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Single-base resolution methylomes of upland cotton (Gossypium hirsutum L.) reveal epigenome modifications in response to drought stress

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Bisulfite treatment of DNA, termed the “gold standard” of DNA methylation research, can convert unmethylated cytosines into uracils while leaving methylated cytosines unchanged, which allows the generation of “methylomes” at single-base resolution [22]. The bisulfite-converted DNA of drought-tolerant cotton variety ZhongH177 under different treatments, including CK, drought (relative water content (RWC) reached approximately 7% in pots) (Fig. 1a), and re-watering, was sequenced with Illumina Hi-Seq instruments at a coverage that provided information on the methylation states of individual cytosines with high confidence. The percentage of methylated cytosines varied depending on the local sequence context (C, CG, CHG and CHH) and the external treatments (CK, drought and re-watering). The results of WGBS are listed in Table 1. We obtained 228,002,142 and 256,948,717 and 253,873,809 reads after CK, drought and re-watering stresses, respectively. In addition, the mapping rate was 65.28, 67.68 and 67.51%, respectively, showing that the method and the results have high reliability and accuracy. The distribution of methylation sites in each chromosome was analyzed (Fig. 1b and Additional file 1: Figure S1, Additional file 2: Figure S2 and Additional file 3: Figure S3). Among the methylated cytosines, we found that more than half of them were located in asymmetric CHH contexts (Table 1). The total mC in the whole genome under three environments (CK, D and re-watering) was 27.99, 32.34 and 29.95%, respectively, showing an up-regulated methylation level after drought stress and a re-down-regulated trend after recovery from drought stress by re-watering compared with CK. The duplication rate (the percentage of repetitive sequences in all clean sequencing reads) increased approximately 2% after drought stress and re-watering (Table 2), which showed that repetitive sequences may be important in response to drought stress through the alterations of the methylation level and state. Interestingly, we found that methylated cytosines in mCpG, mCHG, mCHH contexts all showed a hyper-methylation pattern after drought stress compared with CK and re-watering, but the alterations in the CHH contexts were more significant than the alterations in other contexts. This finding suggested that methylation levels in asymmetric CHH contexts were dynamically changing with external environments and were mostly correlated with environments. In symmetric CG and CHG contexts, more than half of these cytosines were methylated while only a small proportion were methylated in the CHH context (Table 2).

Fig. 1

Epigenome of Gossypium hirsutum L. a Morphological changes of cotyledons under different treatments. b Density plot of 5-methylcytosine in sequence contexts (mCG, mCHG and mCHH where mC signifies 5-methylcytosine, and H represents A, C or T). The circles from the outside to inside represent mCG, mCHG, mCHH, respectively

Table 1

Bisulfite sequencing summary

CK

349,288,166

228,002,142

65.28

201,543,688 (100)

47,879,971 (23.76)

45,874,296 (22.76)

107,789,421 (53.48)

D

379,630,307

256,948,717

67.68

232,876,499 (100)

50,588,873 (21.72)

48,445,561 (20.80)

13,3842,065 (57.47)

Re_W

376,067,028

253,873,809

67.51

215,698,912 (100)

49,367,000 (22.89)

47,336,957 (21.95)

118,994,955 (55.17)

Table 2

Statistical results of methylated cytosines in different contexts

CK

10.70

27.99%

58.42%

53.77%

19.50%

D

12.48

32.34%

61.71%

56.78%

24.21%

Re_W

12.24

29.95%

60.22%

55.48%

21.52%

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