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作物杂志,2017, 第1期: 32–37 doi: 10.16035/j.issn.1001-7283.2017.01.006

• 遗传育种·种质资源·生物技术 • 上一篇    下一篇

激光辐射和杂交对高粱DNA甲基化变异的影响

王欢,刘冰,白子裕,史俊丽,叶锦琳   

  1. 吉林农业大学农学院,130118,吉林长春
  • 收稿日期:2016-08-18 修回日期:2016-12-08 出版日期:2017-02-15 发布日期:2018-08-26
  • 作者简介:王欢,讲师,从事作物逆境生理及分子生物学研究
  • 基金资助:
    吉林省教育厅“十三五”科学技术研究(2016195)

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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摘要：

选择高粱正交F1杂种和它们纯系亲本作为试验材料,用He-Ne激光器在低强度下处理其萌发种子,用甲基化敏感扩增片断多态性（MSAP）分子标记技术在全基因组范围内检测分析高粱各种基因型DNA甲基化水平和变异模式,旨在探讨激光辐射和杂交对基因组DNA甲基化的交互作用机制。结果表明,激光辐射诱导高粱F1杂种和相应亲本在DNA甲基化水平和模式上发生一定变化,再次证明激光辐射诱导是一种表观遗传诱变手段。从甲基化水平上看,激光辐射诱导使高粱3种基因型整体表现为去甲基化。从甲基化变异模式看,CG位点甲基化变异模式要高于CNG位点甲基化变异模式。比较激光辐射对不同基因型的影响,发现F1杂交种的甲基化总体水平要低于两纯系亲本甲基化总体水平的中间值。总之,通过试验证明激光辐射是一种直接的表观遗传诱变手段,激光辐射和杂交联用诱导高粱产生更复杂的甲基化变异,这两种手段联用可能是一个重要改良作物的手段,值得深入研究。

关键词: 高粱, 激光辐射, DNA胞嘧啶甲基化, 表观变异, MSAP

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

表1

高粱MSAP中接头序列、预扩增引物和选择性扩增引物序列及组合"

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）

图1

He-Ne激光处理高粱DNA甲基化变异总体水平（亲本M和N及其正交杂种MN,A图横坐标数字为不同单珠,B图处理数值为重复平均值±标准误,*表示差异显著）"

图2

He-Ne激光处理高粱不同单株MSAP结果,箭头所指的是随机型变异（EcoRⅠ+HpaⅡ简写为E+H,EcoRⅠ+MspⅠ简写为E+M）"

图3

He-Ne激光处理高粱的甲基化变异形式"

表2

高粱发生胞嘧啶甲基化变异带的可能功能、同源性及限制性酶切位点"

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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