水稻类病斑突变体wy3的鉴定和基因定位

doi:10.16819/j.1001-7216.2016.5176

中国水稻科学 ›› 2016, Vol. 30 ›› Issue (3): 239-246.DOI: 10.16819/j.1001-7216.2016.5176

水稻类病斑突变体wy3的鉴定和基因定位

张宏根, 王茂宇, 张丽佳, 胡雅, 马佳琦, 张翼帆, 汤述翥^*(), 梁国华, 顾铭洪

扬州大学江苏省作物遗传生理重点实验室/教育部植物功能基因组学重点实验室,江苏扬州225009;

收稿日期:2015-11-30 修回日期:2015-12-24 出版日期:2016-05-10 发布日期:2016-05-10
通讯作者: 汤述翥
作者简介:
# 共同第一作者;
基金资助:
国家自然科学基金资助项目(31071384,31000533);江苏省科技支撑计划资助项目(BE2013301);江苏高校优势学科建设工程资助项目

Characterization and Gene Mapping of Lesion Mimic Mutant wy3 in Rice

Hong-Gen ZHANG, Mao-yu WANG, Li-jia ZHANG, Ya HU, Jia-qi MA, Yi-fan ZHANG, Shu-zhu TANG^*(), Guo-hua LIANG, Ming-hong GU

Key Laboratory of Crop Genetics and Physiology of Jiangsu Province / Key Laboratory of Plant Functional Genomics,Ministry of Education,Yangzhou University,Yangzhou 225009,China;

Received:2015-11-30 Revised:2015-12-24 Online:2016-05-10 Published:2016-05-10
Contact: Shu-zhu TANG
About author:
# These authors contributed equally to this work;

摘要/Abstract

摘要：

在粳稻品种武育粳3号栽培群体中获得一个类病斑突变体wy3。该突变体类病斑出现于苗期,分蘖期扩散至整张叶片,属于扩散型类病斑突变体。相比野生型,突变体wy3的株高明显降低,有效分蘖数减少,穗长、每穗粒数、结实率均显著降低。遮光处理表明,突变体wy3类病斑的产生受自然光诱导。台盼蓝染色结果表明,类病斑部位有大量的死亡细胞。突变体wy3的光合色素含量和净光合速率较野生型显著降低,SOD、POD、CAT活性和MDA含量均显著高于野生型。遗传分析表明突变体表型受单隐性核基因控制,采用BSA将该基因初步定位在第2染色体短臂端粒附近。采用F₂群体中1099株类病斑单株将基因定位在标记W2-17和W2-18之间28kb的物理距离内。测序结果表明,突变体wy3中的LOC_Os02g02000编码区(CDS)第375位碱基C缺失,导致翻译提前终止,突变体中该候选基因为OsHPL3的一个新等位基因。

关键词: 水稻, 类病斑突变体, 基因定位

Abstract:

A lesion mimic mutant wy3 was obtained from the progeny of a japonica variety Wuyujing 3 by natural mutation. The lesions were first observed on the leaves at the seedling stage, then spread gradually to the whole leaf at the initial tillering stage. Compared with the wild type(WT), the plant height, the number of effective tiller, panicle length, grain number per panicle and seed setting rate of wy3 were reduced significantly. The shading assay showed that the lesions on leaves of wy3 were induced by natural light. Compared with the wild type, the photosynthetic pigment and the net photosynthetic rate of wy3 were significantly reduced, but the SOD activity, POD activity, CAT activity and MDA content of wy3 were significantly higher than those of WT. Trypan blue staining experiments showed that there were a large number of dead cells in the mutant lesion. Genetic analysis suggested that the phenotype of wy3 was controlled by a single recessive nuclear locus, and the target gene was mapped between markers W2-17 and W2-18 with the physical distance of 28 kb on chromosome 2. Sequence analysis revealed that the mutated gene in wy3 had a single nucleotide deletion at the 375th in CDS of LOC_Os02g02000, resulting in a premature termination of translation of the target gene, which is an new allele of OsHPL3.

Key words: rice, lesion mimic mutant, gene mapping

中图分类号:

Q343.5
Q754

张宏根, 王茂宇, 张丽佳, 胡雅, 马佳琦, 张翼帆, 汤述翥, 梁国华, 顾铭洪. 水稻类病斑突变体wy3的鉴定和基因定位[J]. 中国水稻科学, 2016, 30(3): 239-246.

Hong-Gen ZHANG, Mao-yu WANG, Li-jia ZHANG, Ya HU, Jia-qi MA, Yi-fan ZHANG, Shu-zhu TANG, Guo-hua LIANG, Ming-hong GU. Characterization and Gene Mapping of Lesion Mimic Mutant wy3 in Rice[J]. Chinese Journal OF Rice Science, 2016, 30(3): 239-246.

图/表 9

表1 突变体和野生型的农艺与产量性状

Table 1 Performance of agronomic and yield traits of the mutant wy3 and the wild type(WT).

材料 Material	株高 Plant height /cm	分蘖数 No. of tillers	穗长 Panicle length /cm	结实率 Seed-setting rate/%	每穗粒数 Grain number per panicle	粒长 Grain length /mm	粒宽 Grain width /mm	千粒重 1000-grain weight/g
wy3	56.9±0.6^**	3.5±0.6^**	14.8±1.2^**	79.0±0.1^**	89.6±6.8^**	7.5±2.2	3.4±1.4	26.54±0.56
WT	87.5±0.5	10.0±1.7	16.6±0.8	98.3±0.1	123.6±11.0	7.3±2.8	3.3±0.9	26.85±0.62

图1 突变体wy3及其野生型表型 A-苗期植株表型; B-分蘖初期野生型(左)和突变体wy3(右)植株表型; C-成熟期野生型(左)和突变体wy3(右)植株表型; D-野生型(左)和突变体wy3 (右) 穗部表型; E-分蘖期野生型(左)和突变体wy3(右)叶片表型。

Fig.1. Phenotypes of wy3 and wild type(WT). A, Phenotypes of wy3 during seedling stage; B, Phenotypes of WT (left) and wy3(right) during the early tillering stage; C, Phenotypes of WT (left) and wy3(right) during the mature stage; D, Panicle traits of WT (left) and wy3(right); E, Leaves of WT (left) and wy3 (right) during the early tillering stage.

图2 遮光对突变体wy3叶片的影响 A-遮光前 wy3; B-未发生斑点部位遮光15 d后; C-遮光部位恢复光照7 d后; D-野生型。

Fig. 2. Effects of shading on wy3 leaves. A, Leaf of wy3 before shading; B, Leaf without lesion after shading for 15 days; C, Leaf shaded for 7 days then under normal light for 7 days; D, Wild type.

表2 突变体wy3和野生型叶片光合色素含量

Table 2 Pigment contents in leaves of wy3 and wild type(WT).

材料 Material	叶绿素a含量 Chlorophyll a content /(mg·g^-1)	叶绿素b含量 Chlorophyll b content /(mg·g^-1)	叶绿素a/b Chlorophyll a/b	类胡萝卜素含量 Carotenoid content /(mg·g^-1)
wy3	1.28±0.09^**	0.27±0.11^**	4.68±0.13^**	0.15±0.11^**
WT	2.28±0.03	0.99±0.08	2.31±0.07	0.59±0.04

表3 突变体wy3和野生型武育粳3号分蘖期叶片光合特性

Table 3 Photosynthetic parameters of wy3 and wild type(WT) at the initial tillering stage.

材料 Material	净光合速率 P_n /(mmol·m^-2 s^-1)	蒸腾速率 T_r /(mmol·m^-2 s^-1)	气孔导度 G_s /(mol·m^-2 s^-1)	胞间CO₂浓度 C_i /(mmol·mol^-1)
wy3	14.16±0.27^**	193.27±10.10^**	0.13±0.02^**	2.76±0.23^**
WT	22.46±0.29	276.96±6.30	0.66±0.08	8.93±0.43

图3 武育粳 3号和wy3叶片的台盼蓝染色检测

Fig. 3. Trypan blue staining of leaves of the mutant wy3 and wild type(WT).

图4 突变体wy3与野生型(WT)对照在分蘖期的生理学特性比较 **P<0.01.

Fig. 4. Physiological characteristics of the wild type(WT) and the wy3 mutant during the tillering stage.

图5 水稻类病斑突变体基因定位 A-基因的初步定位; B-基因的精细定位; C-覆盖基因物理图谱; D-基因内变异位点。

Fig. 5. Mapping of the target gene in wy3. A, Primarily mapping of target gene; B, Fine mapping of target gene; C, Physical map of genes; D, The mutation locus of target gene.

表4 本研究开发的多态性STS标记

Table 4 Polymorphic STS markers developed in this study.

标记 Marker	反向引物 Forward primer (5'-3')	正向引物 Reverse primer (5'-3')
W2-10	GAGATGCCAGGAGAATGA	TGTAGCTGAGGGTTTGATA
W2-17	AAATCTGGACCTGAAAGT	TTAGGGAAGATTCTCAAA
W2-18	GCCAGCGAGAAGAAGAAG	GAGGATGTGGTCGGGTGT
W2-3	GCAGCACGGACTACAAGA	CAATTCTGCCATGACCAA
W2-13	TAGTGTCGCCCCTTTTAA	CATCACAGCAAACAAGCA

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水稻类病斑突变体wy3的鉴定和基因定位

Characterization and Gene Mapping of Lesion Mimic Mutant wy3 in Rice

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