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作物学报 ›› 2020, Vol. 46 ›› Issue (9): 1303-1311.doi: 10.3724/SP.J.1006.2020.03004

• 作物遗传育种·种质资源·分子遗传学 •    下一篇

玉米拟轮枝镰孢菌穗腐病抗性基因的挖掘

闻竞1, 沈彦岐1, 韩四平1, 邢跃先2, 张叶1, 王梓钰1, 李世界1, 杨小红3, 郝东云1, 张艳1,*()   

  1. 1 吉林省农业科学院农业生物技术研究所,吉林长春 130033
    2 吉林省农业科学院玉米研究所,吉林公主岭 136100
    3 中国农业大学农学院,北京 100193
  • 收稿日期:2020-01-18 接受日期:2020-04-15 出版日期:2020-09-12 网络出版日期:2020-04-26
  • 通讯作者: 张艳
  • 基金资助:
    本研究由国家自然科学基金项目资助(31701504)

Exploration of specific gene(s) for ear rot resistance to Fusarium verticilloides in maize

WEN Jing1, SHEN Yan-Qi1, HAN Si-Ping1, XING Yue-Xian2, ZHANG Ye1, WANG Zi-Yu1, LI Shi-Jie1, YANG Xiao-Hong3, HAO Dong-Yun1, ZHANG Yan1,*()   

  1. 1 Agro-Biotechnology Research Institute, Jilin Academy of Agricultural Sciences, Changchun 130033, Jilin, China
    2 Maize Research Institute, Jilin Academy of Agricultural Sciences, Gongzhuling 136100, Jilin, China
    3 College of Agronomy and Biotechnology, China Agricultural University, Beijing 100193, China
  • Received:2020-01-18 Accepted:2020-04-15 Published:2020-09-12 Published online:2020-04-26
  • Contact: Yan ZHANG
  • Supported by:
    National Natural Science Foundation of China(31701504)

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摘要：

玉米穗腐病是一种严重危害玉米生产的真菌性病害, 而目前在世界范围内玉米育种上应用的大多数自交系缺少对穗腐病的抗性。玉米穗腐病抗性位点的挖掘和抗病基因的克隆, 对玉米穗腐病的遗传改良至关重要。本研究旨在通过转录组测序和全基因组关联分析的方法进行玉米拟轮枝镰孢菌穗腐病抗性位点的挖掘并初步确定候选基因。抗病自交系法A和感病自交系掖81162的转录组测序结果表明, 人工接种拟轮枝镰孢菌后7 d两个自交系的差异表达基因有10,761个。通过全基因组关联分析共检测到5个与穗腐病抗性显著相关的SNP, 这些SNP分布在1号和9号染色体上。通过比对B73 RefGen_v3并注释, 发现SNP位点附近涉及的基因包括酰基激活酶1过氧化物酶体、蛋白磷酸酶2C 48、镁转运蛋白、受体蛋白激酶CRINKLY4和锌指CCCH域蛋白19。将在转录组测序中获得差异表达基因和全基因组选择中关联到的基因进行比对, 发现全基因组关联分析中关联到的锌指CCCH域蛋白19同时也是转录组测序中获得的差异表达基因, 表明锌指CCCH域蛋白19可能与玉米拟轮枝镰孢菌穗腐病的抗性相关。本研究结果不仅能为抗病基因的克隆和玉米的抗病分子育种提供一定的理论依据和重要的遗传资源, 而且能为玉米和病原菌的相互作用机理的解析奠定基础。

关键词: 玉米, 拟轮枝镰孢菌, 穗腐病, 抗性基因

Abstract:

Ear rot in maize, caused by fungal pathogens, poses a grave threat to maize production, and current inbred lines in use generally lack resistance to ear rot. It is essential to explore the resistant loci and corresponding resistance genes for improvement of the resistance to ear rot in maize by molecular marker-assisted breeding and biotechnology breeding. The purpose of this study was to explore the resistance loci of ear rot caused by Fusarium verticilloides and preliminary identify candidate genes by transcriptome analysis and genome-wide association. The result of transcriptome analysis showed that there were 10,761 differentially expressed genes between inbred lines Fa A and Ye 81162 at seven days after artificial inoculation with Fusarium verticilloides. A total of five SNPs significantly associated with ear rot resistance were detected by genome-wide association analysis, and these SNPs were distributed on chromosomes 1 and 9. By comparing B73 RefGen_v3 and annotating, it was found that the genes involved near the SNP site included acyl activating enzyme 1 peroxisome, protein phosphatase 2C 48, magnesium transporter, receptor protein kinase CRINKLY4 and zinc finger CCCH domain protein 19. The zinc finger CCCH domain protein 19 detected in genome-wide association just was the differentially expressed gene from transcriptome analysis, indicating that Zinc finger CCCH domain protein 19 probably is related to resistance to Fusarium verticilloides ear rot in maize. The results not only provide theoretical basis and important genetic resources for resistance gene cloning and molecular breeding of maize, but also lay a foundation for analysing the interaction between maize and pathogen.

Key words: maize, Fusarium verticilloides, ear rot, resistance genes

表1

玉米穗腐病病情分级标准"

病情评级Disease severity 发病百分比Percentage of symptom
1 发病面积占雌穗总面积0~1% Percentages of lesion area: 0-1%
3 发病面积占雌穗总面积2%~10% Percentages of lesion area: 2%-10%
5 发病面积占雌穗总面积11%~25% Percentages of lesion area: 11%-25%
7 发病面积占雌穗总面积26%~50% Percentages of lesion area: 26%-50%
9 发病面积占雌穗总面积51%~100% Percentages of lesion area: 51%-100%

表2

引物序列"

基因名称
Gene name		 上游引物序列
Forward primer sequence (5'-3')		 下游引物序列
Reverse primer sequence (5'-3')		 产物长度
Product length (bp)
actin CCTTCATTGGCATGGAATCT GCAACCACCTTCACCTTCAT 128
β-tubulin2 AGACCGGTCAGTGCGGTAAC CGTGCTCGCCAGAGAGATGGT 70
CCCH domain protein 19 CAGGTGAGTTGTGGACATGG CTCCAAGCACAAGCAAACAA 197

图1

掖81162样品中β-tubulin2基因的RT-PCR分析 M: DL2000 marker; 1~6: 接菌前和接菌后1 d、2 d、3 d、4 d、7 d的玉米籽粒样品。"

图2

人工接种拟轮枝镰孢菌后β-tubulin2基因的相对表达量"

图3

人工接种拟轮枝镰孢菌后法A和掖81162的差异表达基因分析 R: 抗病; S: 感病; R-0: 接菌前的法A; R-7d: 接菌后7 d的法A; S-0: 接菌前的掖81162; S-7d: 接菌后7 d的掖81162。"

图4

玉米拟轮枝镰孢菌穗腐病抗性全基因组关联分析的Manhattan图和QQ-plot图"

表3

人工接种拟轮枝镰孢菌后β-tubulin2基因的相对表达量"

自交系
Inbred line		 β-tubulin2基因的相对表达量 Relative expression of β-tubulin2 gene
接菌前
Before inoculation		 1 d 2 d 3 d 4 d 7 d
法A Fa A 0±0.00 0.0004±0.00 0.0002±0.00 0.0003±0.00 0.0002±0.00 1.006E-07±0.00
掖81162 Ye 81162 0±0.00 2.3894±0.93 0.6537±0.14 3.1675±0.38 3.0244±0.65 4.2477±0.26

表4

拟轮枝镰孢菌穗腐病抗性的相关SNP位点涉及的基因信息"

标记
Marker		 注释
Annotation		 染色体
Chromosome		 描述
Description
chr1.S_7569254 GRMZM2G110616 1 酰基激活酶1过氧化物酶体
Probable acyl-activating enzyme 1 peroxisomal
chr1.S_8288770 GRMZM5G891266 1 蛋白磷酸酶2C 48 Probable protein phosphatase 2C 48
chr1.S_8353697 GRMZM2G018706 1 镁转运蛋白10 Magnesium transporter 10
chr9.S_154383692 GRMZM2G092776 9 受体蛋白激酶CRINKLY4 Receptor protein kinase CRINKLY4
chr9.S_154384544 GRMZM2G393471 9 锌指CCCH域蛋白19 Zinc finger CCCH domain-containing protein 19

图5

人工接种拟轮枝镰孢菌后锌指CCCH域蛋白19基因的相对表达量"

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