of amino acids; *p*I: isoelectric point; green : chitin binding domain (CBD); blue : glycoside hydrolase 19 super family; orange : chitinase glycoside hydrolase 19; and : Barwin. : glycoside hydrolase 19 super family; orange **Figure 1.** Domain architecture of CaChi classes I–VII in pepper and other plant species. The logos of domain organization were generated by Pfam database (Available online: http://pfam.xfam.org/search#tabview=tab0), and then further amendments were made with PhotoScape X. the aa: the number of amino acids; *p*I: isoelectric point; green : chitin binding domain (CBD); blue : glycoside hydrolase 19 super family; orange : chitinase glycoside : chitinase glycoside hydrolase 19; and **Figure 1.** Domain architecture of CaChi classes I–VII in pepper and other plant species. The logos of domain organization were generated by Pfam database (Available online: http://pfam.xfam.org/search#tabview=tab0), and then further amendments were made with PhotoScape X. the aa: the number of amino acids; *p*I: isoelectric point; green : chitin binding domain (CBD); blue : glycoside hydrolase 19 super family; orange : chitinase glycoside hydrolase 19; and : Barwin. : Barwin.

#### *2.2. Construction of Phylogenetic Tree, Exon/Intron Structure and Conserved Motif Analysis 2.2. Construction of Phylogenetic Tree, Exon/Intron Structure and Conserved Motif Analysis* To better understand the similarities and differences among the pepper and other plants chitin-*2.2. Construction of Phylogenetic Tree, Exon/Intron Structure and Conserved Motif Analysis 2.2. Construction of Phylogenetic Tree, Exon/Intron Structure and Conserved Motif Analysis 2.2. Construction of Phylogenetic Tree, Exon/Intron Structure and Conserved Motif Analysis*

hydrolase 19; and : Barwin.

hydrolase 19; and : Barwin.

hydrolase 19; and : Barwin.

*2.2. Construction of Phylogenetic Tree, Exon/Intron Structure and Conserved Motif Analysis* To better understand the similarities and differences among the pepper and other plants chitinbinding protein genes, an unrooted phylogenetic tree was created using 162 chitin genes protein sequences from various plant species (Figure 2). These sequences used in the construction of phylogenetic tree were mainly from *Aegilops tauschii*, *Arabidopsis thaliana*, *Artemisia annua*, *Brassica napus*, *Brassica rapa*, *Bromus inermis*, *Bupleurum kaoi*, *Capsicum annuum*, *Capsicum baccatum*, *Capsicum chinense*, *Carica papaya*, *Cryptomeria japonica*, *Dionaea muscipula*, *Drosera rotundifolia*, *Euonymus europaeus*, *Gossypium barbadense*, *Gossypium hirsutum*, *Glycine max*, *Hevea brasiliensis*, *Hippophae rhamnoides*, *Hordeum vulgare*, *Limonium bicolor*, *Linum usitatissimum*, *Lupinus albus*, *Malus domestica*, *Mikania micrantha*, *Momordica charantia*, *Nepenthes khasiana*, *Nepenthes maxima*, *Nicotiana attenuate*, *Nicotiana benthamiana*, *Olea europaea*, *Oryza sativa*, *Persea americana*, *Pisum sativum*, *Psophocarpus tetragonolobus*, *Rehmannia glutinosa*, *Saccharum officinarum*, *Secale cereal*, *Sesamum indicum*, *Solanum tuberosum*, *Sorghum bicolor*, *Triphyophyllum peltatum*, *Triticum aestivum*, *Urtica dioica*, *Vitis vinifera*, *Zea diploperennis*, and *Zea mays.* The analysis shows that 16 CaChi were clearly classified into four distinct classes according to their sequence relatedness with previous research. Three CaChi (*CaChiI1*, *CaChiI2* and *CaChiI3*) were clustered in class I, seven CaChi (*CaChiIII1*, *CaChiIII2*, *CaCHiIII3*, *CaChiIII4*, *CaChiIII5*, *CaChiIII6* and *CaChiIII7*) in were clustered class III, two CaChi-genes (*CaChiIV1* and *CaChiIV2*) were clustered in class IV, and four CaChi (*CaChiVI1*, *CaChiVI2*, *CaChiVI3* and *CaChiVI4*) were clustered in class VI (Figure 2). Each class is highlighted with a different color following the To better understand the similarities and differences among the pepper and other plants chitinbinding protein genes, an unrooted phylogenetic tree was created using 162 chitin genes protein sequences from various plant species (Figure 2). These sequences used in the construction of phylogenetic tree were mainly from *Aegilops tauschii*, *Arabidopsis thaliana*, *Artemisia annua*, *Brassica napus*, *Brassica rapa*, *Bromus inermis*, *Bupleurum kaoi*, *Capsicum annuum*, *Capsicum baccatum*, *Capsicum chinense*, *Carica papaya*, *Cryptomeria japonica*, *Dionaea muscipula*, *Drosera rotundifolia*, *Euonymus europaeus*, *Gossypium barbadense*, *Gossypium hirsutum*, *Glycine max*, *Hevea brasiliensis*, *Hippophae rhamnoides*, *Hordeum vulgare*, *Limonium bicolor*, *Linum usitatissimum*, *Lupinus albus*, *Malus domestica*, *Mikania micrantha*, *Momordica charantia*, *Nepenthes khasiana*, *Nepenthes maxima*, *Nicotiana attenuate*, *Nicotiana benthamiana*, *Olea europaea*, *Oryza sativa*, *Persea americana*, *Pisum sativum*, *Psophocarpus tetragonolobus*, *Rehmannia glutinosa*, *Saccharum officinarum*, *Secale cereal*, *Sesamum indicum*, *Solanum tuberosum*, *Sorghum bicolor*, *Triphyophyllum peltatum*, *Triticum aestivum*, *Urtica dioica*, *Vitis vinifera*, *Zea diploperennis*, and *Zea mays.* The analysis shows that 16 CaChi were clearly classified into four distinct classes according to their sequence relatedness with previous research. Three CaChi (*CaChiI1*, *CaChiI2* and *CaChiI3*) were clustered in class I, seven CaChi (*CaChiIII1*, *CaChiIII2*, *CaCHiIII3*, *CaChiIII4*, *CaChiIII5*, *CaChiIII6* and *CaChiIII7*) in were clustered class III, two CaChi-genes (*CaChiIV1* and *CaChiIV2*) were clustered in class IV, and four CaChi (*CaChiVI1*, *CaChiVI2*, *CaChiVI3* and *CaChiVI4*) were clustered in class VI (Figure 2). Each class is highlighted with a different color following the previous chitin-binding protein genes classification [32,33]. binding protein genes, an unrooted phylogenetic tree was created using 162 chitin genes protein sequences from various plant species (Figure 2). These sequences used in the construction of phylogenetic tree were mainly from *Aegilops tauschii*, *Arabidopsis thaliana*, *Artemisia annua*, *Brassica napus*, *Brassica rapa*, *Bromus inermis*, *Bupleurum kaoi*, *Capsicum annuum*, *Capsicum baccatum*, *Capsicum chinense*, *Carica papaya*, *Cryptomeria japonica*, *Dionaea muscipula*, *Drosera rotundifolia*, *Euonymus europaeus*, *Gossypium barbadense*, *Gossypium hirsutum*, *Glycine max*, *Hevea brasiliensis*, *Hippophae rhamnoides*, *Hordeum vulgare*, *Limonium bicolor*, *Linum usitatissimum*, *Lupinus albus*, *Malus domestica*, *Mikania micrantha*, *Momordica charantia*, *Nepenthes khasiana*, *Nepenthes maxima*, *Nicotiana attenuate*, *Nicotiana benthamiana*, *Olea europaea*, *Oryza sativa*, *Persea americana*, *Pisum sativum*, *Psophocarpus tetragonolobus*, *Rehmannia glutinosa*, *Saccharum officinarum*, *Secale cereal*, *Sesamum indicum*, *Solanum tuberosum*, *Sorghum bicolor*, *Triphyophyllum peltatum*, *Triticum aestivum*, *Urtica dioica*, *Vitis vinifera*, *Zea diploperennis*, and *Zea mays.* The analysis shows that 16 CaChi were clearly classified into four distinct classes according to their sequence relatedness with previous research. Three CaChi (*CaChiI1*, *CaChiI2* and *CaChiI3*) were clustered in class I, seven CaChi (*CaChiIII1*, *CaChiIII2*, *CaCHiIII3*, *CaChiIII4*, *CaChiIII5*, *CaChiIII6* and *CaChiIII7*) in were clustered class III, two CaChi-genes (*CaChiIV1* and *CaChiIV2*) were clustered in class IV, and four CaChi (*CaChiVI1*, *CaChiVI2*, *CaChiVI3* and *CaChiVI4*) were clustered in class VI (Figure 2). Each class is highlighted with a different color following the previous chitin-binding protein genes classification [32,33]. To better understand the similarities and differences among the pepper and other plants chitinbinding protein genes, an unrooted phylogenetic tree was created using 162 chitin genes protein sequences from various plant species (Figure 2). These sequences used in the construction of phylogenetic tree were mainly from *Aegilops tauschii*, *Arabidopsis thaliana*, *Artemisia annua*, *Brassica napus*, *Brassica rapa*, *Bromus inermis*, *Bupleurum kaoi*, *Capsicum annuum*, *Capsicum baccatum*, *Capsicum chinense*, *Carica papaya*, *Cryptomeria japonica*, *Dionaea muscipula*, *Drosera rotundifolia*, *Euonymus europaeus*, *Gossypium barbadense*, *Gossypium hirsutum*, *Glycine max*, *Hevea brasiliensis*, *Hippophae rhamnoides*, *Hordeum vulgare*, *Limonium bicolor*, *Linum usitatissimum*, *Lupinus albus*, *Malus domestica*, *Mikania micrantha*, *Momordica charantia*, *Nepenthes khasiana*, *Nepenthes maxima*, *Nicotiana attenuate*, *Nicotiana benthamiana*, *Olea europaea*, *Oryza sativa*, *Persea americana*, *Pisum sativum*, *Psophocarpus tetragonolobus*, *Rehmannia glutinosa*, *Saccharum officinarum*, *Secale cereal*, *Sesamum indicum*, *Solanum tuberosum*, *Sorghum bicolor*, *Triphyophyllum peltatum*, *Triticum aestivum*, *Urtica dioica*, *Vitis vinifera*, *Zea diploperennis*, and *Zea mays.* The analysis shows that 16 CaChi were clearly classified into four distinct classes according to their sequence relatedness with previous research. Three CaChi (*CaChiI1*, *CaChiI2* and *CaChiI3*) were clustered in class I, seven CaChi (*CaChiIII1*, *CaChiIII2*, *CaCHiIII3*, *CaChiIII4*, *CaChiIII5*, *CaChiIII6* and *CaChiIII7*) in were clustered class III, two CaChi-genes (*CaChiIV1* and *CaChiIV2*) were clustered in class IV, and four CaChi (*CaChiVI1*, *CaChiVI2*, *CaChiVI3* and *CaChiVI4*) were clustered in class VI (Figure 2). Each class is highlighted with a different color following the previous chitin-binding protein genes classification [32,33]. To better understand the similarities and differences among the pepper and other plants chitinbinding protein genes, an unrooted phylogenetic tree was created using 162 chitin genes protein sequences from various plant species (Figure 2). These sequences used in the construction of phylogenetic tree were mainly from *Aegilops tauschii*, *Arabidopsis thaliana*, *Artemisia annua*, *Brassica napus*, *Brassica rapa*, *Bromus inermis*, *Bupleurum kaoi*, *Capsicum annuum*, *Capsicum baccatum*, *Capsicum chinense*, *Carica papaya*, *Cryptomeria japonica*, *Dionaea muscipula*, *Drosera rotundifolia*, *Euonymus europaeus*, *Gossypium barbadense*, *Gossypium hirsutum*, *Glycine max*, *Hevea brasiliensis*, *Hippophae rhamnoides*, *Hordeum vulgare*, *Limonium bicolor*, *Linum usitatissimum*, *Lupinus albus*, *Malus domestica*, *Mikania micrantha*, *Momordica charantia*, *Nepenthes khasiana*, *Nepenthes maxima*, *Nicotiana attenuate*, *Nicotiana benthamiana*, *Olea europaea*, *Oryza sativa*, *Persea americana*, *Pisum sativum*, *Psophocarpus tetragonolobus*, *Rehmannia glutinosa*, *Saccharum officinarum*, *Secale cereal*, *Sesamum indicum*, *Solanum tuberosum*, *Sorghum bicolor*, *Triphyophyllum peltatum*, *Triticum aestivum*, *Urtica dioica*, *Vitis vinifera*, *Zea diploperennis*, and *Zea mays.* The analysis shows that 16 CaChi were clearly classified into four distinct classes according to their sequence relatedness with previous research. Three CaChi (*CaChiI1*, *CaChiI2* and *CaChiI3*) were clustered in class I, seven CaChi (*CaChiIII1*, *CaChiIII2*, *CaCHiIII3*, *CaChiIII4*, *CaChiIII5*, *CaChiIII6* and *CaChiIII7*) in were clustered class III, two CaChi-genes (*CaChiIV1* and *CaChiIV2*) were clustered in class IV, and four CaChi (*CaChiVI1*, *CaChiVI2*, *CaChiVI3* and *CaChiVI4*) were clustered in class VI (Figure 2). Each class is highlighted with a different color following the previous chitin-binding protein genes classification [32,33]. To better understand the similarities and differences among the pepper and other plants chitin-binding protein genes, an unrooted phylogenetic tree was created using 162 chitin genes protein sequences from various plant species (Figure 2). These sequences used in the construction of phylogenetic tree were mainly from *Aegilops tauschii*, *Arabidopsis thaliana*, *Artemisia annua*, *Brassica napus*, *Brassica rapa*, *Bromus inermis*, *Bupleurum kaoi*, *Capsicum annuum*, *Capsicum baccatum*, *Capsicum chinense*, *Carica papaya*, *Cryptomeria japonica*, *Dionaea muscipula*, *Drosera rotundifolia*, *Euonymus europaeus*, *Gossypium barbadense*, *Gossypium hirsutum*, *Glycine max*, *Hevea brasiliensis*, *Hippophae rhamnoides*, *Hordeum vulgare*, *Limonium bicolor*, *Linum usitatissimum*, *Lupinus albus*, *Malus domestica*, *Mikania micrantha*, *Momordica charantia*, *Nepenthes khasiana*, *Nepenthes maxima*, *Nicotiana attenuate*, *Nicotiana benthamiana*, *Olea europaea*, *Oryza sativa*, *Persea americana*, *Pisum sativum*, *Psophocarpus tetragonolobus*, *Rehmannia glutinosa*, *Saccharum officinarum*, *Secale cereal*, *Sesamum indicum*, *Solanum tuberosum*, *Sorghum bicolor*, *Triphyophyllum peltatum*, *Triticum aestivum*, *Urtica dioica*, *Vitis vinifera*, *Zea diploperennis*, and *Zea mays.* The analysis shows that 16 CaChi were clearly classified into four distinct classes according to their sequence relatedness with previous research. Three CaChi (*CaChiI1*, *CaChiI2* and *CaChiI3*) were clustered in class I, seven CaChi (*CaChiIII1*, *CaChiIII2*, *CaCHiIII3*, *CaChiIII4*, *CaChiIII5*, *CaChiIII6* and *CaChiIII7*) in were clustered class III, two CaChi-genes (*CaChiIV1* and *CaChiIV2*) were clustered in class IV, and four CaChi (*CaChiVI1*, *CaChiVI2*, *CaChiVI3* and *CaChiVI4*) were clustered in class VI (Figure 2). Each class is highlighted with a different color following the previous chitin-binding protein genes classification [32,33].

previous chitin-binding protein genes classification [32,33].

**Figure 2.** The phylogenic tree of chitin-binding protein family genes in pepper and other plant species. The phylogenetic tree was built using the neighbor-joining method and diagram was drawn using online iTOL (Available online: https://itol.embl.de/). The number of chitin-binding protein family genes were divided in I–VII well conserved groups. **Figure 2.** The phylogenic tree of chitin-binding protein family genes in pepper and other plant species.The phylogenetic tree was built using the neighbor-joining method and diagram was drawn using online iTOL (Available online: https://itol.embl.de/). The number of chitin-binding protein family genes were divided in I–VII well conserved groups.

The members within certain class exhibited higher identity percentage of the amino acids sequences (Figure 3a). The exon/intron structure analysis showed that out of 16 CaChi, 8 CaChi (50%) had no introns while 8 CaChi (50%) contained only one intron (Figure 3c). The conserved motifs of CaChi proteins were identified by online MEME server (Available online: ). A sum of ten putative different motifs were obtained (Table S3). Motif 1 was found in all CaChi while motif 5 was found in most CaChi (except *CaChiIV1*, *CaChiIV2*, *CaChiIII2*, and *CaChiIII4*). Motif 6 was present in 50% of the CaChi (*CaChiIII1*, *CaChiIII2*, *CaChiIII3*, *CaChiIII4*, *CaChiIII6*, *CaChiIII7*, *CaChiIV1* and *CaChiIV2*). Motifs 2 and 10 were present in five and four CaChi, respectively. Motifs 3, 4 and 9 each were found in three different sequence of CaChi while motifs 7 and 8 each existed in two CaChi (Figure 3b and Table S3). The members within certain class exhibited higher identity percentage of the amino acids sequences (Figure 3a). The exon/intron structure analysis showed that out of 16 CaChi, 8 CaChi (50%) had no introns while 8 CaChi (50%) contained only one intron (Figure 3c). The conserved motifs of CaChi proteins were identified by online MEME server (Available online: ). A sum of ten putative different motifs were obtained (Table S3). Motif 1 was found in all CaChi while motif 5 was found in most CaChi (except *CaChiIV1*, *CaChiIV2*, *CaChiIII2*, and *CaChiIII4*). Motif 6 was present in 50% of the CaChi (*CaChiIII1*, *CaChiIII2*, *CaChiIII3*, *CaChiIII4*, *CaChiIII6*, *CaChiIII7*, *CaChiIV1* and *CaChiIV2*). Motifs 2 and 10 were present in five and four CaChi, respectively. Motifs 3, 4 and 9 each were found in three different sequence of CaChi while motifs 7 and 8 each existed in two CaChi (Figure 3b and Table S3).

*Int. J. Mol. Sci.* **2018**, *19*, 2216 6 of 26

**Figure 3.** Phylogenetic relationship, domain organization and conserved motifs analysis of chitinbinding proteins family genes in pepper. (**a**) Phylogenetic analysis and classification of pepper genes. The phylogenetic tree was constructed via online iTOL (Available online: https://itol.embl.de/). (**b**) Motif analysis of pepper CaChi proteins. Motifs, numbered 1–10, were identified using MEME 4.11.2 software and are illustrated by different colors. Amino acid sequence of each motif is shown in Table S3. (**c**) Exon/intron structures of pepper chitins genes. Yellow boxes represent exons and introns are represented by black lines between two exons. *2.3. Chromosomal Location and Genes Duplication* **Figure 3.** Phylogenetic relationship, domain organization and conserved motifs analysis of chitin-binding proteins family genes in pepper. (**a**) Phylogenetic analysis and classification of pepper genes. The phylogenetic tree was constructed via online iTOL (Available online: https://itol.embl.de/). (**b**) Motif analysis of pepper CaChi proteins. Motifs, numbered 1–10, were identified using MEME 4.11.2 software and are illustrated by different colors. Amino acid sequence of each motif is shown in Table S3. (**c**) Exon/intron structures of pepper chitins genes. Yellow boxes represent exons and introns are represented by black lines between two exons. **Figure 3.** Phylogenetic relationship, domain organization and conserved motifs analysis of chitinbinding proteins family genes in pepper. (**a**) Phylogenetic analysis and classification of pepper genes. The phylogenetic tree was constructed via online iTOL (Available online: https://itol.embl.de/). (**b**) Motif analysis of pepper CaChi proteins. Motifs, numbered 1–10, were identified using MEME 4.11.2

CaChi were distributed across 7 out of 12 chromosomes of the pepper. Intriguingly, all CaChi

software and are illustrated by different colors. Amino acid sequence of each motif is shown in Table

#### According to the chromosomal location of the chitin-binding protein genes in pepper, the 16 *2.3. Chromosomal Location and Genes Duplication* S3. (**c**) Exon/intron structures of pepper chitins genes. Yellow boxes represent exons and introns are represented by black lines between two exons.

members are random and non-randomly distributed across the chromosomes (Figure 4). The results showed that chromosome 3 had the highest number of CaChi (31.25%) as compared to other chromosomes. There were four genes (25%) on chromosome 7, while chromosomes 8 and 10 each have two genes. The remaining chromosomes (4, 6 and 12) each contained one gene. The duplication analysis showed that *CaChiIII1* have segmental duplication with *CaChiIII6* which occurred on chromosomes 3 and 7, respectively (Figure 4). *CaChiIII7* has two segmental duplication events with *CaChiIII2* and *CaChiIII5. CaChiIV1* also exhibited two segmental duplication events with *CaChiI1* and *CaChiIV2*. Moreover, one tandem duplication event was observed between *CaChiVI2* and *CaChiVI3*, which occurred on chromosome 8. Taken together, our findings suggest that, in the expansion of pepper CaChi genes, tandem and segmental duplication have an important contribution. According to the chromosomal location of the chitin-binding protein genes in pepper, the 16 CaChi were distributed across 7 out of 12 chromosomes of the pepper. Intriguingly, all CaChi members are random and non-randomly distributed across the chromosomes (Figure 4). The results showed that chromosome 3 had the highest number of CaChi (31.25%) as compared to other chromosomes. There were four genes (25%) on chromosome 7, while chromosomes 8 and 10 each have two genes. The remaining chromosomes (4, 6 and 12) each contained one gene. The duplication analysis showed that *CaChiIII1* have segmental duplication with *CaChiIII6* which occurred on chromosomes 3 and 7, respectively (Figure 4). *CaChiIII7* has two segmental duplication events with *CaChiIII2* and *CaChiIII5. CaChiIV1* also exhibited two segmental duplication events with *CaChiI1* and *CaChiIV2*. Moreover, one tandem duplication event was observed between *CaChiVI2* and *CaChiVI3*, which occurred on chromosome 8. Taken together, our findings suggest that, in the expansion of pepper CaChi genes, tandem and segmental duplication have an important contribution. *2.3. Chromosomal Location and Genes Duplication* According to the chromosomal location of the chitin-binding protein genes in pepper, the 16 CaChi were distributed across 7 out of 12 chromosomes of the pepper. Intriguingly, all CaChi members are random and non-randomly distributed across the chromosomes (Figure 4). The results showed that chromosome 3 had the highest number of CaChi (31.25%) as compared to other chromosomes. There were four genes (25%) on chromosome 7, while chromosomes 8 and 10 each have two genes. The remaining chromosomes (4, 6 and 12) each contained one gene. The duplication analysis showed that *CaChiIII1* have segmental duplication with *CaChiIII6* which occurred on chromosomes 3 and 7, respectively (Figure 4). *CaChiIII7* has two segmental duplication events with *CaChiIII2* and *CaChiIII5. CaChiIV1* also exhibited two segmental duplication events with *CaChiI1* and *CaChiIV2*. Moreover, one tandem duplication event was observed between *CaChiVI2* and *CaChiVI3*, which occurred on chromosome 8. Taken together, our findings suggest that, in the expansion of pepper CaChi genes, tandem and segmental duplication have an important contribution.

**Figure 4.** Chromosomal localization of CaChi of pepper plant, where the red shading box represents the tandem duplicated region. While the red, green and blue lines connection displaying segmentally duplicated genes.

#### *2.4. Cis-Acting Elements and Gene Ontology (GO) Analysis of CaChi 2.4. Cis-Acting Elements and Gene Ontology (GO) Analysis of CaChi*

To examine the possible *cis*-acting elements involvement in the stimulation of defense-related genes, the 1.5 kb upstream region from the start codon of all the CaChi genes were analyzed with Plant CARE online server. The silico analysis revealed that *Cis-*elements conferring responsiveness to plant hormones, biotic and abiotic stresses were found in the promoters of the CaChi. As shown in Figure 5 and Table S4, the heat stress elements (HSE) were identified in the promoters of all CaChi (except *CaChiI2*, *CaChiI3* and *CaChiVI3*), in which the HSE in the promoters of the *CaChiIV1* and *CaChiVI2* were highest (4) followed by *CaChiIII2*, *CaChiIII6* and *CaChiIV1* (each have 2). MeJA-responsiveness elements (CGTCA-motif) were found in the promoter region of 10 CaChi, where *CaChiIII1* had the highest number (5) of elements followed by *CaChiIII4* (3), while *CaChiIII5* and *CaChiIV2* each have two elements. *Cis*-acting elements involved in abscisic acid (ABA) responsiveness elements (ABRE), salicylic acid responsiveness (TCA-element) and ethylene-responsive element (ERE) were found in the promoter regions of six CaChi. The MYB binding site involved in drought-inducibility (MBS), resistance and stress responsiveness (TC-rich repeats), GA-responsive element (GARE-motif) and fungal elicitor-responsive element (W-box) were found in the promotor regions of 10, 8, 6 and 7 CaChi genes, respectively. The *cis*-acting element involved in low temperature sensitivity (LTR) was found in the promoter region of four CaChi. In addition, GA-responsive element (P box) and auxin-responsive elements (TGA-element and AuxRR-core) were also found in some of the CaChi promoter regions. All of the anticipated *cis*-elements were involved in response to signaling molecules and stresses. To examine the possible *cis*-acting elements involvement in the stimulation of defense-related genes, the 1.5 kb upstream region from the start codon of all the CaChi genes were analyzed with Plant CARE online server. The silico analysis revealed that *Cis-*elements conferring responsiveness to plant hormones, biotic and abiotic stresses were found in the promoters of the CaChi. As shown in Figure 5 and Table S4, the heat stress elements (HSE) were identified in the promoters of all CaChi (except *CaChiI2*, *CaChiI3* and *CaChiVI3*), in which the HSE in the promoters of the *CaChiIV1* and *CaChiVI2* were highest (4) followed by *CaChiIII2*, *CaChiIII6* and *CaChiIV1* (each have 2). MeJAresponsiveness elements (CGTCA-motif) were found in the promoter region of 10 CaChi, where *CaChiIII1* had the highest number (5) of elements followed by *CaChiIII4* (3), while *CaChiIII5* and *CaChiIV2* each have two elements. *Cis*-acting elements involved in abscisic acid (ABA) responsiveness elements (ABRE), salicylic acid responsiveness (TCA-element) and ethylene-responsive element (ERE) were found in the promoter regions of six CaChi. The MYB binding site involved in droughtinducibility (MBS), resistance and stress responsiveness (TC-rich repeats), GA-responsive element (GARE-motif) and fungal elicitor-responsive element (W-box) were found in the promotor regions of 10, 8, 6 and 7 CaChi genes, respectively. The *cis*-acting element involved in low temperature sensitivity (LTR) was found in the promoter region of four CaChi. In addition, GA-responsive element (P box) and auxin-responsive elements (TGA-element and AuxRR-core) were also found in some of the CaChi promoter regions. All of the anticipated *cis*-elements were involved in response to

*Int. J. Mol. Sci.* **2018**, *19*, 2216 7 of 26

**Figure 4.** Chromosomal localization of CaChi of pepper plant, where the red shading box represents

Gene ontology (GO) enrichment analysis of CaChi were predicted by the gene ontology slim analysis using Blast2GO tool. The analysis comprised three categories, i.e., biological process, molecular function, and cellular component same as mentioned by Di et al. (2018) [34]. Our results showed that chitin catabolic processes and cell wall macromolecule catabolic processes, defense response to fungus, bacterium and response to stress were the highly regulated functions having role in biological process which support the function of the CaChi in the cell. In addition, prediction of molecular functions of CaChi proteins indicated that they had mostly involved in chitin binding capacity, chitinase activity and small molecules binding while cellular component analysis revealed that CaChi mostly localized in extracellular region. Furthermore, they can accumulate in subcellular parts of the cell such as vacuole, chloroplast, vacuole and plasma membrane (Figure 6). signaling molecules and stresses. Gene ontology (GO) enrichment analysis of CaChi were predicted by the gene ontology slim analysis using Blast2GO tool. The analysis comprised three categories, i.e., biological process, molecular function, and cellular component same as mentioned by Di et al. (2018) [34]. Our results showed that chitin catabolic processes and cell wall macromolecule catabolic processes, defense response to fungus, bacterium and response to stress were the highly regulated functions having role in biological process which support the function of the CaChi in the cell. In addition, prediction of molecular functions of CaChi proteins indicated that they had mostly involved in chitin binding capacity, chitinase activity and small molecules binding while cellular component analysis revealed that CaChi mostly localized in extracellular region. Furthermore, they can accumulate in subcellular parts of the cell such as vacuole, chloroplast, vacuole and plasma membrane (Figure 6).


**Figure 5.** *Cis*-acting regulatory elements in the promoter regions of CaChi genes. The *cis*-element positions in the individual CaChi promoter region was inferred from the Plant CARE website (Available online: http://bioinformatics.psb.ugent.be/webtools/plantcare/html/). The different number of *cis* regulatory elements represent in different colors. **Figure 5.** *Cis*-acting regulatory elements in the promoter regions of CaChi genes. The *cis*-element positions in the individual CaChi promoter region was inferred from the Plant CARE website (Available online: http://bioinformatics.psb.ugent.be/webtools/plantcare/html/). The different number of *cis* regulatory elements represent in different colors.

raised.

**Figure 6.** Gene ontology analysis of CaChi proteins in three categories (Biological processes, molecular functions and cellular component) using Blast2Go program. Different colors which are indicated near the graphics show different biological process, molecular functions and cellular component of pepper chitin-binding protein family genes. **Figure 6.** Gene ontology analysis of CaChi proteins in three categories (Biological processes, molecular functions and cellular component) using Blast2Go program. Different colors which are indicated near the graphics show different biological process, molecular functions and cellular component of pepper chitin-binding protein family genes.

#### *2.5. Expression Analysis of CaChi under* Phytophthora capsici *Strains Inoculation 2.5. Expression Analysis of CaChi under* Phytophthora capsici *Strains Inoculation*

To examine the transcription levels of CaChi against the virulent (HX-9) and avirulent (PC) strain of *Phytophthora capsici*, pepper plants were inoculated with *P. capsici* via root drenching and their expression levels were analyzed by qRT-PCR. The results exposed that, post *P. capsici* inoculation, the CaChi were differentially expressed (Figure 7). Twelve CaChi (75%) were upregulated on different time points, and three members (*CaChiI1*, *CaChiIII3* and *CaChiVI4*) (18.75%) were downregulated to both strains on maximum time points. Initially, *CaChiI1* and *CaChiVI4* exhibited downregulation after inoculation with virulent strain (HX-9), then *CaChiI1* upregulated on 48 hpi and *CaChiVI4* on 72 hpi, which were 1.18 and 1.45, respectively. However, *CaChiIII2***,** *CaChiIII6* and *CaChiVI1* exhibited progressive upregulation at all the time points in both strains, but *CaChiIII2* was reached to peak (29.39) at 48 hpi in HX-9, while *CaChiIII6* showed the highest transcription level after PC post inoculation (48 h), i.e., 26.17. Whereas *CaChiVI2* peaked at 12 hpi in virulent (29.38) and avirulent (29.75), *CaChiIV1* showed the highest expression compared with other CaChi, reaching a maximum at 38.52 (PC) and 45.51 (HX-9) and then slightly downregulated. Meanwhile, in the event of avirulent strain inoculation, *CaChiI2* (2.25), *CaChiIII1* (5.80) *CaChiIII3* (2.30) and *CaChiIII5* (2.59) were not predominantly upregulated but, at 72 hpi, showed slight expression. However, *CaChiIII4* and *CaChiVI2* were upregulated following the same pattern and reached a maximum 19.39 and 29.75, respectively, at 12 hpi. *CaChiI3* and *CaChiIV2* exhibited significant expression only to virulent (HX-9) strain. *CaChiIII7* revealed upregulation for both virulent and avirulent strains and reached a peak (31.10 and 37.13 respectively) at 6 hpi, then downregulated, and subsequently upregulated. Six hours post inoculation, *CaChiVI3* exhibited the highest transcription in PC strain and then later it was downregulated at every other time point; however, for HX-9 strain, its transcriptional level was To examine the transcription levels of CaChi against the virulent (HX-9) and avirulent (PC) strain of *Phytophthora capsici*, pepper plants were inoculated with *P. capsici* via root drenching and their expression levels were analyzed by qRT-PCR. The results exposed that, post *P. capsici* inoculation, the CaChi were differentially expressed (Figure 7). Twelve CaChi (75%) were upregulated on different time points, and three members (*CaChiI1*, *CaChiIII3* and *CaChiVI4*) (18.75%) were downregulated to both strains on maximum time points. Initially, *CaChiI1* and *CaChiVI4* exhibited downregulation after inoculation with virulent strain (HX-9), then *CaChiI1* upregulated on 48 hpi and *CaChiVI4* on 72 hpi, which were 1.18 and 1.45, respectively. However, *CaChiIII2***,** *CaChiIII6* and *CaChiVI1* exhibited progressive upregulation at all the time points in both strains, but *CaChiIII2* was reached to peak (29.39) at 48 hpi in HX-9, while *CaChiIII6* showed the highest transcription level after PC post inoculation (48 h), i.e., 26.17. Whereas *CaChiVI2* peaked at 12 hpi in virulent (29.38) and avirulent (29.75), *CaChiIV1* showed the highest expression compared with other CaChi, reaching a maximum at 38.52 (PC) and 45.51 (HX-9) and then slightly downregulated. Meanwhile, in the event of avirulent strain inoculation, *CaChiI2* (2.25), *CaChiIII1* (5.80) *CaChiIII3* (2.30) and *CaChiIII5* (2.59) were not predominantly upregulated but, at 72 hpi, showed slight expression. However, *CaChiIII4* and *CaChiVI2* were upregulated following the same pattern and reached a maximum 19.39 and 29.75, respectively, at 12 hpi. *CaChiI3* and *CaChiIV2* exhibited significant expression only to virulent (HX-9) strain. *CaChiIII7* revealed upregulation for both virulent and avirulent strains and reached a peak (31.10 and 37.13 respectively) at 6 hpi, then downregulated, and subsequently upregulated. Six hours post inoculation, *CaChiVI3* exhibited the highest transcription in PC strain and then later it was downregulated at every other time point; however, for HX-9 strain, its transcriptional level was raised.

*Int. J. Mol. Sci.* **2018**, *19*, 2216 9 of 26

**Figure 7.** Expression profiles of CaChi in response to different strains of *Phytophthora capsici* (PC and HX-9). The samples were collected at different time points (0, 6, 12, 24, 48 and 72 hpt) and were analyzed by qRT-PCR. Mean values and SDs for three replicates are shown. Small letters (a–e) represent significant differences (*p* < 0.05). **Figure 7.** Expression profiles of CaChi in response to different strains of *Phytophthora capsici* (PC and HX-9). The samples were collected at different time points (0, 6, 12, 24, 48 and 72 hpt) and were analyzed by qRT-PCR. Mean values and SDs for three replicates are shown. Small letters (a–e) represent significant differences (*p* < 0.05).

#### *2.6. Expression Profile of CaChi in Response to Abiotic Stresses 2.6. Expression Profile of CaChi in Response to Abiotic Stresses*

To examine the expression levels of the CaChi in response to abiotic stresses, eight representative genes (*CaChiI3*, *CaChiIII2*, *CaChiIII4*, *CaChiIII6*, *CaChiIII7*, *CaChiIV1*, *CaChiIV2* and *CaChiVI2*) were selected from the CaChi, in which at least one gene was selected from each class on the basis of their *cis-*acting elements response and expression to *P. capsici*. Then, they were subjected to NaCl, mannitol and cold stresses (Figure 8). *CaChiI3* showed no response to cold and NaCl stress while in response to mannitol it was gradually upregulated, reaching a maximum at 24 hpt (7.78), and then downregulated. *CaChiIII2* showed a slight upregulation at 6 hpt in response to NaCl while exhibited concomitant up- and downregulation in response of mannitol stress. In the case of cold stress, no expression was recorded. In response to NaCl, *CaChiIII4* initially exhibited no response, then upregulated at 6 hpt, reached a maximum at 12 hpt (16.84) and then showed a slight downregulation at 24 and 48 hpt, whereas no significant response was observed in response to cold and mannitol (Figure 8). *CaChiIII6* was not regulated by mannitol stress, whereas abrupt changes were observed to NaCl stress; however, highest expression was noticed at 6 hpt (21.32) in response to cold stress, where the expression was reduced in later hours. *CaChiIII7* was gradually upregulated in response to NaCl, reached a maximum at 6 hpt (5.53) and then downregulated. In response to mannitol, a slight up regulation was noted at 12 hpt and then downregulated similarly. In cold stress, significant expression (7.32) was observed in all time points. The transcript level of *CaChiIV1* was highly induced by mannitol, cold and NaCl at 12, 24 and 48 hpt, which were more than 12, 16 and 15 folds, respectively. *CaChiIV2* was initially downregulated by NaCl and mannitol stress and then exhibited an abrupt upregulation in response to NaCl at 12 hpt (3.34) and again downregulated. In response to mannitol stress, no significant expression occurred. In cold stress, it shown initial abrupt To examine the expression levels of the CaChi in response to abiotic stresses, eight representative genes (*CaChiI3*, *CaChiIII2*, *CaChiIII4*, *CaChiIII6*, *CaChiIII7*, *CaChiIV1*, *CaChiIV2* and *CaChiVI2*) were selected from the CaChi, in which at least one gene was selected from each class on the basis of their *cis-*acting elements response and expression to *P. capsici*. Then, they were subjected to NaCl, mannitol and cold stresses (Figure 8). *CaChiI3* showed no response to cold and NaCl stress while in response to mannitol it was gradually upregulated, reaching a maximum at 24 hpt (7.78), and then downregulated. *CaChiIII2* showed a slight upregulation at 6 hpt in response to NaCl while exhibited concomitant up- and downregulation in response of mannitol stress. In the case of cold stress, no expression was recorded. In response to NaCl, *CaChiIII4* initially exhibited no response, then upregulated at 6 hpt, reached a maximum at 12 hpt (16.84) and then showed a slight downregulation at 24 and 48 hpt, whereas no significant response was observed in response to cold and mannitol (Figure 8). *CaChiIII6* was not regulated by mannitol stress, whereas abrupt changes were observed to NaCl stress; however, highest expression was noticed at 6 hpt (21.32) in response to cold stress, where the expression was reduced in later hours. *CaChiIII7* was gradually upregulated in response to NaCl, reached a maximum at 6 hpt (5.53) and then downregulated. In response to mannitol, a slight up regulation was noted at 12 hpt and then downregulated similarly. In cold stress, significant expression (7.32) was observed in all time points. The transcript level of *CaChiIV1* was highly induced by mannitol, cold and NaCl at 12, 24 and 48 hpt, which were more than 12, 16 and 15 folds, respectively. *CaChiIV2* was initially downregulated by NaCl and mannitol stress and then exhibited an abrupt upregulation in response to NaCl at 12 hpt (3.34) and again downregulated. In response to mannitol stress, no

upregulation and then smoothly downregulated (Figure 8). Responding to NaCl stress, *CaChiVI2* was

significant expression occurred. In cold stress, it shown initial abrupt upregulation and then smoothly downregulated (Figure 8). Responding to NaCl stress, *CaChiVI2* was gradually upregulated, peaked at 6 hpt (9.47) then smoothly downregulated. In response to cold and mannitol, it was upregulated and reached a maximum at 6 hpt (5.86) and 48 hpt (5.40), respectively. *Int. J. Mol. Sci.* **2018**, *19*, 2216 10 of 26 gradually upregulated, peaked at 6 hpt (9.47) then smoothly downregulated. In response to cold and mannitol, it was upregulated and reached a maximum at 6 hpt (5.86) and 48 hpt (5.40), respectively.

**Figure 8.** Expression profiles of CaChi genes in response abiotic stresses. The inducible expression patterns performed by qRT-PCR under Sodium chloride (NaCl) and Mannitol. Mean values and SDs for three replicates are shown. Small letters (a–e) represent significant differences (*p* < 0.05). **Figure 8.** Expression profiles of CaChi genes in response abiotic stresses. The inducible expression patterns performed by qRT-PCR under Sodium chloride (NaCl) and Mannitol. Mean values and SDs for three replicates are shown. Small letters (a–e) represent significant differences (*p* < 0.05).

#### Phyto-hormones and plant signaling molecules, such as MeJA, SA and ABA, are involved in *2.7. Expression Profile of CaChi in Response to Hormonal Treatments*

*2.7. Expression Profile of CaChi in Response to Hormonal Treatments*

various stress signaling pathways [35–38]. The above selected eight CaChi genes were also exposed to exogenous hormonal (MeJA, SA and ABA) treatments to explore the response of these target genes. We investigated the expression profiles of CaChi-genes in AA3 leaves. As shown in Figure 9, post SA Phyto-hormones and plant signaling molecules, such as MeJA, SA and ABA, are involved in various stress signaling pathways [35–38]. The above selected eight CaChi genes were also exposed to exogenous hormonal (MeJA, SA and ABA) treatments to explore the response of these target genes. We investigated the expression profiles of CaChi-genes in AA3 leaves. As shown in Figure 9, post SA and ABA treatment, five CaChi (*CaChiI3*, *CaChiIII2*, *CaChiIII6*, *CaChiIV1* and *CaChiVI2*) were significantly upregulated (>10, 11, 18, 6, 8 folds against SA and >6, 24, 52, 10 and 14 folds against ABA, respectively) at different time points, while *CaChiIV1* and *CaChiIV2* were gradually upregulated over time and maximum expressions at 48 (10 folds) and 12 hpt (two folds) were recorded in response

to MeJA treatment. The *CaChiIII4* was initially upregulated 1 hpt after ABA treatment (>5 folds), and then irregular changes were noticed, while, in response to MeJA and SA, the expression level was very low or even not obvious, except for SA where an abrupt upregulation (2.29) was noted at all given time points. *CaChiIII7* responded to MeJA and reached a peak at 48 hpt (10.60), whereas the response to ABA was antagonistic-like initially where an upregulation and then smooth decline were observed. Expression was not induced by SA treatment. The transcript levels of *CaChiIV1* gene were induced by MeJA, steadily increased and reached a peak (10.90) at 48 hpt. In the case of SA treatment, it was gradually upregulated, reached a peak (6.82) at 6 hpt, downregulated at 12 and 24 hpt, and again upregulated at 48 hpt. After ABA treatment, transcription level was high at 1 (7 folds) and 12 hpt (10 folds). The *CaChiIV2* was upregulated by SA, reached to peak at 48 hpt (8.76). After ABA application, it abruptly upregulated at 1 and 12 hpt (>9 and 16 folds, respectively), while it showed no significant response to MeJA. significantly upregulated (>10, 11, 18, 6, 8 folds against SA and >6, 24, 52, 10 and 14 folds against ABA, respectively) at different time points, while *CaChiIV1* and *CaChiIV2* were gradually upregulated over time and maximum expressions at 48 (10 folds) and 12 hpt (two folds) were recorded in response to MeJA treatment. The *CaChiIII4* was initially upregulated 1 hpt after ABA treatment (>5 folds), and then irregular changes were noticed, while, in response to MeJA and SA, the expression level was very low or even not obvious, except for SA where an abrupt upregulation (2.29) was noted at all given time points. *CaChiIII7* responded to MeJA and reached a peak at 48 hpt (10.60), whereas the response to ABA was antagonistic-like initially where an upregulation and then smooth decline were observed. Expression was not induced by SA treatment. The transcript levels of *CaChiIV1* gene were induced by MeJA, steadily increased and reached a peak (10.90) at 48 hpt. In the case of SA treatment, it was gradually upregulated, reached a peak (6.82) at 6 hpt, downregulated at 12 and 24 hpt, and again upregulated at 48 hpt. After ABA treatment, transcription level was high at 1 (7 folds) and 12 hpt (10 folds). The *CaChiIV2* was upregulated by SA, reached to peak at 48 hpt (8.76). After ABA application, it abruptly upregulated at 1 and 12 hpt (>9 and 16 folds, respectively), while it showed no significant response to MeJA.

and ABA treatment, five CaChi (*CaChiI3*, *CaChiIII2*, *CaChiIII6*, *CaChiIV1* and *CaChiVI2*) were

**Figure 9.** Expression profiles of CaChi in response hormones application. The inducible expression patterns performed by qRT-PCR under Salicylic acid (SA) and methyl-jasmonate (MeJA). Mean values and SDs for three replicates are shown. Small letters (a–f) represent significant differences (*p* < 0.05).
