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http://journals.tubitak.gov.tr/biology/

Turkish Journal of Biology Turk J Biol(2015) 39: 396-406© TÜBİTAKdoi:10.3906/biy-1411-61

Whole-genome DNA methylation analysis in cotton (Gossypium hirsutum L.)under different salt stresses

Xuke LU, Xiaojie ZHAO, Delong WANG, Zujun YIN, Junjuan WANG,Weili FAN, Shuai WANG, Tianbao ZHANG, Wuwei YE*

State Key Laboratory of Cotton Biology, Institute of Cotton Research of Chinese Academy of Agricultural Sciences, Anyang, Henan, P.R. China

* Correspondence: yew158@163.com

1. IntroductionNowadays, soil salinization has become a worldwide problem, and salinized land has reached approximately 1 × 109 hm2 around the globe (http://www.fao.org/statistics/en/); unfortunately, the area of soil salinization continues to expand rapidly. Soil salinization is a key factor in sustainable agricultural development. The total available land in China accounts for merely 1.3 × 108 hm2, of which 3.5 × 107 hm2 is salinized land. Even worse, the area of secondary salinization brought about by human activities and improper irrigation modes is on the rise (Yu et al., 2012). Cotton is a pioneering crop that grows on saline and alkaline land; therefore, salt resistance in cotton remains a hot topic in the field, making the research on cotton’s salt resistance significant (Gonzalgo and Jones, 1997b; Xu et al., 2013).

DNA methylation, an important indicator of genetic modification, is not only closely associated with numerous

life events such as the growth and development of animals and plants, plant defense, and adversity stress, but it also plays an important role in regulating various processes in living organisms. Previous studies carried out in plants and animals have demonstrated the link between DNA methylation and genetic/mutagenic effects during cellular activities, including differential expression of genes, cell differentiation, chromatin deactivation, genome imprinting, and canceration (Gonzalgo and Jones, 1997a; Pfeifer, 2006). A study of Arabidopsis thaliana showed that gene transcription is influenced by DNA methylation: short methylated genes are poorly expressed, and loss of methylation in a gene leads to enhanced transcription (Zilberman et al., 2007). Generally, the methylation level of active genes is lower than that of their inactive counterparts (Finnegan et al., 1993; Zhao et al., 2010). Under normal circumstances, 20% to 30% of cytosine

Abstract: Salt stress, one of the most important abiotic stresses, is a serious constraint on cotton production. Cytosine methylation in nuclear DNA, an epigenetic modification found in plants, animals, and other organisms, imparts an impressive wealth of heritable information upon the DNA code. Although the cotton reference genome sequence is available to the public, the global DNA methylation data under different salinity stresses are still not available. Here, Zhong07 and ZhongS9612, salt-tolerant and salt-sensitive cultivars, respectively, were selected and methylation-sensitive amplification polymorphism (MSAP) technology was adopted to evaluate DNA methylation level alterations under different salt stresses in cotton. The findings indicated that different salt stresses exerted distinct effects on cotton seedling growth: specifically, both the neutral salt NaCl and alkalescent salt NaHCO3 showed relatively weak effects, while the alkaline salt Na2CO3 resulted in overt harm to seedlings, significantly darkening their caudexes and roots. MSAP analysis showed that after NaCl, NaHCO3, and Na2CO3 treatments, the DNA methylation levels of both leaves and roots decreased first before rising again. The trend in the roots for both type B (methylation) and C (demethylation) was identical to that observed in leaves; however, methylation levels had a different trend with the varying pH values of the salt, showing that the variation of the methylation level and status were mainly induced by the varying PH values of the salt. The analysis of transition type indicated that the main transition type was from hemimethylation to complete methylation (type iii), accounting for 38.10% of the total transitions, showing that complete methylation played a vital function in the process of gene transcription and expression after the salt treatments. The methylation levels of leaves differed from those of roots, indicating tissue specificity. Target sequence analysis showed that DNA methylation level induced by salt stress involves various kinds of metabolic pathways, whose synergistic effect helps cope with salt stresses.

Key words: DNA methylation, methylation-sensitive amplification polymorphism, salinity, tissue specificity, synergistic effect

Received: 20.11.2014 Accepted/Published Online: 18.01.2015 Printed: 15.06.2015

Research Article

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residues are methylated in the nuclear genome of flowering angiosperms (Richards, 1997; Zhang et al., 2010), mostly in CpG dinucleotide and CpNpG trinucleotide sequences (Gruenbaum et al., 1981). A report by Zhang et al. (2006) indicated that DNA methylation usually occurs within genes; in addition, more than one-third of genes were found to be methylated, with no more than 5% of genes showing methylation in their promoters, which can lead to tissue-specific gene expression. In Arabidopsis thaliana (Ronemus et al., 1996) and tobacco (Tanaka et al., 1997), demethylation of genetically modified plants results in large morphological changes. However, the methylation differences between different cotton genotypes, various tissues, and different metabolic pathways affected by different salt stresses are largely unknown.

At present, there are many methods to assess DNA methylation, which can be divided into three main categories. Methylation-sensitive amplification polymorphism (MSAP) is a classic method (Gonzalgo and Jones, 1997b; Yang et al., 2004; Li et al., 2010). MSAP is an innovative, improved AFLP technology that adopts two restriction enzymes, namely HpaII and MSPI, both of which have different sensitivities to genomic methylation and recognize the nucleotide sequence 5′-CCGG-3′ (Jaligot et al., 2002). HpaII, however, is not sensitive to the complete methylation (double-strand methylation) of single or double cytosine residues and only cuts hemimethylated 5′-CCGG-3′, whereas MSPI does not cut 5mCCGG but does cut C5mCGG (McClelland et al., 1994). This technology has been widely applied to study methylation in Arabidopsis thaliana, rice, maize, and other species (Ashikawa, 2001; Cervera et al., 2002; Zhao et al., 2007) and has become an important method in methylation research.

From the perspective of salt stress, this study used MSAP technology to detect the changes in DNA methylation levels of different cotton varieties and tissues, aiming to preliminarily unfold the possible molecular mechanisms underlying DNA methylation in response to salt stress, providing a theoretical reference for further research on cotton salt tolerance mechanisms.

2. Materials and methods2.1. Plant materials and treatmentsZhong07 and ZhongS9612, a salt-tolerant and a salt-sensitive variety, respectively, were identified by our laboratory and used in this study. Plump and uniform seeds were selected and rinsed with sterile water after sterilization using 0.1% HgCl2, and they were placed in a sterile culture dish to accelerate germination. The seeds that germinated uniformly were selected and planted with the sand culture method in the greenhouse (14 h/day, 30 °C and 10 h/night, 24 °C) of the Institute of Cotton Research of Chinese Academy of Agricultural Sciences.

At the trefoil stage, NaCl, NaHCO3, and Na2CO3 (0.4% of the sand in the plant pools) were used to perform the treatment for 24 h (Minas et al., 2011), while equal amounts of distilled water were added to the controls. Leaves and full-root samples were collected separately, snap frozen with liquid nitrogen, and stored at –80 °C until use. Treatments were carried out in triplicate and DNA extracted from the three replicates was pooled. High-quality genomic DNA was extracted using the improved CTAB method (Porebski et al., 1997) and residual RNA was removed with RNA polymerase (10 mg mL–1). 2.2. MSAP analysisMSAP analysis was performed as described previously (Zhao et al., 2010). The double digestion combination method was used with EcoRI/HpaII and EcoRI/MspI. Adapters, preamplification, and selective amplification primers used are summarized in Table S1 (on the journal’s website). MSAP analysis was carried out in triplicate.

Enzyme digestion was carried out in a 20-µL reaction system (400 ng DNA, 10 U EcoRI, HpaII, and MspI, 3 h). Ligation was performed in a 20-µL reaction system (200 ng enzyme-digested products, 5 pmol EcoRI linker, 50 pmol HpaII-MspI linker, 10 U T4 ligase, 16 °C, overnight). All products were 10-fold diluted before subsequent use.

Preamplification was performed in a 50-µL reaction system (5 µL of diluted digestion-ligation products; 10 µM preamplification primers E1 and HM1, respectively; 2.5 U Taq polymerase; 1X PCR buffer; 5 µL 2.5 mM dNTP; 94 °C for 2 min, 94 °C for 30 s, 56 °C for 1 min, 72 °C for 1 min, 72 °C for 10 min, 30 cycles; products were diluted 10-fold prior to use). Selective amplification was carried out in a 20-µL reaction system (2 µL preamplification products, 1 µL of each primer (10 µM), 1X PCR buffer, 0.5 U Taq polymerase, 2 µL 2.5 mM dNTP ). The first cycle was carried out at 94 °C for 30 s (denaturation), followed by annealing at 65 °C for 1 min and extension at 72 °C for 1 min. From the 2nd to the 13th cycle, the annealing temperature was decreased by 0.7 °C per cycle, and the remaining steps remained unchanged; from the 14th to the 35th cycle, annealing temperature was kept at 56 °C and the other steps remained unchanged. A final extension was done at 72 °C for 10 min.

The differential fragments in the MSAP gel were excised with sterile surgical knife blades and minced with sterile glass slides. The fragments were then extracted with a Poly-Gel DNA Extraction Kit (Omega BioTek, USA). The resulting products were used for PCR amplification and sequencing.

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3. Results 3.1. Morphological analysis of cotton seedlings under different salt stressesCompared with the controls, the cotyledons and caudexes of both salt-tolerant and salt-sensitive varieties all showed marked changes under different salt stresses (Figure 1). Under neutral salt stress (NaCl), the cotyledons of both varieties had symptoms of mild wilt. The caudexes of the salt-sensitive variety were slightly dark, while those of the salt-tolerant variety did not overtly change. Under stress from the weakly alkaline salt NaHCO3, both salt-tolerant and salt-sensitive varieties suffered slight toxicity, illustrated by their cotyledons turning soft and their caudexes obviously becoming dark and black. ZhongS9612, a salt-sensitive variety, suffered more than its resistant counterpart. Under stress from the alkaline salt Na2CO3, both varieties suffered relatively serious toxicity, indicated by the softening of their cotyledons that even began to fall off; the caudexes were heavily darkened and the entire root tissue began to turn black. With salt type changing from neutral to alkaline, cotton seedlings were not only affected by the stress of saline ions, but also by the toxic effects of an alkaline pH, which together caused cotton seedlings to endure much more damage.3.2. Changes in DNA methylation of cotton genome under different salt stresses3.2.1. MSAP repeatability testAlthough MSAP analysis adopts a group of restriction enzymes (HpaII and MspI) that can both recognize and cut the 5′-CCGG-3′ sequence, their sensitivities to different methylation conditions vary. HpaII is not sensitive to

complete methylation of single or double cytosine residues (double-strand methylation), and it recognizes and cuts only when a single strand is methylated; MspI is sensitive to methylated internal cytosine, but not sensitive to fully methylated external cytosine (McClelland et al., 1994). After digestion with HpaII/EcoRI (H) and MspI/EcoRI (M), genomic DNA often generates four banding types, with only three that can be tested by polyacrylamide gel electrophoresis (Figure 2). In Type I, both lanes have bands, indicating the lack of methylation; in Type II, lane H has a band, but lane M has none, showing a half methylation of locus CCGG; in Type III, lane H has no band, while lane M has one, indicating the full methylation of locus CCGG. According to the banding types of lanes H/M, statistical analysis was carried out.

During the preliminary test, the same variety Zhong07 (CK and NaCl) was analyzed, and six pairs of polymorphic primers were selected randomly to conduct two independent MSAP analyses. After three repetitions to test its repeatability, the results indicated that among the 135 loci amplified, there were 133 and 132 repetitive bands in Zhong07 (CK and NaCl), respectively, accounting for 98.54% and 97.78% of total loci (Table 1). These results indicated a favorable and highly reliable repeatability of the MSAP technology. 3.2.2. Changes of DNA methylation in the cotton genome under different salt stressesA total of 16 primer pairs were used to perform the MSAP analysis. In the leaves of cotton seedlings, 306–342 bands were amplified, with 80–99 methylated, accounting for 24.93%–28.95% of total bands (Table 2). In the root, 302–328 amplified bands were obtained, with 80–94

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Figure 1. Cotton seedlings under different salt stresses. A) Zhong07–dH2O; B) Zhong07–NaCl; C) Zhong07–NaHCO3; D) Zhong07–Na2CO3; E) ZhongS9612–dH2O; F) ZhongS9612–NaCl; G) ZhongS9612–NaHCO3; H) ZhongS9612–Na2CO3.

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methylated, accounting for 24.17%–29.35% of total bands (Table 3).

As shown in Table 2, the methylation levels in Zhong07 and ZhongS9612, salt-tolerant and sensitive cultivars, respectively, all dropped at first before rising and ultimately reaching the maximum with Na2CO3 after different salt stresses (NaCl, NaHCO3, and Na2CO3). The full methylation levels in Zhong07 leaves remained steady after NaCl and NaHCO3 stress, but rose rapidly to a maximum after Na2CO3 stress. However, complete methylation levels in ZhongS9612 leaves increased continuously under salt stress (NaCl→NaHCO3→Na2CO3), reaching a maximum during Na2CO3 treatment.

Tables 2 and 3 also indicated that double-strand methylation (complete methylation) was the main methylation type in the cotton genome, and changes in double-strand methylation after different salt stresses were

Type III

Type II

Type I

H M H M H M

Figure 2. MSAP amplification map of genome DNA of leaves of Zhong07 under NaCl stress. H: HpaII/EcoRI; M: MspI/EcoRI. Type I: unmethylated site; Type II: hemimethylated site; Type III: fully methylated site.

Table 1. Analysis of MSAP repeatability test.

MSAP band typesMSAP band types Number of repeatable bands Number of nonrepeatable bands

H M H M Zhong07 (CK) Zhong07 (S) Zhong07 (CK) Zhong07 (S)

I ■ ■ ■ ■ 105 107 1 0

II ■ ■ 13 11 0 1

III ■ ■ 17 14 1 2

Total bands amplified 135 132 2 3

(Non)repeatable bands ratio (%) 98.54 97.78 1.46 2.32

Table 2. Impact of different salt stresses on the levels of genomic DNA methylation in cotton seeding leaves.

MSAP band types

Leaf samples

Zhong07 ZhongS9612

CK NaCl NaHCO3 Na2CO3 CK NaCl NaHCO3 Na2CO3

I 232 256 242 243 225 227 231 230

II 21 14 16 16 22 16 16 17

III 67 71 72 83 59 64 70 75

Total bands amplified 320 341 330 342 306 307 317 322

Total methylated bands 88 85 88 99 81 80 86 92

The ratio of methylated bands (%) 27.50 24.93 26.67 28.95 26.47 26.06 27.13 28.57

Full methylated ratio (%) 20.94 20.82 21.82 24.27 19.28 20.85 22.08 23.29

The total number of amplified bands = I + II + III; the total number of methylated bands = II + III; the total number of complete methylated bands = III.

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quite significant. These results also indicated disparities between the two cotton genotypes for methylation patterns under different salt stresses. However, methylation levels in the root were slightly higher than that in leaves. With the changes caused by different salt stresses, the changes in methylation levels in roots were more overt compared with those in leaves, demonstrating that change in DNA methylation levels is tissue-specific under different salt stresses, in agreement with previous findings (Gao et al., 2013).

3.2.3. The change in methylation status resulted from different salt stressesMSAP analysis was performed in triplicate with different primer combinations under salt stresses. By comparing MSAP electropherograms, the changes in methylation patterns at the specific CCGG locus were analyzed. A total of 11 banding patterns were obtained for the experimental varieties (Figure 3) and could be divided into polymorphism and monomorphism. Polymorphism refers to a difference between control and treatment in methylation patterns:

Table 3. Impact of different salt stresses on the levels of genomic DNA methylation in cotton seeding roots.

MSAP band types

Root samples

Zhong07 ZhongS9612

CK NaCl NaHCO3 Na2CO3 CK NaCl NaHCO3 Na2CO3

I 230 229 234 219 228 230 232 234

II 20 14 17 16 9 13 14 14

III 68 59 66 75 72 67 74 80

Total bands amplified 318 302 317 310 309 310 320 328

Total methylated bands 88 73 83 91 81 80 88 94

The ratio of methylated bands (%) 27.67 24.17 26.18 29.35 26.21 25.81 27.50 28.66

Full methylated ratio (%) 21.38 19.54 20.82 24.19 23.30 21.61 23.13 24.39

The total number of amplified bands = I + II + III; the total number of methylated bands = II + III, the total number of complete methylated bands = III.

Figure 3. MSAP amplification results between the salt stress and the control. Lanes H0 and M0 are MSAP patterns of control, while lanes H and M are MSAP patterns of salt stress. H0 and H represent digestion with HpaII/EcoRI; M0 and M represent digestion with MspI/EcoRI. A1, A2, A3, B1, B2, B3, B4, C1, C2, C3, and D represent 11 banding patterns.

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3 statuses were obtained, namely methylation (type B), demethylation (type C), and indefinite pattern (type D). Monomorphism refers to identical banding patterns (type A), indicating that the methylation statuses between the control and treatment remained the same. Type B indicates increased methylation levels under salt stress, while type C suggests decreased methylation levels; type D means that DNA methylation statuses in the control and treatment groups are undefined. Detailed numbers of loci are shown in Tables 4 and 5.

As shown in Table 4, DNA methylation status always varied with changes in salt stress compared with the controls. In Zhong07 leaves, the loci with DNA methylation (type B) accounted for 40.00%, 50.00%, and 54.64% of loci

after NaCl, NaHCO3, and Na2CO3 stresses, respectively, and increased with pH values. Meanwhile, the loci with DNA demethylation (type C) accounted for 56.84%, 48.91%, and 43.30% of loci after NaCl, NaHCO3, and Na2CO3 stresses, respectively, presenting a downward trend with increasing pH values. In the salt-sensitive variety ZhongS9612, the loci with DNA methylation (type B) accounted for 51.28%, 52.17%, and 52.63% of loci after NaCl, NaHCO3, and Na2CO3 treatments, while loci with DNA demethylation (type C) accounted for 46.15%, 41.30%, and 42.11% of loci, respectively; both types changed very little with changes in pH values of the salt. In Zhong07 roots, as shown in Table 5, the loci with DNA methylation (type B) accounted for 30.00%, 45.71%, and 52.05% of loci after NaCl, NaHCO3,

Table 4. Impact of different salt stresses on the DNA methylation status of leaves.

Class

TypeMethylation state

Leaf samples

C S Zhong07 ZhongS9612

H M H M C S NaCl NaHCO3 Na2CO3 NaCl NaHCO3 Na2CO3

A1 + + + + CCGGGGCC

CCGG GGCC 212 203 215 208 201 205

A2 + - + - CCGGGGCC

CCGGGGCC

CCGGGGCC

CCGGGGCC 16 15 10 11 18 8

A3 - + - + CCGGGGCC

CCGGGGCC 18 20 20 10 6 14

B1 + + + - CCGGGGCC

CCGGGGCC

CCGGGGCC 7 18 8 9 10 12

B2 - + - - CCGGGGCC

CCGGGGCC 9 12 14 13 16 15

B3 + - - - CCGGGGCC

CCGGGGCC

CCGGGGCC 12 7 15 8 14 9

B4 + + - + CCGGGGCC

CCGGGGCC 10 9 16 10 8 14

C1 - - + + CCGGGGCC

CCGGGGCC 20 16 17 13 15 13

C2 + - + + CCGGGGCC

CCGGGGCC 19 20 18 14 14 15

C3 - + + + CCGGGGCC

CCGGGGCC 15 9 7 9 9 12

D - + + - CCGGGGCC

CCGGGGCC

CCGGGGCC 3 1 2 2 6 5

Total polymorphic bands 95 92 97 78 92 95

Type B band ratio of polymorphic bands (%) 40.00 50.00 54.64 51.28 52.17 52.63

Type C band ratio of polymorphic bands (%) 56.84 48.91 43.30 46.15 41.30 42.11

Type D band ratio of polymorphic bands (%) 3.16 1.09 2.06 2.56 6.52 5.26

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and Na2CO3 stresses, whereas the loci with demethylation (type C) accounted for 68.33%, 51.43%, and 45.21%, respectively. The trend in the roots for both types (B and C) was identical to that observed in leaves; however, the methylation levels had a different trend with varying pH values of the salt. In ZhongS9612 roots, the loci with DNA methylation (type B) accounted for 45.57%, 51.14%, and 55.42% of loci after NaCl, NaHCO3, and Na2CO3 treatments, respectively, whereas those with DNA demethylation (type C) accounted for 48.10%, 45.45%, and 40.96%, respectively. To further investigate the relationship between changes in methylation state/level and the pH value of the salt, we measured the pH values of the sand samples (Table S2; on the journal’s website) in which the cotton seedlings were

planted. The pH value of the sand treated with Na2CO3 was the highest; the sand of Zhong07 had a pH of 10.94 while that of ZhongS9612 was at pH 11.15. These values were higher than what is found in general saline-alkali soils. Thus, we inferred that high pH values in the sand could induce increased DNA methylation levels in cotton seedlings, likely a way of self-protection against adversity. Therefore, the changes in DNA methylation states and levels induced by different salt stresses varied between salt-tolerant and salt-sensitive varieties. In addition, the variation trends of cotton seedlings induced by different salt stresses (neutral salt and alkaline salt) differed as well. Finally, the methylation status changes of cotton seedlings induced by salt stress showed tissue (root and leaf) specificity.

Table 5. Impact of different salt stresses on the DNA methylation status of roots.

Class

TypeMethylation state

Root samples

C S Zhong07 ZhongS9612

H M H M C S NaCl NaHCO3 Na2CO3 NaCl NaHCO3 Na2CO3

A1 + + + + CCGGGGCC

CCGG GGCC 201 210 207 205 202 213

A2 + - + - CCGGGGCC

CCGGGGCC

CCGGGGCC

CCGGGGCC 10 13 12 12 14 17

A3 - + - + CCGGGGCC

CCGGGGCC 31 24 18 14 16 15

B1 + + + - CCGGGGCC

CCGGGGCC

CCGGGGCC 5 7 12 10 9 13

B2 - + - - CCGGGGCC

CCGGGGCC 3 10 10 9 12 9

B3 + - - - CCGGGGCC

CCGGGGCC

CCGGGGCC 3 5 7 8 16 14

B4 + + - + CCGGGGCC

CCGGGGCC 7 10 9 9 8 10

C1 - - + + CCGGGGCC

CCGGGGCC 18 7 13 12 14 10

C2 + - + + CCGGGGCC

CCGGGGCC 15 15 16 15 13 13

C3 - + + + CCGGGGCC

CCGGGGCC 8 14 4 11 13 11

D - + + - CCGGGGCC

CCGGGGCC

CCGGGGCC 1 2 2 5 3 3

Total polymorphic bands 60 70 73 79 88 83

Type B band ratio of polymorphic bands (%) 30.00 45.71 52.05 45.57 51.14 55.42

Type C band ratio of polymorphic bands (%) 68.33 51.43 45.21 48.10 45.45 40.96

Type D band ratio of polymorphic bands (%) 1.67 2.86 2.74 6.33 3.41 3.61

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3.3. Homology analysis of MSAP fragmentsTo study the polymorphic DNA methylation fragments induced by different salt stresses, extraction, reamplification, and sequence analyses were performed, and 21 fragments in total were selected for sequencing and homology search. The results showed a wide distribution of sequences homologous to these fragments.

The above data indicated that great changes took place in methylation types under different salt stresses (Table S3; on the journal’s website). The main transition type was from hemimethylation to complete methylation (type iii), accounting for 38.10% of the total methylation. The proportions of types i and ii were identical, while those of types iv and v were similar. As shown in Table 6 and Figure 4, the transition type from hemimethylation to complete methylation was generated mainly under Na2CO3 stress, while nonmethylation to hemimethylation transitions were mainly obtained under NaHCO3 stress. However, nonmethylation

to complete methylation transition was obtained under stress by the three kinds of salt. Under different salt stresses, changes in methylation type varied and gene expression was regulated by the alteration of its methylation patterns in cotton to adapt to the changing environment; this was a kind of self-protection of cotton seedlings under adversity stress.

Using sequence homology analysis, we found that the sequences highly homologous to discrepancy fragments were mainly involved in S-adenosyl-methionine (SAM) (M2), heat shock protein (M1), transmembrane transport (M5, M6, M7, M8), photosynthesis (M3, M12, M21), plant growth regulators (M4, M18), matter energy metabolism (M11, M17, M21), differential expression correlation (M14, M15, M16), protein binding (M19), signal transduction (M9), and fiber elongation development (M13).

These results also showed that genes related to salt stress covered extensive aspects, including salt-tolerant genes, energy metabolism, ionic transmembrane

Table 6. Sequence analysis of MSAP polymorphic fragments.

Spot No. Homologous sequence Length (bp) Accession No. CombinationE/HM Pattern

M1 S-Adenosyl methionine synthetase, mRNA 756 bp HM370495.1 H02-E10 III→I

M2 Heat shock protein 70, mRNA 948 bp X73961.1 H02-E10 I→III

M3 Oxygen-evolving protein of photosystem II 637 bp X99320.1 H02-E10 II→III

M4 5-Similar to PIN1-like auxin transport protein 352 bp CD486631.1 H02-E11 I→III

M5 Putative tricarboxylate transport protein 405 bp AI731741.1 H02-E11 I→II

M6 Putative transport protein subunit 287 bp CD486002.1 H02-E11 II→I

M7 Transport protein SEC61 254 bp AA336816.1 H02-E11 II→III

M8 Transport protein SEC61, GAMMA subunit 239 bp DT046749.1 H04-E13 I→II

M9 Peroxisomal targeting signal 2 receptor, mRNA 659 bp AF430070.1 H04-E13 II→III

M10 WD-repeat protein GhTTG1, mRNA 782 bp AF530907 H04-E10 I→III

M11 Calcineurin B-like protein2 (CBL2), mRNA 813 bp AY887897.1 H03-E10 II→I

M12 Gossypium hirsutum isolate GH160_435901 retrotransposon Gorge3 273 bp HM626744.1 H03-E10 I→III

M13 Cotton fiber, mRNA 573 bp AM412562.1 H04-E04 II→III

M14 CembMN06 differential display of cotton genotype, leaf 252 bp JG294134.1 H04-E04 I→II

M15 CRNS461 cotton root NaCl-treated suppression subtractive hybridization 676 bp GW691497.1 H04-E11 II→III

M16 CRNS454 cotton root NaCl-treated suppression subtractive hybridization 479 bp GW691490.1 H04-E11 I→III

M17 ATP synthase subunit alpha (atpA) gene, partial cds 867 bp HQ620729.1 H04-E11 II→III

M18 Putative growth regulator 598 bp AAT64033.1 H04-E13 I→II

M19 Binding protein, mRNA 783 bp BT002392.1 H04-E13 II→III

M20 Alternative oxidase 1A, mRNA 869 bp NM_113135.3 H16-E04 I→II

M21 Ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene; chloroplast 759 bp GU981720.1 H16-E11 II→III

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transport, photosynthesis, signal transduction, and many other pathways. Changes in DNA methylation status impacted various biological pathways, and the plants used the synergistic effects of diversified pathways to cope with different salt stresses. To further evaluate their expression, 4 homologous fragments (M1, M6, M12, and M19) were selected for real-time PCR analysis (Figure 5). Fragments M1 and M6 showed relatively high expression under salt stress compared with controls, and fragments M12 and M19 showed reduced expression under salt stress. Some genes were expressed exclusively under control or salt stress conditions. Therefore, salt stress could induce changes

in methylation patterns, modulating gene expression to adapt to the environment.

4. DiscussionDNA methylation of cytosine is important in epigenetics and plays a crucial role in chromatin structure and gene expression regulation (Richards, 1997; Sadri and Hornsby, 1996). During the growth and development process of plants, DNA methylation participates in regulating growth, differentiation, adversity stress, aging, and other biological events (Sadri and Hornsby, 1996). All environmental stimuli, including light, heat, drought, salt, and pathogenic infection, may induce DNA methylation level change in plants. Previous studies have shown that DNA methylation is closely related to gene expression regulation; methylation in DNA may induce gene silencing, while demethylation is beneficial for gene expression (Razin and Cedar, 1991). Therefore, understanding DNA methylation changes in plant crops under different salt stresses can lead to a better understanding of expression regulation and molecular mechanisms of adaptations to adversity stress.

Zhong07 and ZhongS9612, salt-tolerant and sensitive cultivars, respectively, were used as models to carry out a comprehensive analytical comparison in methylation statuses/levels between cotton varieties with different salt

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Figure 4. The pattern of DNA methylation transformation and its percentages. i, ii, iii, iv, and v represent five kinds of DNA methylation transformation.

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Figure 5. Relative expression of homologous fragments. A) Zhong07 leaf; B) ZhongS9612 leaf; C) Zhong07 root; D) ZhongS9612 root. M1, M6, M12, and M19 are homologous fragments chosen from 21 fragments amplified and sequenced.

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tolerance levels. During the seedling stage, to some extent, the methylation levels in both varieties declined after NaCl treatment, which corroborated previous findings (Cantoni, 1951). The decline of DNA methylation levels after treatment with neutral salt NaCl at a low concentration could induce the expression of relevant salt-tolerant genes in the cotton genome. The methylation levels showed an increasing trend after treatment with the alkaline salts NaHCO3 and NaCO3, which were maximized under NaCO3 stress; this may be caused by the toxic effect of the high pH of alkaline salts, which exerted a serious impact on the normal growth of cotton seedlings, altering the expression of most genes. This has not been described in previous studies. It is speculated that the changes observed in the methylation status of relevant loci under 0.4% NaCl stress can help generate or activate relevant mechanisms of resistance, minimizing the toxic effects from stress to ensure the normal growth of plants. When the external stress continuously strengthens, such as in alkaline environments caused by 0.4% NaHCO3 and 0.4% Na2CO3, it will have a serious impact on the normal growth of cotton seedlings; this is followed by the plant occluding or terminating the expression of relevant genes by changing the genome DNA methylation status. Meanwhile, the plant maintains low growth and development by decreasing energy consumption, which is also a basic defense system when facing adversity. Under different salt (neutral and alkaline) stresses, the changes of methylation levels in different tissues (lamina and root) are basically consistent with previous data (Zhang et al., 2006).

Interestingly, significant differences in the methylation status were observed after different salt stresses. After treatment with the neutral salt NaCl, demethylation was more abundant than methylation in Zhong07 (salt-tolerant variety) loci, in agreement with previous findings (Tan, 2010). However, the variation in ZhongS9612, a salt-

sensitive variety, was different from that of the salt-tolerant variety; this may be caused by variety differences (lack of relevant salt tolerance genes in salt-sensitive varieties). After alkaline salt (NaHCO3 and Na2CO3) treatment, both varieties presented more methylation loci than demethylation loci, which is possibly because alkaline salt greatly changes the growing environment of cotton seedlings. Previous studies have shown that the fragments targeted by DNA methylation under salt stress are distributed in both the coding and noncoding regions at similar proportions. Meanwhile, these sequences involve a number of pathways of cotton growth and metabolism, such as, for example, S-adenosyl-L-methionine (SAM), which is highly homogenous to M1. SAM is an important intermediate metabolite (Cantoni, 1951; Morel et al., 1999) widely distributed in plants and animals; it plays roles in trans-methyl, trans-sulfur, and trans-aminopropyl group formation, and it participates in the synthesis and energy metabolic pathways of multiple substances (Roje, 2006). Salt stress, one of the most important abiotic stresses, can cause plants to receive and transmit signals to cellular machinery, thereby activating adaptive responses; this is of fundamental importance to biology (Xiong et al., 2002). In normal plant growth and development, the function of plant genomes depends on chromatin and marks, including DNA methylation and posttranslational modifications of histones and small RNAs (Henderson and Jacobsen, 2007). This also indicates that cotton seedlings deal with adversity stress at the whole genome level and through the synergistic effects of various metabolic pathways.

AcknowledgmentsThis work was supported by grants from the program of Creation and Application of New Germplasm of Salt Tolerance in Cotton (Grant No. 2014ZX0800504B). We also appreciate the reviewers for their patience.

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Table S1. Linker and primer sequences.

Adaptor and primerSequence

EcoRI (E) HpaII/MspII (HM)

Adapter 1 5′-CTCGTAGACTGCGTACC-3′ 5′-GACGATGAGTCTAGAA-3′

Adapter 2 5′-CATCTGACGCATGGTTAA-3′ 5′-CTACTCAGATCTTGC-3′

Preamp primer 5′-GTAGACTGCGTACCAATTCA-3′ 5′-GATGAGTCTAGAACGGT-3′

Selective primer 5′-GTAGACTGCGTACCAATTCACA-3′(H02) 5′-GATGAGTCTAGAACGGTAT-3′(E04)

5′-GTAGACTGCGTACCAATTCACT-3′(H03) 5′-GATGAGTCTAGAACGGTAG-3′(E10)

5′-GTAGACTGCGTACCAATTCACG-3′(H04) 5′-GATGAGTCTAGAACGGTAC-3′(E11)

5′-GTAGACTGCGTACCAATTCAGA-3′(H16) 5′-GATGAGTCTAGAACGGTCG-3′(E13)

Table S2. The pH values of the sand treated with different types of salt.

TreatmentVariety CK NaCl NaHCO3 Na2CO3

Zhong07 7.12 7.22 8.83 10.94

ZhongS9612 6.96 7.10 8.95 11.15

The pH value was measured with a pH meter; the ratio of water to sand was 2.5:1.

Table S3. Methylation type changes and its percentage under salt stress.

Type Number Percentage (%)

i. nonmethylation → hemimethylation (I→II) 5 23.81

ii. nonmethylation → full methylation (I→III) 5 23.81

iii. hemimethylation → full methylation (II→III) 8 38.10

iv. full methylation → nonmethylation (III→I) 1 4.76

v. hemimethylation → nonmethylation (II→I) 2 9.52