1.1 Effect of heterogeneous grafting on survival rate
The potato was grafted using the "split grafting method". Seven days after grafting, the survival rate of self-root grafting was 100%, and the survival rates of heterogeneous graft Q/F and F/Q were 93.9% and 90.9%, respectively (Table 1).
Table 1. Statistics of potato graft survival rate
|
Graft combination
|
Number of plants
|
Deaths/strain
|
Survival rate/%
|
1
|
Q/F
|
33
|
2
|
93.9
|
2
|
F/Q
|
33
|
3
|
90.9
|
3
|
F/F
|
33
|
0
|
100
|
4
|
Q/Q
|
33
|
0
|
100
|
1.2 Resistance identification results of potato late blight after grafting
Thirty-five days after grafting, in vitro leaf grafting was inoculated on F/F, F/Q, Q/Q, and Q/F scion leaves. The control group had ungrafted F and Q seeded on the same day, and the resistance statistics were performed on the seventh day. The ungrafted F resistance index was susceptible (S), and the self-root grafted F/F resistance index was also S. However, the disease index was slightly lower than F: 76.6, which was 56.7. Compared with F/F and F, the resistance index of F/Q had been improved, which was due to medium resistance (MR). Hence, the ungrafted Q resistance index had high resistance (HR), and the self-root grafted Q/Q resistance index was also of HR. Compared to Q and Q/Q, the Q/F resistance index decreased to disease resistance (R). (Table 2)
Table 2. Statistics on the effects of grafting on potato late blight resistance
Grafting combination
|
Total grafting/strain
|
Number of outbreaks/strain
|
Incidence /%
|
Illness index
|
Resistance index
|
F
|
30
|
5
|
16.7
|
76.6
|
S
|
F/F
|
30
|
4
|
13.3
|
56.7
|
S
|
F/Q
|
30
|
4
|
13.3
|
33.3
|
MR
|
Q
|
30
|
1
|
3.3
|
3.3
|
HR
|
Q/Q
|
30
|
1
|
3.3
|
3.3
|
HR
|
Q/F
|
30
|
2
|
6.6
|
11.1
|
R
|
Note: (1) I: Immune, DI = 0; (2) HR: High resistance, 0 ≤ DI ≤ 10; (3) R: Disease resistance, 10 ≤ DI ≤ 30; (4) MR: Medium resistance, 30 ≤ DI ≤ 50 (5) S: Suffering from illness, DI > 50
1.3 Transcriptome sequencing and assembly
Based on the transcriptome sequencing and data assembly analysis of the high-throughput sequencing platform (IlluminaHiseq 4000), the third leaves from the top of the grafted combination F/Q, Q/F, and ungrafted F, Q, were sequenced separately. Raw reads of 6.5 × 107, 5.7 × 107, 6.1 × 107, 5.5 × 107 were obtained, and the original sequences were filtered to obtain 6.3 × 107, 5.6 × 107, 6.1 × 107, and 5.4 × 107 clean reads. The data error rates were 0.03%; Q20 was 97.90%, 97.96%, 98.02%, and 97.54%, and GC content was 42.38%, 42.31%, 42.24%, and 42.18%. After obtaining clean reads, the clean reads were aligned to the reference genome sequence by Hierarchical Indexing for Spliced Alignment of Transcripts (HISAT). The average ratio of each sample was 87.61%, 86.09%, 82.43% and 85.78%. The uniform ratio between samples indicated that the clean reads data between samples were comparable. To conclude, the transcriptome sequencing results were credible.
1.4 Screening of differentially expressed genes (DEGs)
DEGs with a gene expression fold > 1 and a p-value of < 0.05 after multiple checks were selected for screening in both groups. A total of 8022 DEGs were screened (Fig. 1). By comparing F/Q with F and Q/F with Q, it was found that F/Q versus F had 3152 genes up-regulated and 4389 genes down-regulated; Q/F versus Q had 329 genes up-regulated and 151 genes down-regulated.
1.5 Differential Gene Ontology analysis
According to the Gene Ontology (GO) classification display (Fig. 2), GO functional analysis mainly included three categories, which were used to describe the biological process (BP), cellular component (CC), and molecular function (Molecular function, MF). In F/Q versus F, BP mainly annotated genes of photosynthesis. Differential genes in CC were higher in thylakoids, photosynthetic membranes, and photosynthetic systems. Molecular function was mainly concentrated in enzyme inhibitor activity, hydrolase activity, hydrolyzed O-glycosyl compounds, endopeptidase modulator activity, endopeptidase inhibitor activity, peptidase modulator activity, and in the peptidase inhibitor activity gene. Among them, the up-regulated genes were mainly enriched in cell recognition, pollination, pollen pistil interaction, multicellular biological processes, pollen recognition, etc. in BP, as well as ubiquitin protein transferase activity, ubiquitin-like protein transferase activity, peptidase activity, pattern binding, and polysaccharide binding genes in MF. Down-regulated genes were enriched in photosynthesis, translation, peptide biosynthesis processes, amide biosynthesis processes in BP, ribosomes, thylakoids, thylakoid parts, photosynthetic membranes, photosystems, and ribosomal structural components, structural molecule activity, enzyme inhibitor activity, hydrolase activity, genes acting on glycosyl bonds. In Q/F versus Q, BP mainly concentrated on response to injury and stress. The differential genes of CC were concentrated in the cell wall and external packaging structure. MF was mainly annotated to enzyme inhibitor activity, enzyme regulation activity, endopeptidase inhibitor activity, peptidase inhibitor activity, regulating molecular function and other genes. Among them, the up-regulated genes were mainly enriched in the two types of genes corresponding to stress and protein folding of BP, apoplasts, extracellular regions, cell walls, external packaging structures in CC, folding protein binding, transferase activity, and transfer in MF Alkyl or aryl (except methyl) genes.
1.6 Differential gene KEGG analysis
The KEGG pathway analysis was performed on the differential genes in the two grafted combinations F/Q, Q/F scions and the non-grafted F and Q leaves of the control group (Fig. 3). The results showed that among the top 20 pathways with the highest enrichment, when F/Q was compared with F, the KEGG pathway, enriched by differentially up-regulated genes, mainly included the mRNA monitoring pathway, autophagy-other, glutathione metabolism, plant-pathogen interaction, plant hormone signal transduction, nitrogen metabolism, and the MAPK signaling pathway. Down-regulated differential genes were mainly enriched in ribosomes and photosynthesis-antenna protein, and involved in photosynthesis, phenylpropane biosynthesis, cyanoamino acid metabolism, steroid biosynthesis, Pentose, and glucuronide conversion. Compared with Q and Q/F, the up-regulated differential genes were mainly concentrated in the protein processing pathway in the endoplasmic reticulum. Down-regulated differential genes were involved in fatty acid elongation, arginine and proline metabolism, and diterpenoid biosynthetic pathways.
1.6.1 Effect of grafting on gene expression related to plant-pathogen interactions
The KEGG pathway analysis showed that 36 plant-pathogen interaction-related genes in the grafting combination F/Q scion were up-regulated compared to leaves in the same part of F. Among them were ten calcium-dependent protein kinases (CDPKs), five cyclic nucleotide-gated ion channel proteins (CNGC), three chitin trigger receptor receptor kinases (CERKs), two LRR receptor serine/Threonine protein kinases (LRR-LRKs), and two WRKY transcription factors. However, no such gene was found in the results of the KEGG analysis of the differential genes between the graft combination Q/F and its control Q.
1.6.2 Effects of grafting on the gene expression of MAPK signaling pathways in plants
The KEGG pathway analysis showed that 31 MAPK signal pathway-related genes in the grafting combination of the F/Q scion were up-regulated compared to leaves in the same part of F. Among these were six mitogen-activated protein kinases (MAPKs), five ethylene-insensitive proteins, three abscisic acid receptors PYL. There were no significant differences between the grafted combination of Q/F and the non-grafted Q, in the genes of this pathway
1.6.3 Effects of grafting on the expression of plant hormone signaling related genes
The jasmonate (JA) pathway and the salicylic acid (SA) pathway were the two main pathways involved in plant-induced defense. In F/Q versus F, there were 19 differential genes enriched to the JA pathway, of which four were up-regulated genes and 15 were down-regulated genes. In F/Q versus F, there were 19 differential genes enriched to the JA pathway, of which four were up-regulated genes and 15 were down-regulated genes. There were four JA synthetases in the up-regulated genes and 12 TIFY family genes in the down-regulated genes. There were 37 differential genes enriched in the SA pathway, of which 36 were up-regulated genes; one was a down-regulated gene, and six were up-regulated genes, NPR family genes, 13 were TGA family genes, and 17 were disease-associated proteins.
1.6.4 Effect of grafting on the expression of fatty acid chain elongation related genes
Very long chain fatty acids (VLCFAs) refer to fatty acids with a hydrocarbon chain length of more than 18 carbon atoms. VLCFAs are not only the main components of the structure of biological membranes, but also participate in life activities in the body, as signal molecules. They play a significant role in the growth, development, and resistance to the external environment. During the synthesis of VLCFAs, the first condensation reaction is a rate-limiting step. The reaction is catalyzed by β-ketoacyl CoA synthetase, which is encoded by the 3-ketoacyl-CoA synthase (KCS) gene, which determines the carbon chain length [20]. The down-regulated differential genes in the grafted combination Q/F and ungrafted Q leaves are mainly concentrated in the fatty acid elongation pathway, and all 29 are KCS family genes. However, no significant differential expression of this pathway gene has been found in F/Q versus F.