Genome-wide identification and annotation of HMs genes
A total of 117 putative HMs genes were identified in the grape genome having 48 HMTs, 22 HDMs, 33 HATs, and 14 HDACs genes. According to previous studies [31-33], all of the genes were divided into 11 subfamilies based on the conserved domains. HMTs have 43 SDG and 5 PRMT genes; HDMs contain 15 JMJ and 7 HDMA genes; HATs have 28 HAG, 2 HAM, 2 HAC, and 1 HAF gene; and HDACs have 10, 2, and 2 genes of HDA, SRT, and HDT, respectively. These genes were named according to their families and their position on chromosomes. The predicted length of proteins in the SDG, PRMT, JMJ, HDMA, HAG, and HDA subfamilies ranges from 184-2199, 337-651, 131-1905, 492-984, 157-667, and 349-698 amino acids, respectively. Detailed information about genes including accession number, gene ID, chromosomal position, length of coding sequence, and length of protein sequence are presented in Table 1.
Phylogenetic and exon-intron analysis
To illustrate the phylogenetic history and to classify the HMs gene into different subfamilies, phylogenetic trees were constructed between Arabidopsis and grapevine HMs protein sequences (Fig. 1). Moreover, to deeply study the phylogenetic realtionships unrooted trees were generated among VvHMS genes of same group, and intron-exon analysis was performed (Figure 2). For generation of phylogenetic trees among VvHMS genes of same group, we followed the previous trend. Phylogenetic trees were constructed only among members of three well studied subfamilies.
All 43 SDG genes were divided into seven classes on the basis of phylogenetic tree constructed between Arabidopsis and grape (Fig. 1A). Class V was further subdivided into subclass I and subclass II. Class I was found more conserved with respect to gene numbers, as grape and Arabidopsis contained same; 3 number of genes. Further, different classes contained different number of exons but almost same number of exons with in the same class e.g. class I. However, all five members of PRMTs contained different number of exons, suggesting that PRMTs class is less conserved. Same number of HMTs genes were found in Arabidosis and grapevine. These results suggest that HMTs family is more conserved as compared to other families.
A phylogenetic tree was constructed between JMJ proteins of grapevine and Arabidopsis. According to the Fig. 1B, JMJ grapevine and Arabidopsis proteins were clustered into 4 and 5 different clades, respectively. Class I (JMJ- domain only), has only one member which is from Arabidopsis. Class II (KDM3), class III (KDM4), and class V (JMJD6) all have equal number of proteins in both species 6, 3, and 2, respectively. However, class IV (KDM5) contained 5 and 3 proteins of Arabidopsis and grapes, respectively. These results suggest that class II, III, and V are more conserved with respect to protein number. Further, exon-intron distribution and size was not same in the same class. However, all duplicated gene pairs have similar exon-intron structure (VvJMJ2/VvJMJ13, VvJMJ4/VvJMJ11, and VvJMJ12/VvJMJ14), with multiple exons and introns, but have difference in size and distribution of exons. Grapevine and Arabidopsis contained 7 and 4 HDMAs proteins, respectively.
VvHATs and VvHDACs:
In HATs family, HAG subgroup contained the highest (28) number of genes among all groups. In HATs group more difference in number of genes was noticed between grape and Arabidopsis as compared to other groups, suggesting that HATs group have underwent more changes during the process of evolution. Tandemly duplicated gene pairs (VvHAG3/VvHAG4 and VvHAG7/VvHAG8) showed conserved exon-intron distribution. Moreover, the HAG proteins have high bootstrap values, suggesting high similarity with each other. According to the phylogenetic analysis (Fig. 1C), genes of VvHDA were further divided into three classes. Interestingly, class III contained only 2 members, one from each grape and Arabidopsis.
GO (Gene ontology) analysis of HMs
We performed in silico GO analysis of grapevine genes at biological, cellular, and molecular level (Additional file 2: Fig. S1). Prediction of biological process suggested the involvement of HMs genes in regulation of gene expression, cellular component organization, flower development, response to biotic and abiotic stimulus, protein modification processes, and transcription process. Most of the HMs proteins were found in cytoplasm, nucleus, and intracellular organelles. HMs genes might have a role in binding (chromatin, DNA, and protein) transcription and transferase regulation activities at molecular level.
Chromosomal distribution and expansion pattern of HMs genes in grapevine
All 117 HMs genes were unevenly distributed on 20 chromosomes (Fig. 3). Chromosome seven contained the highest number of genes (11) while chromosome nineteen contained the lowest only one gene. The 43 VvSDG genes were randomly distributed on fourteen chromosomes, because chromosomes 2, 3, 6, 9, 17, and 19 has no genes. Four chromosomes 1, 4, 16, and 18 contained equal number of genes; five. These four chromosomes contained almost 50% of genes. For all other gene families, same trend of uneven distribution was noticed (Fig. 3). Tandem duplication was calculated according to the criteria described in materials and methods.
Only three events of tandem duplication (VvHAG3/VvHAG4, VvHAG7/VvHAG8, and VvSDG39/VvSDG40) were observed among six genes on three chromosomes. Two duplication pairs belonged to HAG family, suggesting that this family is less conserved during the evolutionary process. We also calculated the segmental duplication among HMs grapevine genes. Total 15 genes underwent segmental duplication in the form of eight pairs; VvSDG4/VvSDG30, VvSDG11/VvSDG35, VvSDG29/VvSDG15, VvSDG31/VvSDG15, VvJMJ2/VvJMJ13, VvJMJ11/VvJMJ4, VvJMJ12/VvJMJ14, and VvHDT1/VvHDT2. Interestingly, VvSDG15 paired with two genes, VvSDG29 and VvSDG31. These results suggest that both tandem and segmental duplication (21%) events have played a role in the expansion of HMs genes. These results can provide clues for evolutionary history and functional analysis. The ratio between Ka and Ks provides help to describe the evolutionary process . Furthermore, Ka/Ks ratios of all duplicated genes were less than 1, suggesting that all genes have purifying selection pressure (Additional file 3: Table S2).
Evolutionary relationships between grapes and Arabidopsis
The roles of HMs genes have been well studied in model plants e.g. Arabidopsis and rice. Therefore, to gain insights about the origin and potential functions of HMs genes in grapevine and to explore the evolutionary history, we performed synteny analysis of grapes and Arabidopsis.
As shown in Fig. 4, a total of 69 orthologous pairs were identified between HMs genes of Arabidopsis and grapes comprising of 24, 3, 7, 3, 18, 2, 1, 3, 2, and 6 pairs of SDGs, PRMTs, JMJs, HDMAs, HAGs, HAMs, HACs, HDAs, SRTs, and HDTs, respectively (Additional file 4: Table S3). However, HAFs genes have no syntenic block. There were 24 syntenic blocks of SDG genes having 21 Arabidopsis and 19 grapevine genes. Moreover, there were four pairs of SDG, two pairs of PRMT, one pair of JMJ, four pairs of HAG, and two pairs of HDT, where a single grapevine gene paired with more than one gene of Arabidopsis. There were also five HMs Arabidopsis (3 SDG and 2 HDT) genes, which formed pair with two grapevine genes. All of the pairs contained members from the same subfamily, suggesting that they have a common origin before speciation.
Transcriptomic analysis of HMs genes during seed development
To get some clues about the potential roles of HMs grapevine genes in seed development, transcriptomic analysis of HMs grapevine genes was performed (Fig. 5 and Additional file 5: Table S4). The samples were taken at three different stages of seed development from seeded and seedless progenies. Most of the HMs genes were highly expressed during successive stages of seed formation in both seeded and seedless progenies. However, most of the HDA family members were expressed relatively very low. There were some exceptions as well, for example, VvJMJ14 showed up-regulation during the later stages of seed development in seeded progenies as compared to seedless. In contrast, VvSDG30, VvHDA1, and VvHAG23 were highly expressed during all stages of seed development in seedless cultivars, suggesting their potential role in ovule abortion. Therefore, we speculate that HMS genes have potential roles in seed development and ovule abortion. We observed that some of the grapevine HMs gene pairs generated from duplication events showed similar expression patterns in gene pairs. For example, most of the duplicated gene pairs of JMJ family were conserved with respect to exon-intron distribution and expression pattern. However, there were some exceptions as well, VvSDG4/VvSDG30 and VvSDG15-VvSDG29/VvSDG31 (Segmentally), i.e. VvSDG4 and VvSDG30 has significantly different expression during progressive stages of seed development. Further with regard to duplicated genes, JMJ subfamily is more conserved as compared to SDG subfamily.
Expression pattern of HMs genes during different stages of seed development in different grape cultivars
To verify the reliability of transcriptome data, and to explore whether the expression pattern of VvHMs genes is widespread in different grape varieties, we selected 15 HMs genes and analyzed the expression pattern in different seeded (‘Kyoho’ and ‘Muscat kyoho’) and seedless (‘Flame seedless’ and ‘Crimson seedless’) grape cultivars during successive stages of seed development. The genes were selected according to the results of transcriptome data of seed development (Fig. 5). These genes were comprised of representatives of all subfamilies including differently (VvHAC1, VvHAG23, VvHAM2, VvHDA1, VvHDA8, VvHDT1, VvJMJ14, VvJMJ15, VvSDG20, VvSDG32, and VvSDG38 ) and ubiquitously (VvHAG4, VvHDMA4, VvPRMT4, and VvSRT2) expressed genes during different stages of seed development in seeded and seedless cultivars. As shown in Fig. 6, VvJMJ14, VvJMJ15, and VvSDG38, were significantly highly expressed during later stages of seed development in seeded cultivars. VvSDG20 showed significantly higher expression during all stages of seed development in seeded cultivars compared to seedless ones. Further, the change ratio between seeded and seedless cultivars was relatively higher in VvSDG20 as compared to other differentially expressed genes. Based on the results, we speculate that these genes might have a role in seed development. In contrast to these results, some genes were signficantly highly expressed in some stages of seed formation in seeedless cultivars, for example, VvHAG23, VvHDA8, and VvPRMT4. Moreover, VvHAG4 showed remarkable difference in the expression pattern during all stages of seed development in seedless cultivars than seeded ones. These results suggest that these genes might have a role in ovule abortion. In general, the results of qRT-PCR are consistent with the results of transcriptome.
Expression profiling of HMs genes in response to E. necator inoculation and hormone treatments
To investigate the potential role of HMs genes against powdery mildew infection, we performed qRT-PCR analysis of fifteen HMs genes (same genes as for seed study) after infecting ‘Shang-24’ with Erysiphe necator. At 12 hpi the expression of three genes (VvHAC1, VvHAG4, and VvHAG23) was significantly up-regulated but subsequently down-regulated at 24 hpi (Fig. 7). However, the expression of VvHAM2, VvHDA1, VvPRMT4, VvHDT1, VvSDG38, and VvSRT2 was down-regulated at 12 hpi, and up-regulated at 24 hpi. Moreover, some of the genes (VvJMJ14, VvJMJ15, and VvSDG32) showed no significant difference in expression between treated and control plants. These results suggest that some of the HMs genes are responsive to powdery mildew and might have a role in resistance mechanism. We also checked the responses of 15 HMs grapevine genes to ABA, ethylene, JA, and SA applications. In plants treated with ABA, most of the genes were down regulated at 12 hpi (Fig. 8). But VvHAC1, VvHAG23, SDG20, and VvSRT2 genes were significantly up-regulated at 12 hpi. VvHAG4, VvJMJ14, and VvSDG20 showed significantly differential expression at all-time points. However, VvSDG38 gene showed same expression in control and treated plants at all-time points, suggesting that, except this all other genes have link with ABA regulation in grapevine. For JA treated plants, VvHAC1, VvHAG4, VvHDA1, VvHDA8, VvJM14, and VvSDG32 showed significant up-regulation at all-time points. However, VvHAG23 and VvHDMA4 showed significant up-regulation only at 1 hpi. Further, VvHDT1 was significantly down-regulated at 6 and 12 hpi after JA treatment (Fig. 9). In SA treated plants, VvHAC1, VvHAG23, VvHAM2, VvHDA1, VvHDA8, VvHDMA4, VvJMJ14, VvSDG20, and VvSDG32 gene were significantly up-regulated at 1 hpi and most of these genes were down-regulated at 12 hpi (Fig. 10). Furthermore, seven genes (VvHAC1, VvHAG4, VvHAG23, VvHAM2, VvHDA1, VvSDG32, and VvSRT2) showed significantly higher expression at 12 hpi in ethylene treated plants as compared to control (Fig. 11). Moreover, the expression of VvHAG23, VvHAM2, and VvHDA1 was similar with each other at all-time points i.e. same expression at 1 hpi, down-regulation at 6 hpi and subsequent up-regulation at 12 hpi. However, VvJMJ15 and VvSDG20 showed significant down-regulation at 12 hpi. These results suggest that most of the HMs genes have role in defense mechanism of grapevine in response to E. necator inoculation and have involvement in hormone pathways.