Crops ›› 2017, Vol. 33 ›› Issue (1): 32-37.doi: 10.16035/j.issn.1001-7283.2017.01.006

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Effects of Laser Irradiation and Hybridization on the DNA Methylation Alteration in Sorghum (Sorghum bicolor L.)

Wang Huan,Liu Bing,Bai Ziyu,Shi Junli,Ye Jinlin   

  1. Department of Agronomy,Jilin Agricultural University,Changchun 130118,Jilin,China
  • Received:2016-08-18 Revised:2016-12-08 Online:2017-02-15 Published:2018-08-26

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Abstract:

In this study, sorghum (Sorghum bicolor L.) inter-strain hybrids was used as the test organism, and their germinating seeds were treated by the He-Ne laser irradiation, then the level and pattern of the different genotype DNA methylation in the plants were analyzed by methylation-sensitive amplified polymorphism (MSAP) to reveal genome-wide DNA methylation alterations in the recipient sorghum genome. We aimed to probe the possible interactive effect of low-dose laser irradiation and hybridization. Results showed that low-dose laser irradiation induced the whole level and pattern of DNA methylation in plant intraspecific F1 hybrids relative to their pure-line parents. The results can demonstrate that low-dose laser irradiation is epigenetically mutagenic in plants. The first finding suggests that variation of the whole DNA methylation in different genotypes sorghum is accompanied by hypomethylation. The second finding indicates the CG sites variation more than the CNG sites variation in the DNA methylation variation mode. Comparing the effects of laser irradiation on the different genotypes, the DNA methylation level of the F1 hybrids lower than the inter mediate value of the corresponding pure line parents. Therefore, we propose that the combined use of intraspecific hybridization and an epigenetically mutagenic treatment like low-dose laser irradiation could be a useful means to generate heritable epigenetic variations in plants, should be further investigated.

Key words: Sorghum bicolor L., Iaser irradiation, Cytosine DNA methylation, Epigenetic variations, MSAP

Table 1

Sequence of adaptors, pre-amplification primers and selective amplification primers combinations in sorghum MSAP analysis"

命名Name 5'-3'序列5'-3' sequence
接头序列Sequence of adaptors EcoRⅠ-adapter1 5'-CTCGTAGACTGCGTACC-3'
EcoRⅠ-adapter2 5'-AATTGGTACGCAGTC-3'
H/M-adapter1 5'-GATCATGAGTCCTGCT-3'
H/M-adapter2 5'-CGAGCAGGACTCATGA-3'
预扩增引物Pre-amplification primers E-A 5'-GACTGCGTACCAATTCA-3'
H/M-0 5'-ATCATGAGTCCTGCTCGG-3'
选择性扩增引物Selective amplification primers 1H/M-TCT 5'-ATCATGAGTCCTGCTCGGTCT-3'
2H/M-TCG 5'-ATCATGAGTCCTGCTCGGTCG-3'
3H/M-TTC 5'-ATCATGAGTCCTGCTCGGTTC-3'
4H/M-TTG 5'-ATCATGAGTCCTGCTCGGTTG-3'
5H/M-TTA 5'-ATCATGAGTCCTGCTCGGTTA-3'
6H/M-TGT 5'-ATCATGAGTCCTGCTCGGTGT-3'
7H/M-TGA 5'-ATCATGAGTCCTGCTCGGTGA-3'
8H/M-TAC 5'-ATCATGAGTCCTGCTCGGTAC-3'
E-AAC a 5'-GACTGCGTACCAATTCAAC-3'（1,2,3和8引物组合）
E-AAG b 5'-GACTGCGTACCAATTCAAG-3'（3和4引物组合）
E-ACA c 5'-GACTGCGTACCAATTCACA-3'（3和4引物组合）
E-ACG f 5'-GACTGCGTACCAATTCACG-3'（4和5引物组合）
E-AGC g 5'-GACTGCGTACCAATTCAGC-3'（1,3,6和7引物组合）
E-AGG h 5'-GACTGCGTACCAATTCAGG-3'（1,3,4,5和7引物组合）
E-ATC j 5'-GACTGCGTACCAATTCATC-3'（7引物组合）

Fig.1

He-Ne iaser inducing the sorghum methylation level（The parental pure-lines M and N,F1 hybrids MN, the number (in A) of single beads for different genotypes.B as means±SE of replicates,* significant difference）"

Fig.2

Results of MSAP profiles showing the various types of locus-specific DNA methylation inheritance and variation (marked by arrows) in sorghum under the He-Ne laser（EcoRⅠ+HpaⅡ→E+H,EcoRⅠ+MspⅠ→E+M）"

Fig.3

He-Ne laser irradiation on the variation in the major methylation patterns"

Table 2

Putative function,identity and restriction map of the sequenced MSAP profiles showing difference in methylation pattern in different genotype"

MSAP片段
MSAP profiles		 去甲基化变异位点
Hypomethyl
ated site		 基因型和单株个体Genotype and the
different individual		 染色体Chromosome
位置Location
同源性Idendity		 预测功能
Predicted function		 长度（bp）和限制性酶切位点
Size (bp) and restriction map
Z-2 CG or CNG MN-4 Chr.8; 6.1e-32
Sb08g000720.1
Position:634595..636034		 漆酶-23前体
Laccase-23 precursor		 EcoRI H/M
↓ 291 ↓
N-4 CG N-5 Chr.4; 6.8e-130
Sb04g004650.1
Position:4450817..4452596		 未知蛋白
Putative uncharacterized protein		 EcoRI M
↓ 243 ↓
F-13 CG NM-1 Chr.2; 5.7e-80
Sb02g005470.1
Position:6482952..6489780		 未知蛋白
Putative uncharacterized protein		 EcoRI H
↓ 159 ↓
Z-14 CNG MN-2 Chr.3; 1.7e-23
Osa5(12001:8156651-16748019) 165 loci		 高粱–水稻同源片段
Sorghum-Rice syntenic segments		 EcoRI H
↓ 360 ↓
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