ISSR analysis of genomic DNA
Twelve primers with clear amplification bands, good polymorphism and stability were screened from 28 ISSR primers for PCR amplification (Table 4). The ISSR amplification results showed that there were a large number of ISSR markers and abundant polymorphisms (Fig. 2 and S1). A total of 71 bands ranging from 0.15 to 2.0 kb were amplified by 12 primers, with an average of 5.92 markers per primer, of which 64 were polymorphic markers, and the polymorphism rate was 90.14%. The polymorphism of different primers varied, and the number of polymorphic bands ranged from 3 to 9. Repeated experiments showed that ISSR markers had strong stability and abundant polymorphic bands. The Jaccard similarity coefficient of the tested materials was between 0.83 and 1[32]. ISSR analysis was performed on the leaves of susceptible and nonsusceptible plants and the DNA of floral organs with different degrees of susceptibility. The results showed that ISSR bands could be amplified from the genomic DNA of leaves and floral organs of the tested materials, and each pair of primers generated approximately seven bands. The genetic similarity coefficient analysis indicated that there was no significant difference in the genomic DNA sequence of leaves and floral organs between susceptible and nonsusceptible plants, suggesting that the disease did not cause changes in the genomic DNA sequence of plants.
Table 4
Annealing temperature and amplification results of the 12 primers
Primer | Sequence | Number of loci | Number of polymorphic loci | Polymorphic proportion |
810 | GAG AGA GAG AGA GAG AT | 6 | 6 | 100.00 |
813 | CTC TCT CTC TCT CTC TT | 9 | 8 | 88.89 |
816 | CAC ACA CAC ACA CAC AT | 6 | 5 | 83.33 |
817 | CAC ACA CAC ACA CAC AA | 5 | 5 | 100.00 |
818 | CAC ACA CAC ACA CAC AG | 6 | 6 | 100.00 |
819 | GTG TGT GTG TGT GTG TA | 8 | 8 | 100.00 |
820 | GTG TGT GTG TGT GTG TC | 5 | 5 | 100༎00 |
821 | GTG TGT GTG TGT GTG TT | 4 | 4 | 100.00 |
823 | TCT CTC TCT CTC TCT CC | 7 | 5 | 71.42 |
824 | TCT CTC TCT CTC TCT CG | 6 | 5 | 83.33 |
825 | ACA CAC ACA CAC ACA CT | 4 | 3 | 75.00 |
826 | ACA CAC ACA CAC ACA CC | 5 | 4 | 80.00 |
Total | | 71 | 64 | |
Average | | 5.92 | 5.33 | 90.14 |
MSAP analysis of DNA methylation in leaves and floral organs of susceptible and nonsusceptible plants
Both HpaII and MspI are restriction enzymes that recognize the same site, CCGG, but they differ in their sensitivity to methylation sites. HpaII cannot digest the sites containing mCCGG, CmCGG and mCmCGG, but it can recognize the sites where only one cytosine is methylated, while MspI can recognize the internal cytosine methylation on single or double strands, that is, it cannot digest the sites containing mCCGG. Because the two enzymes have different reactions to cytosine methylation at this site, the polymorphic fragment amplified by PCR reflects the methylation status and degree of the modified site. There may be four types of fragments (Table 5). The total number of amplified bands was the sum of the bands of types I, II and III, and the total number of methylated bands was the sum of the bands of types II and III. The total methylation ratio was calculated as the number of bands of type III divided by the total number of amplified bands, and the semimethylation ratio was calculated as the number of bands of type II divided by the total number of amplified bands. The MSAP ratio was calculated as the total number of methylated bands divided by the total number of amplified bands.
Table 5
Methylation status of CCGG loci based on differential sensitivity to both isoschizomers
Types | E + M band patterns | E + H band patterns | CCGG pattern | CCGG methylation pattern |
Ⅰ | 1 | 1 | CCGG GGCC | Unmethylated |
Ⅱ | 0 | 1 | 5mCCGG GGCC | lateral side of single-stranded DNA (Hemi-methylation) |
Ⅲ | 1 | 0 | C5m CGG GGC5m C | medial side of double stranded (Full-methylation) |
Ⅳ | 0 | 0 | 1. 5m C5m CGG GGC5m C5m 2. 5m CCGG GGCC5m 3. CCGG | 1. Double-chain medial and lateral cytosine methylation 2. Double-stranded extrinsic cytosine methylation 3. absence of CCGG site |
Type I: the banding of DNA sample lane by Hpa II digestion, and the banding of DNA sample lane by Msp I digestion, which is unmethylated; Type II: Hpa II digested DNA sample lane with band, Msp I digested DNA sample lane without band, this type is hemimethylation; type III: the use of Hpa II enzyme digestion DNA sample lane without band, the use of Msp I digestion DNA sample lane with band, this type is full methylation; type IV: no band, there are three cases, representing the presence of double-stranded methylation, double-stranded methylation or no CCGG sequence.
Preamplification and selective amplification of genomic DNA
The genomic DNA of cherry susceptible and nonsusceptible plants was digested with EcoRI/HpaII and EcoRI/MSpI enzyme systems, and the digestion products were detected by 1% agarose gel electrophoresis. After 6 h of digestion, the DNA was completely digested without the main band, and the fragments were concentrated in 100–5000 bp. The digested products were ligated for 12 h, and the preamplification reaction was carried out according to the preamplification reaction system. The products were detected by 1% agarose gel electrophoresis. The preamplification effect was good, and the amount of preamplification products was large, mainly concentrated in the 100–2000 bp range (Fig. 3 and S2). A total of 16 pairs of primers were selected for selective amplification. Among them, 12 pairs of primers had good amplification effects, clear and abundant bands, and good amplification repeatability. Each pair of primers showed a certain degree of amplification polymorphism( Fig. 4,S3 and S5).
Analysis of genomic DNA methylation in susceptible and nonsusceptible leaves
We used MSAP to compare the genomic DNA methylation patterns of susceptible and nonsusceptible leaves. We obtained 671 fragments from 12 primer pairs, of which 97 were fully methylated, 85 were semimethylated and 182 were methylated (Table 6). The total methylation rate was 27.30% in susceptible leaves and 26.96% in nonsusceptible leaves. The full methylation rate was 18.40% in susceptible leaves and 10.72% in nonsusceptible leaves. The semimethylation rate was 8.89% in susceptible leaves and 16.23% in nonsusceptible leaves. These results indicated that susceptible leaves had slightly higher levels of total and full methylation and lower levels of semimethylation than nonsusceptible leaves.
We classified the MSAP fragments into four types according to the differences in methylation patterns between susceptible and nonsusceptible leaves: Type A, monomorphic loci, where both leaves had the same methylation status, either single-stranded (A1) or double-stranded (A2); Type B, demethylation loci, where nonsusceptible leaves were methylated but susceptible leaves were demethylated; Type C, hypermethylation loci, where susceptible leaves had higher methylation levels than nonsusceptible leaves; and Type D, hypomethylation loci, where susceptible leaves had lower methylation levels than nonsusceptible leaves but still had some methylation (Fig. 5,S4 and S5).
We identified 14 different MSAP fragment types (Table 7). Among them, type A accounted for 12.8% (19 fragments), type B accounted for 16.8% (25 fragments), type C accounted for 57.6% (86 fragments) and type D accounted for 12.8% (19 fragments). In type A, A2 (double-stranded methylation) was more frequent than A1 (single-stranded methylation), accounting for 57.9%. (Table 8)
Table 6
Genomic DNA methylation of pathological and healthy Chinese cherry plants
| LCK | L1 |
Total number of amplified bands | 345 | 326 |
Full-methylation | 37 | 60 |
Hemi-methylation | 56 | 29 |
Total methylation | 93 | 89 |
Full-methylation rate | 10.7% | 18.4% |
Hemi-methylation rate | 16.2% | 8.9% |
Total methylation rate | 26.9% | 27.3% |
Table 7
Number of different methylation type bands of genomic DNA of pathological and nonpathogenic plants Type A: monomorphic site, type B: demethylation type, type C: hypermethylation type, type D: hypomethylation type.
Types | LCK | L1 |
A | 2 | 2 |
B | 3 | 3 |
C | 6 | 6 |
D | 3 | 3 |
Total | 14 | 14 |
Table 8
Different methylation patterns in infected cherry and healthy cherry
Types | Patterns of band | Numbers of polymorphic bands | Number of polymorphic | Polymorphism rate | Number of total loci |
| LCK | L1 | 2 | | | |
M | H | M | H |
A1 | 1 | 0 | 1 | 0 | 8 | 19 | 12.8% | 149 |
A2 | 0 | 1 | 0 | 1 | 11 | | | |
B1 | 0 | 0 | 1 | 1 | 2 | 25 | 16.8% | |
B2 | 0 | 1 | 1 | 1 | 10 | | | |
B3 | 1 | 0 | 1 | 1 | 13 | | | |
C1 | 0 | 1 | 0 | 0 | 13 | 86 | 57.6% | |
C2 | 1 | 0 | 0 | 0 | 20 | | | |
C3 | 1 | 1 | 0 | 0 | 10 | | | |
C4 | 1 | 1 | 1 | 0 | 4 | | | |
C5 | 1 | 1 | 0 | 1 | 21 | | | |
C6 | 1 | 0 | 0 | 1 | 18 | | | |
D1 | 0 | 0 | 1 | 0 | 10 | 19 | 12.8% | |
D2 | 0 | 0 | 0 | 1 | 6 | | | |
D3 | 0 | 1 | 1 | 0 | 3 | | | |
DNA methylation analysis of susceptible and nonsusceptible floral organs
We performed MSAP analysis to compare the DNA methylation patterns of susceptible and nonsusceptible floral organs. We used 12 primer pairs and obtained 674 fragments, of which 96 were fully methylated, 79 were hemimethylated and 175 were nonmethylated (Table 9). The global methylation level was 28.32% in susceptible floral organs and 23.58% in nonsusceptible floral organs. The fully methylated fragments accounted for 17.99% in susceptible floral organs and 11.04% in nonsusceptible floral organs. The hemimethylated fragments accounted for 10.32% in susceptible floral organs and 12.53% in nonsusceptible floral organs. These results suggested that susceptible floral organs had higher levels of total and full methylation and lower levels of hemimethylation than nonsusceptible floral organs.
We classified the MSAP fragments into four categories based on the differences in methylation patterns between susceptible and nonsusceptible floral organs: Type A, monomorphic fragments, where both floral organs had the same methylation status, either single-stranded (A1) or double-stranded (A2); Type B, demethylated fragments, where nonsusceptible floral organs were methylated but susceptible floral organs were not; Type C, hypermethylated fragments, where susceptible floral organs had higher methylation levels than nonsusceptible floral organs; and Type D, hypomethylated fragments, where susceptible floral organs had lower methylation levels than nonsusceptible floral organs but still had some methylation (Fig. 6 and S3). We identified 14 different MSAP fragment types (Table 7). Among them, type A represented 19.1% (29 fragments), type B represented 29.6% (25 fragments), type C represented 34.9% (86 fragments) and type D represented 16.4% (19 fragments). In type A, A2 (double-stranded methylation) was more prevalent than A1 (single-stranded methylation), representing 55.2%. (Table 10)
Table 9
Genomic DNA methylation levels of susceptible and nonsympathetic flower organs
| FCK | F1 |
Total number of amplified bands | 339 | 335 |
Full-methylation | 61 | 37 |
Hemi-methylation | 35 | 42 |
Total methylation | 96 | 79 |
Full-methylation rate | 17.99% | 11.04% |
Hemi-methylation rate | 10.32% | 12.53% |
Total methylation rate | 28.32% | 23.58% |
Table 10
Different methylation types of genomic DNA in susceptible and nonpathogenic plants
Types | Patterns of band | Number of polymorphic bands | Number of polymorphic | The ratios of polymorphic | Number of total polymerphicloci |
| FCK | F1 | 4 F1 | | | |
M | H | M | H |
A1 | 1 | 0 | 1 | 0 | 13 | 29 | 19.1% | 152 |
A2 | 0 | 1 | 0 | 1 | 16 | | | |
B1 | 0 | 0 | 1 | 1 | 8 | 45 | 29.6% | |
B2 | 0 | 1 | 1 | 1 | 20 | | | |
B3 | 1 | 0 | 1 | 1 | 17 | | | |
C1 | 0 | 1 | 0 | 0 | 9 | 53 | 34.9% | |
C2 | 1 | 0 | 0 | 0 | 12 | | | |
C3 | 1 | 1 | 0 | 0 | 7 | | | |
C4 | 1 | 1 | 1 | 0 | 14 | | | |
C5 | 1 | 1 | 0 | 1 | 4 | | | |
C6 | 1 | 0 | 0 | 1 | 7 | | | |
D1 | 0 | 0 | 1 | 0 | 16 | 25 | 16.4% | |
D2 | 0 | 0 | 0 | 1 | 9 | | | |
D3 | 0 | 1 | 1 | 0 | 3 | | | |
Genomic DNA methylation analysis of floral organs with different degrees of susceptibility in the same plant
We performed methylation-sensitive amplified polymorphism (MSAP) analysis to compare the genomic DNA methylation patterns of floral organs with different degrees of susceptibility in the same plant. We amplified 1744 fragments from 12 primer pairs, of which 154 were fully methylated, 199 were semimethylated and 353 were methylated (Table 11). The total methylation rate varied from 15.61–25.21%. The full methylation rate ranged from 6.07–11.81%. The semimethylation rate varied from 9.54–13.45%. These results indicated that the total methylation rate and full methylation rate decreased, while the semimethylation rate increased with increasing susceptibility.
Different degrees of susceptibility of the same plant exhibited different methylation types. We identified 14 different methylation types in the DNA of floral organs, which could be classified into 4 methylation difference types, namely, A, B, C and D, as shown in Table 12. The DNA methylation types of floral organs with different degrees of susceptibility in the same plant are shown in Table 13. The results of the pairwise comparison between different degrees of susceptibility are shown in Table 14. We detected a total of 1504 methylation differential sites in different degrees of susceptibility in the same plant, of which 273, 345, 583 and 303 were A, B, C and D methylation types, respectively. The frequency of occurrence was 11.2%, 22.9%, 38.8% and 20.1%. Among all methylation types, the C type accounted for the highest proportion, followed by the B type, D type and A type. These results suggested that a large number of hypermethylation or hypermethylation and demethylation mutations occurred in the genome of the same plant when different symptoms appeared. In addition, the frequency of submethylation mutations was also high.
Table 11
Genomic DNA methylation levels of the same susceptible plants
| F1a | F1b | F1c | F1d | F1e |
Total number of amplified bands | 349 | 330 | 333 | 386 | 346 |
Full-methylation | 41 | 39 | 25 | 28 | 21 |
Hemi-methylation | 47 | 41 | 37 | 41 | 33 |
Total methylation | 88 | 80 | 62 | 69 | 54 |
Full-methylation rate | 11.75% | 11.81% | 7.51% | 7.25% | 6.07% |
Hemi-methylation rate | 13.47% | 12.42% | 11.11% | 10.62% | 9.54% |
Total methylation rate | 25.21% | 24.24% | 18.61% | 17.88% | 15.61% |
a-f indicates that the degree of susceptibility gradually deepened.
Table 12
Number of different methylation types of different genomic DNAs in the same plant
Types | Varieties of band patterns |
5 F1a | 6 F1b | 7 F1c | 8 F1d | 9 F1e |
A | 2 | 2 | 2 | 2 | 2 |
B | 3 | 3 | 3 | 3 | 3 |
C | 6 | 6 | 6 | 6 | 6 |
D | 3 | 3 | 3 | 3 | 3 |
总计Total | 14 | 14 | 14 | 14 | 14 |
A: monomorphic site; B: demethylation type; C: hypermethylation type; D: hypomethylation type.
Table 13
Different methylation types of different genomic DNAs of the same plant
Types | Patterns of band |
| F1a | F1b |
A1 | 1 | 0 | 1 | 0 |
A2 | 0 | 1 | 0 | 1 |
B1 | 0 | 0 | 1 | 1 |
B2 | 0 | 1 | 1 | 1 |
B3 | 1 | 0 | 1 | 1 |
C1 | 0 | 1 | 0 | 0 |
C2 | 1 | 0 | 0 | 0 |
C3 | 1 | 1 | 0 | 0 |
C4 | 1 | 1 | 1 | 0 |
C5 | 1 | 1 | 0 | 1 |
C6 | 1 | 0 | 0 | 1 |
D1 | 0 | 0 | 1 | 0 |
D2 | 0 | 0 | 0 | 1 |
D3 | 0 | 1 | 1 | 0 |
Table 14
Different planting genomic DNA different methylation types in the same plant
Types | Numbers of polymorphic bands | Number of polymorphic | The ratios of polymorphic | Number of total polymerphicloci |
| F1a F1b | F1a F1c | F1a F1d | F1a F1e | F1b F1c | F1b F1d | F1b F1e | F1c F1d | F1c F1e | F1d F1e | | | |
A1 | 7 | 11 | 11 | 15 | 18 | 17 | 17 | 22 | 20 | 26 | 164 | 273 | 11.2% | 1504 |
A2 | 11 | 8 | 10 | 14 | 16 | 10 | 11 | 10 | 8 | 11 | 109 | | | |
B1 | 5 | 7 | 9 | 9 | 7 | 12 | 15 | 9 | 10 | 10 | 93 | 345 | 22.9% | |
B2 | 16 | 17 | 18 | 21 | 10 | 12 | 12 | 7 | 7 | 12 | 132 | | | |
B3 | 15 | 17 | 14 | 14 | 11 | | 7 | 14 | 13 | 3 | 120 | | | |
C1 | 13 | 16 | 15 | 5 | 10 | 15 | 8 | 7 | 4 | 8 | 101 | 583 | 38.8% | |
C2 | 16 | 15 | 17 | 10 | 13 | 12 | 7 | 11 | 18 | 9 | 128 | | | |
C3 | 8 | 8 | 9 | 14 | 3 | 4 | 6 | 8 | 5 | 3 | 68 | | | |
C4 | 11 | 16 | 10 | 11 | 11 | 7 | 10 | 6 | 9 | 13 | 104 | | | |
C5 | 17 | 7 | 10 | 10 | 4 | 13 | 9 | 14 | 17 | 12 | 113 | | | |
C6 | 11 | 6 | 7 | 10 | 2 | 3 | 13 | 8 | 4 | 5 | 69 | | | |
D1 | 17 | 20 | 16 | 19 | 22 | 16 | 18 | 15 | 20 | 13 | 176 | 303 | 20.1% | |
D2 | 1 | 3 | 6 | 10 | 2 | 7 | 11 | 1 | 15 | 16 | 72 | | | |
D3 | 9 | 8 | 6 | 9 | 4 | 3 | 9 | 1 | 5 | 2 | 55 | | | |
Results of DNA methylation analysis of the genome and floral organs of susceptible and nonsusceptible plants
We compared the differences in DNA methylation levels and patterns of leaves, floral organs of susceptible and nonsusceptible plants and floral organs of the same plant with different degrees of susceptibility. We found that there were some differences in DNA methylation levels and patterns between susceptible and nonsusceptible leaves, floral organs and floral organs of the same plant with different degrees of susceptibility. The total methylation rate and full methylation rate of DNA in leaves and floral organs of susceptible plants were higher than those in leaves and floral organs of nonsusceptible plants, while the semimethylation rate was lower than that of the control. Among the floral organs of the same plant with different degrees of susceptibility, the total methylation rate and semimethylation rate showed a downward trend with increasing susceptibility. Our analysis suggested that the occurrence of phytoplasma disease could cause a decrease in the semimethylation rate of plants, resulting in gene expression variation and plant morphological variation. In addition, we identified 14 different methylation types in the DNA of floral organs, which could be classified into 4 methylation difference types, namely, A, B, C and D. We detected a total of 1504 methylation differential sites in different degrees of susceptibility in the same plant, of which the C type accounted for the highest proportion, followed by the B type, D type and A type. These results indicated that a large number of hypermethylation or hypermethylation and demethylation mutations occurred in the genome of the same plant when different symptoms appeared. In addition, the frequency of submethylation mutations was also high.