Genome-Wide Identification and Expression of Chitinase Class I Genes in Garlic (Allium sativum L.) Cultivars Resistant and Susceptible to Fusarium proliferatum

Vegetables of the Allium genus are prone to infection by Fusarium fungi. Chitinases of the GH19 family are pathogenesis-related proteins inhibiting fungal growth through the hydrolysis of cell wall chitin; however, the information on garlic (Allium sativum L.) chitinases is limited. In the present study, we identified seven class I chitinase genes, AsCHI1–7, in the A. sativum cv. Ershuizao genome, which may have a conserved function in the garlic defense against Fusarium attack. The AsCHI1–7 promoters contained jasmonic acid-, salicylic acid-, gibberellins-, abscisic acid-, auxin-, ethylene-, and stress-responsive elements associated with defense against pathogens. The expression of AsCHI2, AsCHI3, and AsCHI7 genes was constitutive in Fusarium-resistant and -susceptible garlic cultivars and was mostly induced at the early stage of F. proliferatum infection. In roots, AsCHI2 and AsCHI3 mRNA levels were increased in the susceptible and decreased in the resistant cultivar, whereas in cloves, AsCHI7 and AsCHI5 expression was decreased in the susceptible but increased in the resistant plants, suggesting that these genes are involved in the garlic response to Fusarium proliferatum attack. Our results provide insights into the role of chitinases in garlic and may be useful for breeding programs to increase the resistance of Allium crops to Fusarium infections.


Introduction
Plants belonging to the Allium genus are widely cultivated for their culinary and medicinal properties [1]. Throughout their life cycle, onions (including garlic), like all other agricultural plants, are infected with fungi, in particular Fusarium spp., which are the most viable and destructive soil-dwelling crop pathogens causing Fusarium basal rot (F. oxysporum f. sp. cepae) and bulb rot (F. proliferatum) [2][3][4][5].
F. oxysporum f. sp. cepae is responsible for 60% of the world's garlic crop losses. This species produces chlamydospores that can survive in the soil for many years and cause decease symptoms at all pre-and post-harvest stages, infecting the roots and basal plates [5]. The mycotoxigenic species F. proliferatum is able to colonize the roots of garlic plants, remaining a latent infection during growth, and develop rot during storage, affecting almost 30% of bulbs and producing a wide range of toxins [4]. Observed symptoms include dry brown necrotic spots, and sometimes white mycelium and water-soaked signs at the clove surface [4].
Fusarium infections are suppressed with fungicides, the use of resistant cultivars, and the intercropping of Allium spp., which is considered an effective method of Fusarium biological control [6,7]. It has been shown that Fusarium growth can be suppressed by antifungal volatiles such as 2-methyl-2-pentenal and organosulfur compounds and non-volatiles such as spirostanol, furostanol, and steroidal saponins produced by Allium roots [8][9][10][11][12], as well as

In Silico Genome-Wide Identification of Class I Chitinase Genes in A. sativum cv. Ershuizao
A total of seven complete sequences of class I chitinase genes were found in the genome and transcriptome of A. sativum cv. Ershuizao (PRJNA606385, PRJNA607255) and annotated as AsCHI1-7 (A. sativum chitinases 1-7) ( Table 1). The AsCHI1 gene is located on chromosome 5 and the other six genes-on chromosome 6. The sizes of the identified genes range from 1118 to 1589 bp, and the coding sequences (CDSs) range from 933 bp in AsCHI3 to 969 bp in AsCHI6 and consist of three exons (Table 1, Figure 1A). addition, the expression of AsCHI1-7 mRNA was determined in various tissues of garlic under normal conditions and in response to F. proliferatum infection. Based on the data obtained, we suggest a role for chitinases in garlic protection against fungal pathogens. Our results provide useful insights into the function of A. sativum chitinases, which can be used in breeding programs to increase the resistance to Fusarium in cultivated Allium spp.

In Silico Genome-Wide Identification of Class I Chitinase Genes in A. sativum cv. Ershuizao
A total of seven complete sequences of class I chitinase genes were found in the genome and transcriptome of A. sativum cv. Ershuizao (PRJNA606385, PRJNA607255) and annotated as AsCHI1-7 (A. sativum chitinases 1-7) ( Table 1). The AsCHI1 gene is located on chromosome 5 and the other six genes-on chromosome 6. The sizes of the identified genes range from 1118 to 1589 bp, and the coding sequences (CDSs) range from 933 bp in AsCHI3 to 969 bp in AsCHI6 and consist of three exons (Table 1, Figure 1A).   Phylogenetic analysis of the AsCHI protein sequences revealed three main clades: the first comprising closely related AsCHI4, AsCHI5, and AsCHI6 (99.1-99.4% identity), the second-AsCHI1 and AsCHI7 (95.3% identity), and the third-AsCHI2 and AsCHI3 (90.6% identity with 30 variable residues) as well as the only known class I chitinase from Arabidopsis thaliana (AT3G12500) ( Figure 1A).

Structural and Phylogenetic Analyses of Class I Chitinases Predicted in Garlic
The sizes of the translated AsCHI homologues were 310 (AsCHI3), 319 (AsCHI1, AsCHI2, and AsCHI7), 321 (AsCHI4, and AsCHI5), and 322 (AsCHI6) amino acids (aa) ( Table 1). The predicted physicochemical properties of AsCHI proteins are presented in Table 2. AsCHI aliphatic indexes showing the relative number of hydrophobic residues were from 52.73 to 63.42 and AsCHI hydrophobicity indexes (grand average hydropathy-GRAVY) calculated as the sum of hydrophobicity values of all residues divided by sequence length were negative, indicating the hydrophilic nature of the translated proteins.
Functional annotation in gene ontology (GO) terms predicted that the AsCHI proteins had chitinase activity and chitin-binding ability, and were involved in the catabolism of chitin, cell wall macromolecules, polysaccharides and carbohydrates, and in plant response to fungal infection ( Table 2).

In Silico Analysis of the Expression of A. sativum Class I Chitinases
Next, we analyzed the expression of class I chitinase genes in various tissues (roots, bulbs, pseudostem, leaves, buds, flowers, and sprouts) of A. sativum cv. Ershuizao based on transcriptomics data (PRJNA607255 and GSE145455 [38]). The analysis of gene expression in bulbs was more detailed than for other tissues, and included eight developmental stages (from 192 to 227 days old), since this underground part of the plant is most susceptible to Fusarium infection with visible symptoms. A less detailed analysis of other tissue types, in addition to the above, is due to the presence of transcriptome data for these tissues only for one stage [38]. Figure 3 shows that AsCHI2/AsCHI3 and AsCHI4-6 had a similar expression pattern within each clade, whereas those of AsCHI1 and AsCHI7 were rather different. AsCHI4-6 showed maximum expression in the pseudostem and shoots, and AsCHI2/AsCHI3-in The total ratio of basic (Arg, His, and Lys) to acidic (Asp and Glu) residues in putative AsCHI proteins was 27/17 (AsCHI1), 24/18 (AsCHI2), 19/19 (AsCHI3), 34/30 (AsCHI4, 34/31 (AsCHI5), 34/31 (AsCHI6), and 26/23 (AsCHI7). Considering the pI value determined for each studied chitinase (Table 2), it can be proposed that AsCHI1 and AsCHI2are basic enzymes, while the rest of the proteins are acidic rather than basic.
Functional annotation in gene ontology (GO) terms predicted that the AsCHI proteins had chitinase activity and chitin-binding ability, and were involved in the catabolism of chitin, cell wall macromolecules, polysaccharides and carbohydrates, and in plant response to fungal infection ( Table 2).

In Silico Analysis of the Expression of A. sativum Class I Chitinases
Next, we analyzed the expression of class I chitinase genes in various tissues (roots, bulbs, pseudostem, leaves, buds, flowers, and sprouts) of A. sativum cv. Ershuizao based on transcriptomics data (PRJNA607255 and GSE145455 [38]). The analysis of gene expression in bulbs was more detailed than for other tissues, and included eight developmental stages (from 192 to 227 days old), since this underground part of the plant is most susceptible to Fusarium infection with visible symptoms. A less detailed analysis of other tissue types, in addition to the above, is due to the presence of transcriptome data for these tissues only for one stage [38]. Figure 3 shows that AsCHI2/AsCHI3 and AsCHI4-6 had a similar expression pattern within each clade, whereas those of AsCHI1 and AsCHI7 were rather different. AsCHI4-6 showed maximum expression in the pseudostem and shoots, and AsCHI2/AsCHI3-in the pseudostem. The AsCHI1 gene was strongly expressed in the roots and AsCHI7-in the roots, pseudostem, and flowers. the pseudostem. The AsCHI1 gene was strongly expressed in the roots and AsCHI7-in the roots, pseudostem, and flowers.
During bulb development, the expression of AsCHI2, AsCHI3, AsCHI5, and AsCHI6 was quite weak, whereas that of AsCHI4 showed a bell shape, increasing up to stage 4 and then decreasing. The expression of AsCHI7, which was almost absent at stages 1 and 2, gradually increased, reaching a maximum at stage 7, whereas that of AsCHI1 peaked at stage 3 ( Figure 3). It should be noted that all genes, except AsCHI1, had a high mRNA expression level in the pseudostem, whereas only AsCHI1 and AsCHI7 were strongly expressed in the roots.

Promoter Analysis of Garlic Class I Chitinase Genes
The AsCHI1-7 sequences upstream of the initiation codon (~1.0 kb), which predictably included the promoter and 5′UTR, were amplified and analyzed for hormone-and stress-responsive elements. The search for sites important for gene transcription showed that the AsCHI1-7 regulatory regions contained 13 hormone-responsive elements (ABRE, ABRE3a, ABRE4, CARE, AUXRR-core, TGA-box, TGA-element, CGTCA-motif, TCA-element, TATC-box, P-box, GARE-motif, and ERE), and 12 stress-responsive elements (ARE, DRE1, DRE core, LTR, MBS, STRE, F-box, AT-rich sequence, TC-rich repeats, W-box, Wun-motif, and WRE3) ( Table 3). Among them, four elements (methyl jasmonate (MeJA)-responsive CGTCA motif, ethylene (ET) responsive ERE, anaerobic induction essential ARE, and stress responsive STRE) were common for all analyzed genes which, however, differed in their numbers. During bulb development, the expression of AsCHI2, AsCHI3, AsCHI5, and AsCHI6 was quite weak, whereas that of AsCHI4 showed a bell shape, increasing up to stage 4 and then decreasing. The expression of AsCHI7, which was almost absent at stages 1 and 2, gradually increased, reaching a maximum at stage 7, whereas that of AsCHI1 peaked at stage 3 ( Figure 3). It should be noted that all genes, except AsCHI1, had a high mRNA expression level in the pseudostem, whereas only AsCHI1 and AsCHI7 were strongly expressed in the roots.

Functional Description
Annotation Motif

Number of Elements Found in Gene Promoters
AsCHI1 AsCHI2 AsCHI3 AsCHI4 AsCHI5 AsCHI6 AsCHI7 Hormone responsive Cis-acting elements, involved in abscisic acid response Cis-acting elements involved in auxin response AsCHI1 contained six abscisic acid (ABA)-responsive elements (ABRE), whereas the other genes contained one or two; ABRE3a and ABRE4 as well as dehydration-responsive DRE1 were found only in the AsCHI1 gene (Table 3).
AT-rich sequence (maximal elicitor-mediated activation) was present only in AsCHI2, whereas ABA-responsive CARE and auxin-responsive TGA-box were found in AsCHI2 and AsCHI3, and defense and stress responsive TC-rich repeats-in AsCHI6 and AsCHI7. WRE3 involved in wound and pathogen responses was detected in AsCHI2, AsCHI5, and AsCHI7, and salicylic acid (SA)-responsive elements were found in AsCHI2, AsCHI3, and AsCHI7 (Table 3).

Class I Chitinase Gene Expression in cv. Sarmat and Strelets Infected with F. proliferatum
Data on chitinase genes obtained for cv. Ershuizao were used to elucidate the possible roles of the identified genes in two garlic cultivars known to be resistant and susceptible, respectively, to Fusarium infection.
Garlic cv. Sarmat and Strelets resistant and susceptible to Fusarium rot, respectively, were infected with F. proliferatum, and analyzed for disease symptoms after 24 and 96 h. In cv. Sarmat, there were no symptoms of infection, whereas in cv. Strelets, the roots showed white fungal mycelium cover after 96 h (Figure 4). White mycelium is the reported symptom of Fusarium rot. Consequently, the external growth of the pathogen is observed only in the case of root colonization and indicates the sensitivity of the cultivar to this infection, while the tissues of the resistant cultivar are able to suppress the growth of fungi. Garlic cv. Sarmat and Strelets resistant and susceptible to Fusarium rot, respectively, were infected with F. proliferatum, and analyzed for disease symptoms after 24 and 96 h. In cv. Sarmat, there were no symptoms of infection, whereas in cv. Strelets, the roots showed white fungal mycelium cover after 96 h (Figure 4). White mycelium is the reported symptom of Fusarium rot. Consequently, the external growth of the pathogen is observed only in the case of root colonization and indicates the sensitivity of the cultivar to this infection, while the tissues of the resistant cultivar are able to suppress the growth of fungi.
As sequences of some chitinase genes were very similar, common primer pairs were used to amplify AsCHI4-6 and AsCHI1/AsCHI7. In these cases, the resulting products were sequenced to determine preferentially expressed genes. Analysis of AsCHI1-7 expression in roots, stems, and cloves of infected and control plants revealed that AsCHI1/AsCHI7, AsCHI2, and AsCHI3 were transcribed in all analyzed organs, whereas AsCHI4-6-only in cloves (Figures 5 and 6). In Sarmat roots, the chitinase genes were significantly upregulated at 24 and 96 h after infection and in Strelets roots, the upregulation was observed only at 24 h, whereas at 96 h the expression of some genes was decreased (AsCHI1/AsCHI7), slightly increased (AsCHI2), or was the same (AsCHI3) compared to the control ( Figure 5). In the stems, the activation of class I chitinase genes was observed in both cultivars at 24 h, but in cv. Sarmat it almost doubled that in cv. Strelets; however, at 96 h the expression of all AsCHI genes was significantly downregulated. In the cloves, the transcription of the analyzed genes was overall higher in all infected samples compared to the control. The mRNA expression of AsCHI2 and AsCHI3 increased with time; in cv. Strelets, it markedly exceeded that in cv. Sarmat As sequences of some chitinase genes were very similar, common primer pairs were used to amplify AsCHI4-6 and AsCHI1/AsCHI7. In these cases, the resulting products were sequenced to determine preferentially expressed genes.
Analysis of AsCHI1-7 expression in roots, stems, and cloves of infected and control plants revealed that AsCHI1/AsCHI7, AsCHI2, and AsCHI3 were transcribed in all analyzed organs, whereas AsCHI4-6-only in cloves (Figures 5 and 6). In Sarmat roots, the chitinase genes were significantly upregulated at 24 and 96 h after infection and in Strelets roots, the upregulation was observed only at 24 h, whereas at 96 h the expression of some genes was decreased (AsCHI1/AsCHI7), slightly increased (AsCHI2), or was the same (AsCHI3) compared to the control ( Figure 5). In the stems, the activation of class I chitinase genes was observed in both cultivars at 24 h, but in cv. Sarmat it almost doubled that in cv. Strelets; however, at 96 h the expression of all AsCHI genes was significantly downregulated. In the cloves, the transcription of the analyzed genes was overall higher in all infected samples compared to the control. The mRNA expression of AsCHI2 and AsCHI3 increased with time; in cv. Strelets, it markedly exceeded that in cv. Sarmat where the expression level at 24 h was almost the same as in the control. However, the transcription of AsCHI1/AsCHI7 and AsCHI4-6 had different dynamics in the two cultivars, increasing with time in cv. Strelets and decreasing in cv. Sarmat ( Figure 5).    In control plants, the expression of the AsCHI genes was also time-dependent ( Figure 6). In both analyzed cultivars, AsCHI1/AsCHI7 transcription increased from 24 to 96 h, except for cv. Strelets cloves, where it decreased. Compared to 24 h, at 96 h AsCHI2 and AsCHI3 were upregulated in the stems but downregulated in the roots and cloves, whereas AsCHI4-6 was slightly upregulated in the cloves of cv. Sarmat but significantly downregulated in those of cv. Strelets. In the stems of uninfected cv. Strelets, the levels of AsCHI2, AsCHI3, and AsCHI1/AsCHI7 expression were higher than in those of cv. Sarmat ( Figure 6).

Expression and Characterization of AsCHI CDSs in A. sativum cv. Sarmat and Strelets
To determine which genes of AsCHI1/AsCHI7 and AsCHI4-6 clades were expressed in the tissues of the two garlic cultivars, we performed sequencing of PCR-amplified In control plants, the expression of the AsCHI genes was also time-dependent ( Figure 6). In both analyzed cultivars, AsCHI1/AsCHI7 transcription increased from 24 to 96 h, except for cv. Strelets cloves, where it decreased. Compared to 24 h, at 96 h AsCHI2 and AsCHI3 were upregulated in the stems but downregulated in the roots and cloves, whereas AsCHI4-6 was slightly upregulated in the cloves of cv. Sarmat but significantly downregulated in those of cv. Strelets. In the stems of uninfected cv. Strelets, the levels of AsCHI2, AsCHI3, and AsCHI1/AsCHI7 expression were higher than in those of cv. Sarmat ( Figure 6).

Expression and Characterization of AsCHI CDSs in A. sativum cv. Sarmat and Strelets
To determine which genes of AsCHI1/AsCHI7 and AsCHI4-6 clades were expressed in the tissues of the two garlic cultivars, we performed sequencing of PCR-amplified products, Plants 2021, 10, 720 13 of 21 which revealed that only AsCHI7 and AsCHI5 were transcribed in the roots, stems, and cloves of both cultivars.

Analysis of Promoters in Class I Chitinase Genes Differentially Expressed in cv. Sarmat and Strelets after F. proliferatum Infection
We next amplified and analyzed the promoters (~1.0 kb before the start codon) of the AsCHI2, AsCHI3, and AsCHI7 genes, which were differently regulated in cv. Sarmat and Strelets in response to F. proliferatum infection. The AsCHI2 promoter differed from the corresponding cv. Ershuizao sequence by four nucleotide substitutions, and between Sarmat and Strelets sequences by 1 bp indel at position −451 relative to cv. Ershuizao AsCHI2 promoter.
The AsCHI3 promoter differed from the corresponding cv. Ershuizao sequence by nine nucleotide substitutions and three indels (1, 3, and 20 bp). The 20 bp indel (TTGCTGACGT-GAGAAAGGCA) was present at position −365 in cv. Sarmat and Strelets but at position −874 in cv. Ershuizao, which can be explained by inaccuracies in the assembly of the A. sativum genome. The AsCHI7 promoter was identical in both cv. Sarmat and Strelets, and differed from the corresponding cv. Ershuizao sequence by 12 SNPs and two indels (2, and 3 bp).
The search for sites important for AsCHI2, AsCHI3, and AsCHI7 transcription in cv. Sarmat and Strelets revealed the same cis-regulatory elements as in the AsCHI2 promoter of the corresponding genes in cv. Ershuizao (Table 3). Compared to cv. Ershuizao, the AsCHI3 promoter of cv. Sarmat and Strelets acquired two additional elements, ABRE and P-box, at position −365 in cv. Ershuizao AsCHI3 promoter and lost the W-box element; the AsCHI7 promoter of cv. Sarmat and Strelets acquired additional TC-rich repeat.

Discussion
The genus Allium (family Amaryllidaceae, order Asparagales) comprises 971 species, including economically important cash crops such as diploid garlic A. sativum, which has one of the largest genomes (16.24 Gb vs. 157 Mb of A. thaliana genome) among vegetables [38,41,42]. Significant pre-and post-harvest losses of garlic regularly occur worldwide because of diseases caused by soil fungi of the Fusarium genus [43]. The antifungal activity of class I chitinases indicate their role in the mechanism of garlic resistance to Fusarium rot, which can make these enzymes a promising target in crop breeding programs.
In the present study, we identified and characterized seven class I chitinase genes in the A. sativum cv. Ershuizao genome [38]); among them, four were amplified from garlic cv. Sarmat and Strelets resistant and susceptible to Fusarium rot, respectively. All the genes were orthologues of the only known A. thaliana class I chitinase gene (PR3; OAP02400.1), suggesting redundancy or, conversely, functional divergence among AsCHI1-7, such as involvement in differential responses to various stresses and pathogens. Analysis of chromosomal localization showed that the garlic genome contained two clusters of tandemly arranged chitinase genes, AsCHI4-7 and AsCHI/AsCHI2, which may be a result of tandem duplication. All AsCHI1-7 genes had only two introns (Figure 1), which is similar to the structure of Brassica rapa chitinase genes shown to be induced at the early stage of clubroot infection [44]. This finding confirms the hypothesis about the rapid regulation of stress-related genes during stress, which is considered to be partly due to a low number of introns [45,46].
All putative AsCHI1-7 proteins contained chitin-binding and catalytic domains, suggesting functional conservation of garlic class I enzymes in regard to chitin hydrolysis. The presence of N-terminal signal peptides indicates the secretion of mature proteins, which is supported by GO analysis predicting the involvement of AsCHI1-7 in the secretory pathway ( Table 2) and is in agreement with the subcellular localization of A. thaliana chitinases [47]. The structure of putative AsCHI1-7 chitinases may also indicate participation in the response to fungal pathogens, as apoplastic chitinases were shown to be induced at the early stage of infection and block the intercellular hyphae growth, acting together with pathogen elicitors to initiate downstream defense pathways [48].
The expression of chitinase genes is triggered by phytohormones (e.g., MeJA and SA) and pathogen attack [49][50][51]. Thus, 20 hormone-and stress-related cis-regulatory elements were found in the promoters of the Cucumis sativus chitinase genes [52]. Cisacting elements associated with response to SA, MeJA, auxin, ET, and GA as well as TC-rich repeats involved in defense and stress response were identified in the promoters of B. rapa class I chitinase genes [44]. Similar to C. sativus, but unlike B. rapa, the AsCHI1-7 promoters contained elements associated with the response to ABA as well as those related to immune defense and response to elicitors and various stresses such as anaerobic conditions, dehydration, low temperature, salinization, heavy metals, and wounding (Table 3). Among the detected stress response elements, the fungal elicitor-responsive W-box was reported to be associated with the induction of chitinase gene expression by a transcription factor implicated in host antifungal defense [53]. It is assumed that other stress-responsive elements such as STRE and WUN-motif mediate pathogen-and/or elicitor-inducible transcription of chitinase genes [52,54].
In plants, pathogen attacks induce systemic or local acquired resistance associated with antagonistic SA and JA defense signaling, respectively [21,55]. The SA pathway leads to the accumulation of PR1, PR2, and PR5 proteins, whereas the JA pathway-to that of PR3, PR4 and PR12 proteins [21]. Thus, in garlic an attack by necrotrophic Fusarium fungi should trigger JA signaling, including the activation of class I chitinases belonging to PR3, PR4, and PR11 families, because the AsCH1-7 promoters contain MeJA-responsive elements, indicating the participation of AsCHI1-7 enzymes in local acquired resistance. However, SA-responsive elements found in the promoters of AsCHI2, AsCHI3, and AsCHI7 genes may also suggest their role in systemic acquired resistance.
ET is responsible for the defense against necrotrophic pathogens [55], and the presence of the ET-responsive element ERE found in all AsCHI1-7 promoters (Table 3) suggests the possibility of their induction by ET after Fusarium attack.
Infection with pathogens can also modulate ABA homeostasis in host plants [44]. For example, infection with necrotrophic pathogen Botrytis cinereal promotes ABA biosynthesis in Arabidopsis [56], whereas increased ABA biosynthesis in tomatoes in response to bacterial attack inhibits pathogen penetration through stomatal closure [57]. Four AsCHI genes contain ABA-responsive promoter elements; among them, AsCHI1 contains 14 (Table 3).
An important role in mediating biotic stress response is played by the crosstalk of JA with auxins and GAs, which promotes plant growth [58]. Among the AsCHI gene promoters, GA-responsive elements were absent only in AsCHI1 and AsCHI7; however, the AsCHI7 promoter had six auxin-responsive elements. Thus, AsCHI1 and AsCHI7 may not participate in GA-mediated defense, but may be involved in ABA-and auxin-mediated response, respectively, to Fusarium.
A possible role of the identified chitinases in the protection of garlic against Fusarium was revealed by examining the AsCHI1-7 expression pattern in tissues of control and infected bulbs of garlic cultivars differing in the resistance to Fusarium basal rot. The results indicated that three genes, AsCHI1, AsCHI4, and AsCHI6, were not functional in cv. Sarmat and Strelets, since their mRNA could not be detected irrespectively of F. proliferatum infection; in contrast, AsCHI4 and AsCHI1 transcripts were observed in the pseudostem and roots, respectively, of cv. Ershuizao (Figure 3). It is possible that these genes are expressed in a cultivar-specific manner and involved in responses to pathogens other than Fusarium spp. AsCHI2, AsCHI3, and AsCHI7 were transcribed constitutively, whereas AsCHI5 expression was specific to cloves (( Figure 6).
All genes were differentially regulated by infection, which is consistent with the induction of chitinase expression in plants under biotic stresses [21]. In both resistant and susceptible cultivars, AsCHI2, AsCHI3, and AsCHI7 genes showed early induction, except for AsCHI2 and AsCHI3 in cv. Sarmat cloves, where these genes were upregulated later ( Figure 5). The most pronounced difference between the response of the resistant and susceptible cultivars to Fusarium was the time-dependent upregulation of AsCHI2 and AsCHI3 expression in cv. Sarmat roots and their downregulation in cv. Strelets roots. Furthermore, AsCHI7 and AsCHI5 mRNA levels decreased in the cloves of cv. Sarmat while increasing in those of cv. Strelets ( Figure 5). These results suggest that the expression of AsCHI2, As-CHI3, AsCHI5, and AsCHI7 may underlie the difference between resistant and susceptible cultivars in the response to Fusarium infection. The identical promoter sequences of these genes in resistant and susceptible garlic plants suggest that their expression after infection is controlled at the level of the activity of transcriptional regulators.
The results obtained can be useful for breeding programs with the aim of increasing the resistance of onion crops to Fusarium infections, since chitinases, which have antifungal activity and are differently expressed in cultivars resistant and susceptible to fungi, may be involved in plant defense from pathogens. The protective effect can be achieved by homologous and heterologous overexpression of chitinase genes, which has already been tried by many researchers. For example, overexpression of the Momordica charantia chitinase gene McCHIT1 in rice (Oryza sativa subsp. indica) significantly increased the resistance of transgenic lines to Sheath blight compared to wild-type plants [59]. Heterologous overexpression of the Coniothyrium minitans chitinase gene CmCH1 led to the upregulation of defense-related genes and enzymes in soybean plants, and thus enhanced their resistance to Sclerotinia sclerotiorum infection [60].

In Silico Identification and Structural Characterization of A. sativum Class I Chitinase Genes
The search for class I chitinase genes was performed in the A. sativum cv. Ershuizao transcriptome sequence (NCBI accession number: PRJNA607255) and whole-genome shotgun contigs (NCBI accession number: PRJNA606385) [38]; partial sequences of A. sativum class I chitinases (M94105.1 and M94106.1) were used as references. We selected sequences that contained start and stop codons and full-length CBD1 and GH19 domains characteristic of class 1 chitinases but did not have premature stop codons in CDSs.

In Silico mRNA Expression Analysis
The expression of class I chitinase genes in garlic tissues was determined using A. sativum cv. Ershuizao RNA-Seq data (FPKM; ID: PRJNA607255) [38] and visualized using Heatmapper [65].

Gene Identification
To amplify full-length chitinase genes from garlic cultivars, gene-specific primers were designed based on A. sativum cv. Ershuizao transcriptomic data (NCBI accession number: PRJNA607255) ( Table 5); manual revision of sequence polymorphisms and additional evaluation were performed using Primer3 (http://frodo.wi.mit.edu/primer3/, accessed on 15 October 2020). Genomic DNA was isolated from young leaves of a single plant of each cultivar accession as previously described [66] and used as a template (100 ng) for PCR amplification at the following conditions: initial denaturation at 94 • C for 5 min, 35 cycles of denaturation at 94 • C for 30 s, primer annealing at 55 • C for 30 s, and extension at 65 • C for 2 min, and final extension at 65 • C for 5 min. The amplified PCR products of the expected size were purified by using the QIAEX ® II Gel Extraction kit (QIAGEN, Hilden, Germany), cloned in the pGEM ® -T Easy (Promega, Madison, WI, USA), and sequenced (3-5 clones for each accession) on ABI Prism 3730 DNA Sequencer (Applied Biosystems, Waltham, MA, USA) using the designed primers.

Plants, Fungi, and Fusarium Infection Assay
Accessions of garlic cv. Sarmat and Strelets resistant and susceptible to Fusarium rot, respectively, were kindly provided by the Federal Scientific Vegetable Center (Moscow region, Russia) and F. proliferatum was kindly provided by the Group of Experimental Mycology, Winogradsky Institute of Microbiology (Research Center of Biotechnology of the RAS, Moscow, Russia).
The choice of cultivars for comparative analysis was based not only on the difference in resistance to fungal diseases, but also on the similarity of other varietal morphological characteristics for the possible elimination of their influence on the experimental results. Both cultivars are winter garlic of Russian breeding. These are mid-season, shooting varieties: the leaf is green with a waxy bloom of medium intensity, leaf length/width is 50-51/2.0-2.3 cm; the bulb is round-flat, 65 g; the number of cloves is 5-7 (Strelets) or 7-11 (Sarmat); the structure of the cloves is simple; the color of dry scales is lilacpurple (Strelets) or lilac-white with anthocyanin streaks (Sarmat), leathery scales are brown (Strelets) or light pink (Sarmat), the flesh is white; productivity is 2.0 (Strelets) and 1.9 (Sarmat) kg m −2 . Data on the resistance of cultivars to F. oxysporum infection were obtained from their originators (the Federal Scientific Vegetable Center, Moscow region, Russia). The F. proliferatum strain was previously isolated from garlic bulbs (cv. Strelets); the pathogenicity test showed that the first signs of the decease appear on the surface of the treated cloves after five days of infection (Filyushin et al., Plant Desease, accepted 3 April 2021). Since this strain was identified in cv. Strelets, and, in addition, it was identified for the first time in Russia, it was decided to use this particular strain in the present study.
The number of cloves in a bulb (5-7 for cv. Strelets and 7-11 for cv. Sarmat) determined the number of cloves used per biological replicate in the Fusarium infection experiment. In total, six bulbs of each cultivar were used; six cloves from each bulb were processed and halved for control and experiment.
Cloves of each cultivar were sterilized in 70% ethanol for 3 min, rinsed with sterile water, placed in Petri dishes with wet filter paper, and incubated at +25 • C in the dark. After 36 h, active root growth was observed and half of the cloves of each cultivar were infected by soaking in F. proliferatum conidial suspension (~10 6 conidia mL −1 ) for 5 min (the inoculation procedure was carried out according to [67]). Then, the infected cloves were transferred to fresh Petri dishes and incubated at +25 • C in the dark; uninfected cloves were used as the control. The experiment was performed in three biological replicates. Various tissues (roots, stems (basal plates), and cloves) were collected at 24 (tissues of three out of six treated and control cloves from each replicate) and 96 h (tissues of the remaining three treated and control cloves from each replicate) after inoculation, immediately frozen in liquid nitrogen, and stored at −80 • C. Expression was checked at 24 h and 96 h after inoculation, as it has been reported that the genes of certain pathogenesis-related proteins showed peak expression 1-3 days after inoculation with hemibiotrophic pathogens [52,68].

RNA Extraction and Quantitative Real-Time Reverse Transcription PCR (qRT-PCR)
Total RNA was extracted from individual roots, stems, and cloves (0.5 g of each tissue, pre-ground to powder in liquid nitrogen) using the RNeasy Plant Mini Kit (QIAGEN, Hilden, Germany), purified from genomic DNA (RNase free DNasy set; QIAGEN, Hilden, Germany), qualified by gel electrophoresis, and used for first-strand cDNA synthesis (GoScript Reverse Transcription System; Promega, Madison, WI, USA) with an oligo-dT primer. RNA and cDNA concentrations were quantified by fluorimetry (Qubit ® Fluorometer, Thermo Fisher Scientific, Waltham, MA, USA) and qRT-PCR was performed in a CFX96 Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA) with 3.0 ng of cDNA, SYBR Green RT-PCR mixture (Syntol, Moscow, Russia), and specific primers ( Table 5). The following cycling conditions were used: initial denaturation at 95 • C for 5 min and 40 cycles of denaturation at 95 • C for 15 s and annealing/extension at 60 • C for 40 s.
AsCHI gene expression was normalized using two reference garlic genes, GAPDH [69] and UBQ [70], and qRT-PCR results were statistically analyzed with Graph Pad Prism version 8 (GraphPad Software Inc., San Diego, CA, USA; https://www.graphpad.com/ scientific-software/prism/, accessed on 12 December 2020). The data were expressed as the mean ± standard deviation (SE) based on three technical replicates of three biological replicates for each combination of cDNA and primer pairs. The unequal variance (Welch's) t-test was applied to assess differences in gene expression; p < 0.05 was considered to indicate statistical significance.

Promoter and 5 -UTR Identification and Analysis
The regulatory regions of chitinase genes were amplified by PCR using specific primers designed based on A. sativum cv. Ershuizao transcriptome sequence (Table 5), purified, cloned, and sequenced. The search of specific cis-elements in promoters and 5 -UTRs (1.0 kb regions upstream of the initiation codon) was performed using the PlantCARE database, which provides an evaluation of cis-regulatory elements, enhancers, and repressors [71]; (http: //bioinformatics.psb.ugent.be/webtools/plantcare/html/; accessed on 25 January 2021).

Conclusions
We identified and characterized seven genes encoding class I chitinases in A. sativum cv. Ershuizao and cloned AsCHI2, AsCHI3, AsCHI5, and AsCHI7 homologues from two garlic cultivars resistant and susceptible to Fusarium rot. The chitinase gene promoters contained hormone-and stress-responsive elements, including those associated with responses to fungal pathogens and their elicitors, suggesting that the putative garlic chitinases participate in local acquired resistance, whereas AsCHI2, AsCHI3, and AsCHI7 may be involved in systemic acquired resistance. Upon Fusarium attack, all AsCHI genes can be induced by ET, whereas AsCHI1 and AsCHI7 may be activated by ABA and auxin, respectively. AsCHI1-7 transcriptional profiling in resistant and susceptible garlic cultivars infected with F. proliferatum suggests that the expression of AsCHI2, AsCHI3, AsCHI5, and AsCHI7 genes could define the difference in chitinase-mediated response to Fusarium infection between resistant and susceptible plants. Our results provide useful insights into the functions of class I chitinases in A. sativum and Allium plants in general, and may be used in breeding programs to increase the resistance of Allium crops to Fusarium infections.