全 文 ： ACTA AGRONOMICA SINICA 2009, 35(12): 2150−2158 http://www.chinacrops.org/zwxb/
ISSN 0496-3490; CODEN TSHPA9 E-mail: xbzw@chinajournal.net.cn
(973)(2006CB101708)(B08025)
*
!(Corresponding author): #$, E-mail: cotton@njau.edu.cn; Fax: 025-84395307
%& !()*: E-mail: zhuxxshz@126.com
Received(+,-.): 2009-04-13; Accepted(/0-.1: 2009-07-25.
DOI: 10.3724/SP.J.1006.2009.02150
12 2
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Inheritance of DNA Methylation in Two Cotton Hybrid Derived from
CRI-12
ZHU Xin-Xia1,2, WANG Bao-Hua1, GUO Wang-Zhen1, and ZHANG Tian-Zhen1,*
1 National Key Laboratory of Crop Genetics & Germplasm Enhancement, Nanjing Agricultural University, Nanjing 210095, China; 2 Life Science
College of Shihezi University, Shihezi 832003, China
Abstract: DNA methylation in the form of cytosine methylation is proposed as an evolutionary event, which contributes to ge-
nome evolution and plays an important role in maintaining genome integrity and controlling dynamics of gene activity. Hybridiza-
tion and polyploidization play significant roles in the evolution of higher plants. Compared with animals, cytosine methylation is
more abundant in plants, particularly at genomic regions containing transposons and their derivatives. Accumulated evidence had
suggested that DNA methylation plays important roles in normal plant development. In different stages of plant growth, the
changes in DNA methylation level play an important role in response to variations of its heredity and the environment. Notwith-
standing these interesting findings, the causing factors for, and generality of, methylation dynamics in plants, particularly with
hybrid formation and trans-generational heritability, remained largely vague. However, there is a paucity of evidence to support
any direct link between the epigenetic phenomena of cytosine methylation alteration and gene silencing following hybridization
and polyploidization. In this study, MSAP (methylation-sensitive amplified fragment length polymorphism) was used in this study
to detect the DNA methylation patterns in the 5′-CCGG sites of two cotton hybrids derived from CRI-12 and their parents for
understanding developmental stability and inheritance of cytosine methylation. It was found MSAP ratios, which were the ratios
of MSAP type in the two cotton hybrids were 12.41–20.05%, cytosine methylation profiles were variable, from increase to de-
crease during plant growth and development. Full methylation of internal cytosine (6.90–11.47%) was the dominant in two cotton
hybrids. Meanwhile, the MSAP profiles enable the monitoring of inheritance or variation of parental methylation patterns in hy-
brid progenies. It was found that a great majority (from 96.61% to 98.86%, depending on crosses) of the methylation profiles in
cotton inbred lines transmitted to the inter-strain hybrids; however, from 1.14% to 3.39% of the profiles in the hybrids exhibited
variation from the expected parental additivity. Both inherited and altered methylation profiles can be divided into distinct groups,
and their frequencies are variable among the cross-combinations, and during plant growth and development. Bands in hybrids that
appeared in both groups digested by Hpa II-EcoR I and Msp I-EcoR I, which were inherited from either or both of the maternal
and paternal parent—this type apparently comprised the greatest majority, as all monomorphic bands belong to this type. In addi-
tion, sequencing of differentially methylated fragments and subsequent homology analysis of isolated bands that showed variation
12 :
12
2 DNA 2151
in hybrids indicated that diverse sequences were involved, including known-function cellular genes and mobile elements, Such as
leucine-rich repeat family protein, PDR-like ABC-transporter, putative oligopeptide transporter, GTP-binding protein, similar to
pathogenesis-related protein, DOMON domain-containing protein, putative adenosine phosphosulfate kinase, putative protein,
RNA-directed DNA polymerase. The remaining 14 bands showed no homology to the database sequences. These results clearly
demonstrated the power of the MSAP technique for large-scale DNA methylation detection in the cotton genome, and the com-
plexity of DNA methylation change during plant growth and development. The different methylation levels may be induced by
interspecific hybridization between two cotton hybrids, and indicated a direct relationship between cytosine methylation alteration
and gene expression variation.
Keywords: CRI-12; Hybrids cotton; DNA methylation; MSAP
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¼½ 5mCCGG Hpa II[Msp I«
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K), Hpa II©°(e$F,, . Msp I
2152 35
:©°.:e$F,(L H/M= +/–); TU¨
V»¼½(\K), Hpa II:©°.Ws
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–/+); TU CCGG ¹ºF»¼½Y $(P¨
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表 1 杂交种和亲本在 CCGG位点的胞嘧啶甲基化水平
Table 1 Levels of cytosine methylation at the CCGG sites among hybrids and their parents
总位点数
Total site
苗期
SS
蕾期
BS
花铃期
FS
苗期
SS
蕾期
BS
花铃期
FS
苗期
SS
蕾期
BS
花铃期
FS
苗期
SS
蕾期
BS
花铃期
FS
苗期
SS
蕾期
BS
花铃期
FS
苗期
SS
蕾期
BS
花铃期
FS
父本
Male 81.52 80.52 78.73 9.38 8.61 11.17 8.00 7.81 4.83 0.97 3.06 0.27 18.34 19.48 16.26
母本
Female 84.14 77.80 80.70 7.31 7.70 11.71 5.93 7.13 5.18 2.62 3.51 0.27 15.86 18.35 16.98
中亲值
Mid-parent 82.90 79.16 79.71 8.34 8.15 11.44 6.97 7.47 5.00 1.79 3.28 0.27 17.11 18.91 16.89
湘杂棉 2号
XZM2
F1
725 883 1119
85.10 79.05 82.93 6.90 10.08 11.53 4.69 7.93 5.00 0.83 2.04 0.45 12.41 20.05 17.07
父本
Male 82.51 80.89 77.41 8.88 10.47 10.51 5.87 6.14 4.47 2.73 2.50 0.96 17.49 19.11 15.94
母本
Female 83.33 78.16 79.07 7.24 7.74 11.47 4.64 7.17 5.08 2.60 3.53 0.26 14.48 18.43 16.81
中亲值
Mid-parent 82.92 79.52 78.24 8.06 9.10 10.99 5.26 6.66 4.77 2.66 3.01 0.61 15.98 18.77 16.37
中棉所 28
CRI-28
F1
732 879 1142
82.10 79.75 83.45 7.10 9.90 11.30 6.28 7.51 4.73 3.01 2.62 0.53 16.39 20.02 16.55
SS: seedling stage; BS: budding stage; FS: flowering stage.
非甲基化 CCGG位点比例
Frequency of non-methylated
CCGG sites
内侧胞嘧啶全甲基化比例
Frequency of full methylation
of the internal Cs
外侧胞嘧啶半甲基化比例
Frequency of
hemi-methylation of the ex-
ternal Cs
外侧或内外侧胞嘧啶
全甲基化比例
Frequency of full methylation of
the internal Cs or external Cs
胞嘧啶甲基化位点比例
Frequency of methylated
CCGG sites 亲本和杂交种
Inbreds and hybrids
2154 35
2.2 *+,-./0 DNA
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2 DNA
Table 2 DNA methylation patterns between hybrids and their two parents
Number of sites
2 XZM 2
28 CRI-28
Pattern
Hpa II
P1
Msp I
P1
Hpa II
F1
Msp I
F1
Hpa II
P2
Msp I
P2
SS
BS
FS
SS
BS
FS
Genetic site
M1 – + – + – – 5 4 3 2 2 5
H1 + – + – – – 2 2 5 1 5 6
HM1 + + + + – – 14 5 6 4 11 7
M2 – – – + – + 8 3 2 2 21 3
H2 – – + – + – 3 8 1 3 6 0
HM2 – – + + + + 8 11 5 4 11 7
D – + – + – + 53 65 121 55 61 120
D + – + – + – 42 60 54 34 49 47
D + – + + – + 3 5 1 1 5 3
D – + + + + – 2 16 0 2 7 2
D + + + + + + 604 682 934 604 678 930
Sum 744 861 1132 712 856 1130
Variant site
VM1 – + – – – – 1 1 1 2 3 3
VH1 + – – – – – 0 1 1 1 1 1
VHM1 + + – – – – 0 0 0 0 0 2
VM2 – – – – – + 5 0 3 2 1 3
VH2 – – – – + – 2 4 2 1 3 1
VHM2 – – – – + + 0 4 2 2 5 0
VD + – – – + – 0 3 2 5 2 0
VD – + – – – + 1 1 0 1 2 1
VD – + – – + – 1 1 1 2 2 0
VD + – – – – + 1 1 0 1 2 0
VD + + – – + + 0 1 0 3 0 0
VD – – + – – – 2 4 0 1 1 0
VD – – – + – – 2 8 3 4 1 2
VD – – + + – – 1 0 0 0 1 0
Sum 16 29 15 25 24 13
Total sites 760 890 1147 737 880 1143
+: EcoR I+Hpa II EcoR I+Msp I !#; –$% !&#
+: bands appeared in EcoR I+Hpa II or EcoR I+Msp I digested; –: means no bands in EcoR I+Hpa II or EcoR I+Msp I digested.
SS: seedling stage; BS: budding stage; FS: flowering stage.
12 :
12
2 DNA 2155
3 CCGG
Table 3 Inheritance and variation of cytosine methylation patterns at the CCGG sites among hybrids and their parents
Inheritance pattern and frequency
Maternal
Paternal
Both parentsMaterial
M1 H1 MH1 Sum M2 H2 MH2 Sum D
Total sum
2 XZM2 0.66 0.26 1.84 2.76 1.05 0.39 1.05 2.50 92.63 97.89
SS 28 CRI-28 0.27 0.14 0.54 0.95 0.27 0.41 0.54 1.22 94.44 96.61
2 XZM2 0.45 0.22 0.56 1.24 0.34 0.90 1.24 2.47 93.03 96.74
BS 28 CRI-28 0.23 0.57 1.25 2.05 2.39 0.68 1.25 4.32 90.91 97.27
2 XZM2 0.26 0.44 0.52 1.24 0.17 0.09 0.44 0.71 96.77 98.69
FS 28 CRI-28 0.44 0.52 0.61 1.59 0.26 0.00 0.61 0.88 96.41 98.86
2 XZM2 0.43 0.32 0.90 1.65 0.47 0.43 0.86 1.76 94.97 98.38
Total 28 CRI-28 0.33 0.44 0.80 1.57 0.95 0.33 0.80 2.07 94.58 98.22
()* Variation pattern and frequency
Maternal
Paternal
Both parents
Material
VM1 VH1 VD Sum VM2 VH2 VMH2 Sum VD
Total sum
2 XZM2 0.13 0.00 0.00 0.13 0.66 0.26 0.00 0.92 1.05 2.11
SS 28 CRI-28 0.27 0.14 0.00 0.41 0.27 0.14 0.27 0.68 2.31 3.39
2 XZM2 0.11 0.11 0.00 0.22 0.00 0.45 0.45 0.90 2.13 3.26
BS 28 CRI-28 0.34 0.11 0.00 0.45 0.11 0.34 0.57 1.02 1.25 2.73
2 XZM2 0.09 0.09 0.00 0.18 0.26 0.17 0.17 0.60 0.52 1.31
FS 28 CRI-28 0.26 0.09 0.17 0.52 0.26 0.09 0.00 0.35 0.26 1.14
2 XZM2 0.11 0.07 0.00 0.18 0.29 0.29 0.22 0.79 1.19 2.16
Total 28 CRI-28 0.29 0.11 0.07 0.47 0.22 0.18 0.25 0.66 1.13 2.26
SS: seedling stage; BS: budding stage; FS: flowering stage.
96.74%~98.69% ( 3),
28
96.61%~98.86%,
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Table 4 Sequence of methylated fragments
Sequence No.
Function based on Blastx against the NCBI database
Identity based on Blastx
E
E-value
L1792 Leucine-rich repeat family protein / protein kinase family protein (Arabidopsis thaliana) AT5G48740 6×E–50
L1794 PDR-like ABC-transporter (Glycine max) gb|EF159964.1| 3×E–39
L1795 GTP-binding protein gb|AAA53276.1| 2×E–11
L1798 Similar to pathogenesis-related protein gb|AAF97329.1| 1×E–18
L1828 DOMON domain-containing protein / dopamine binding protein ref|NP_200294.1| 2×E–25
L1836 Putative oligopeptide transporter (Oryza sativa) dbj|BAD81723.1| 5×E–13
L1838 Putative adenosine phosphosulfate kinase (Arabidopsis thaliana) gb|AAM62496.1| 5×E–26
L1839 Putative protein emb|CAA18481.1| 8×E–13
L1840 RNA-directed DNA polymerase (Reverse transcripase) gb|ABN06136.1| 7×E–27
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