水稻矮秆多分蘖突变体bf370的遗传分析和基因定位

doi:10．3969/j．issn．1001G7216．2015．04．004

摘要/Abstract

摘要：

通过中子辐射诱变早籼稻品种红矮B,获得矮秆多分蘖突变体bf370。该突变体与野生型相比表现为植株矮化,分蘖极多。bf370在全生育期内的分蘖数达200个左右,是野生型分蘖数量的14倍以上。遗传分析表明该矮秆多分蘖突变体表型受一对隐性核基因控制。利用突变体bf370与日本晴杂交构建的F₂群体将突变基因定位到第1染色体长臂Indel 4 与 Indel 10 之间398 kb区域内。测序分析发现,与野生型相比突变体该区段内的D10基因在第2外显子上缺失66 bp碱基,导致D10蛋白RPE65结构域22个氨基酸缺失。结合D10其他突变体表型推断,bf370表型极有可能由D10突变所致。

关键词: 水稻, 矮秆突变体, 多分蘖, 遗传分析, 基因定位

Abstract:

A dwarf and high-tillering mutant bf370 was obtained from the indica rice variety Hong’ai B by neutron radiation. Compared with the wild type, the mutant bf370 was characterized by dwarf plant and more tillers. The number of tillers in bf370 was about 200, which was 13 times more than that in the wild type. Genetic analysis indicated that this phenotype was controlled by a single recessive nuclear gene, which was mapped to a 398 kb region between the marker Indel 4 and Indel 10 on the long arm of chromosome 1 using the F₂ mapping population generated from the cross between mutant bf370 and Nipponbare. Sequencing analysis showed a 66 nucleotide deletion in the second exon of D10, resulting in a 22 amino acids deletion in the predicted RPE65 domain of D10 protein in the bf370 mutant. Combined with the phenotype of other d10 mutants, the phenotype of bf370 was likely caused by a 66 nucleotide deletion in D10.

Key words: rice, dwarf mutant, high-tillering, genetic analysis, mapping

中图分类号:

胡运高, 杨国涛, 郭连安, 钦鹏, 陈永军, 李仕贵. 水稻矮秆多分蘖突变体bf370的遗传分析和基因定位[J]. 中国水稻科学, 2015, 29(4): 357-362.

Yun-gao HU, Guo-tao YANG, Lian-an GUO, Peng QIN, Yong-jun CHEN, Shi-gui LI. Genetic Analysis and Mapping of a Dwarf and High-tillering Mutant bf370 in Rice[J]. Chinese Journal OF Rice Science, 2015, 29(4): 357-362.

图/表 7

图1 红矮B和bf370的形态性状比较(A-红矮B与突变体bf370的植株表型; B-红矮B与突变体bf370的节间长度模型,P、Ⅰ、Ⅱ、Ⅲ和Ⅳ分别代表穗、倒Ⅰ、Ⅱ、Ⅲ、Ⅳ节间; C-红矮B与突变体bf370植株的分蘖数量动态变化。)

Fig.1. Morphological characterization of Hong’ai B and the mutant bf370. (A, Phenotypic comparison between Hong’ai B and the mutant bf370; B, Plant height and internode length of Hong’ai B and the mutant bf370. P,Ⅰ,Ⅱ,Ⅲ, Ⅳ indicates the panicle and the upper four internodes, respectively; C, Dynamic changes of tiller number for Hong’ai B and the mutant bf370.)

表1 红矮B与bf370的农艺性状比较

Table 1 Comparison of agronomic traits between Hong’ai B and the mutant bf370.

性状 Character	红矮B Hong’ai B	bf370
株高 Plant height/cm	81.0±1.1	40.1±1.5^**
分蘖数 Number of tillers per plant	13.4±1.3	192.6±12.9^**
穗长 Panicle length/cm	22.1±0.3	6.7±0.6^**
每穗粒数No. of grains per panicle	116.2±6.9	9.7±1.0^**
结实率 Seed-setting rate/%	81.7±1.0	48.5±2.2^**
千粒重 1000-grain weight/g	31.5±0.9	19.9±0.4^**

表 2 水稻矮秆多分蘖突变体bf370的遗传分析

Table 2 Genetic analysis of rice dwarf and high-tillering mutant bf370.

组合 Cross	F₁表型 Phenotype of F₁ plants	F₂ 群体 F₂ population
组合 Cross	F₁表型 Phenotype of F₁ plants	总植株数 No. of plants	野生型 Wild type			矮秆多分蘖株数 No. of dwarf and high-tillering plants
bf370/日本晴 bf370/Nipponbare	野生型 Wild type	479	344	137		3.03	3.84

表 2 水稻矮秆多分蘖突变体bf370的遗传分析

Table 2 Genetic analysis of rice dwarf and high-tillering mutant bf370.

组合 Cross	F₁表型 Phenotype of F₁ plants	F₂ 群体 F₂ population
组合 Cross	F₁表型 Phenotype of F₁ plants	总植株数 No. of plants	野生型 Wild type			矮秆多分蘖株数 No. of dwarf and high-tillering plants
bf370/日本晴 bf370/Nipponbare	野生型 Wild type	479	344	137		3.03	3.84

图2 BF370基因的图位克隆和分析(A- BF370的精细定位,n为F2群体中的突变单株数量; B-D10基因的结构和突变位点,黑框代表外显子,黑线代表内含子,白三角代表突变体bf370中第2个外显子66个核苷酸缺失; C-D10蛋白的氨基酸序列和预测的PRE65结构域(黑色底线),黑框代表突变体bf370中22个氨基酸缺失。)

Fig. 2. Mapping-based cloning and analysis of BF370. (A, Fine mapping of BF370, n indicates the mutant plants in F2 population; B, Structure and allelic variation of the D10, black boxes show exons, black wires show introns, and triangle shows 66 nucleotides deletion in the second exon in bf370; C, The amino acid sequence of D10 protein and predicted PRE65 domain (black underline), black box represents 22 amino acids deletion in bf370.)

图3 红矮B与突变体bf370中的D10基因编码区突变位点序列比对

Fig. 3. Sequence alignment of mutation site of D10 coding region between Hong’ai B and the mutant bf370.

图4 标记Indel11在22个F2矮秆多分蘖单株中的分离情况(M-标记; Nip-日本晴; 1~22表示来源于亲本日本晴与bf370构建的F2群体的22个不同矮秆多分蘖单株。)

Fig. 4. Genotype analysis of the 22 F2 plants with dwarf and high-tillering phenotype using the InDel marker 11. (M, Marker; Nip, Nipponbare; 1-22 represent 22 dwarf and high-tillering individual plants derived from the cross of Nipponbare×bf370.)

表3 定位BF370的部分重要标记

Table 3 Some important markers for BF370 mapping.

引物 Primer	正引物(5'-3') Forward primer(5'-3')	反引物(5'-3') Reverse primer(5'-3')
RM265	CGAGTTCGTCCAAGTGAGC	CATCCACCATTCCACCAATC
RM297	TCTTTGGAGGCGAGCTGAG	CGAAGGGTACATCTGCTTAG
RM5497	AGCAGACCAATGAACCATGAACC	GGTTTGGCTAAACTCATGAGAGAGG
RM11229	TGACAGAAACAAAGCGGAAGG	TCCAAACCGCTATTCTTGTAGC
Indel1	CGGTTGCTCGCTTTCTGG	CACCCGAAGCCTTCCACT
Indel2	AGGTTTGGTCTCGGGTTT	ACACGCCATAGCAAGGAA
Indel4	TTCCCGTTACCCGCCTAT	ATTACCGGGCCAACAACA
Indel9	AAAGGAAAGCGGATTACAGA	ACACCCTAACTGGTTCGTTGTA
Indel10	CGCTCATAAACATCCCAACC	CCCACTGCTATCAGCCTCAA
Indel11	CACGGTCGGCAAGTTCCAGT	GTGCATGTAGCTGCCGGACT

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