水稻显性脆秆突变体Bc18的鉴定和基因定位

doi:10.16819/j.1001-7216.2016.5162

摘要/Abstract

摘要：

从三交组合Ⅱ-32B//协青早B/Dular的F₂群体中获得了1个脆性突变体,整个植株表现全生育期脆性。根据该突变体的表型,将其命名为Bc18(Brittle culm 18)。为了更好地鉴定该突变体,用正常茎秆强度品种中9B作轮回亲本与Bc18杂交,创制了Bc18脆秆近等基因系中脆B和中9B。表型鉴定显示,突变体Bc18在生育期、株高、单株穗数、每穗粒数、结实率和千粒重等主要农艺和产量性状上与野生型中9B 无显著差别,但茎、叶的机械强度分别下降了70.70%和47.16%。细胞壁组分分析表明,突变体Bc18茎、叶的纤维素和木质素含量与野生型中9B 无显著差异,但半纤维素含量分别提高了31.84%和17.35%。6个杂交组合F₂和12个回交BC₁F₁群体的遗传分析证明Bc18 脆性突变由单显性基因控制。采用图位克隆技术,构建了Bc18/02428和Bc18/9311的F₂定位群体,并利用网上公布的SSR标记和新设计的InDel标记,最终将Bc18基因定位在第1染色体长臂端InDel标记PBC22与PBC33之间约154 kb的区间内。

关键词: 水稻, Bc18, 显性脆秆, 机械强度, 半纤维素含量, 基因定位

Abstract:

A brittle culm mutant was obtained from the F₂ population of three-way cross Ⅱ-32B//Xieqingzao B/Dular and named as Brittle culm 18 ( Bc18 ) according to its phenotypes. Each part of plant showed brittleness during the whole growth period. In order to identify the mutant, near isogenic line populations of Zhongcui B and Zhong 9B were created with Bc18 mutant as the donor of brittle gene and Zhong 9B, a normal culm-strength variety, as the receptor and recurrent parent. Compared with wild type, Bc18 significantly declined by 70.70% and 47.16% in mechanical strength of culm and leaves, respectively. No significant differences were found in terms of growth duration, plant height, panicle number per plant, spikelet number per panicle, seed setting rate and 1000-grain weight. Cell wall components analyses showed that cellulose content and lignin content in Bc18 had no significant difference as compared with those of wild type, while hemicellulose content in culm and leaf dramatically increased by 31.84% and 17.35%, respectively. Genetic analyses of six F₂ and twelve BC₁F₁ backcross populations revealed that the phenotype of Bc18 was controlled by a single dominant gene. Bc18/02428 and Bc18/9311 F₂ populations were developed for Bc18 gene mapping. By means of map-based cloning technology, with some SSR markers published online and new designed InDel markers, Bc18 was localized between InDel marker PBC22 and PBC33 at a physical distance of about 154 kb on the long arm of chromosome 1. This work laid the foundation for cloning Bc18 gene in the future.

Key words: rice ( Oryza sativa L. ), Bc18, dominant brittle culm, mechanical strength, hemicellulose content, gene mapping

中图分类号:

彭应财, 刘文真, 傅亚萍, 王鹤潼, 胡国成, 陈温福, 徐正进. 水稻显性脆秆突变体Bc18的鉴定和基因定位[J]. 中国水稻科学, 2016, 30(2): 127-135.

Ying-cai PENG, Wen-zhen LIU, Ya-ping FU, He-tong WANG, Guo-cheng HU, Wen-fu CHEN, Zheng-jin XU. Characterization and Gene Mapping of a Dominant Brittle Culm Mutant Bc18 in Rice (Oryza sativa L.)[J]. Chinese Journal OF Rice Science, 2016, 30(2): 127-135.

图/表 9

表1 突变体Bc18及其野生型的主要特征特性(2008年,杭州)

Table 1 Major agronomic traits of Bc18 and its wild type in Hangzhou, 2008.

材料 Material	播始历期 DSH/d	株高 PH/cm	茎秆粗度 ODS/mm	茎秆壁厚 TS/mm	叶长 LL/cm	叶宽 LW/cm	叶厚 TL/mm
Bc18	64	92.83±1.75 a	5.24±0.52 a	1.69±0.46 a	47.20±4.13 a	1.14±0.11 a	0.28±0.05 a
WT	64	92.66±2.28 a	5.47±0.79 a	1.49±0.48 a	43.20±7.25 a	1.26±0.11 a	0.23±0.04 a

表2 突变体Bc18与及其野生型的产量与产量构成(2008-2009年,杭州)

Table 2 Yields and yield components of Bc18 and its wild type in Hangzhou, 2008-2009.

材料 Material	年份 Year	单株有效穗 No. of effective panicles per plant	每穗总粒数 Spikelet number per panicle	每穗实粒数 Filled grain number per panicle	结实率 Seed-setting rate/%	穗长 Panicle length /cm	千粒重 1000-grain weight /g	产量 Yield /(kg·666.7m^-2)
Bc18	2008	11.93±0.50	149.16±3.33	116.30±6.33	77.96±3.47	22.39±0.73	24.17±0.30	431.06±10.25
	2009	13.55±0.07	151.63±10.95	116.47±14.05	76.68±3.73	24.06±1.39	24.03±0.32	425.76±24.30
	平均 Average	12.74±0.29 a	150.40±7.14 a	116.39±10.19 a	77.32±3.60 a	23.22±1.06 a	24.10±0.31 a	428.41±17.28 a
WT	2008	12.43±0.78	156.07±8.40	118.42±7.94	75.85±1.99	23.23±1.03	24.38±0.18	441.67±9.46
	2009	13.10±0.42	153.44±5.08	110.36±1.62	71.95±1.33	23.83±2.44	24.25±0.21	440.15±27.40
	平均 Average	12.77±0.60 a	154.75±6.74 a	114.39±4.78 a	73.90±1.66 a	23.53±1.73 a	24.32±0.19 a	440.91±18.43 a

图1 突变体Bc18和野生型的表型 A、B、C和D分别为突变体Bc18易折断的茎秆、叶片、叶鞘和稻穗; 箭头表示突变体Bc18各个部分易断裂之处。

Fig. 1. Phenotypes of Bc18 and its wild type. A, B, C and D show broken culm, leaf, leaf sheath and panicle of Bc18, respectively; Arrows indicate easily broken position of Bc18; WT, Wild type.

图2 突变体Bc18和野生型的机械强度 10个测定数据的平均值;** 表示在0.01水平上差异显著;横栏表示标准误。

Fig. 2. Mechanical strength of Bc18 and its wild type. The average values are means of 10 measurements; ** significant at 0.01 level; Bars are standard errors; WT, Wild type.

图3 突变体Bc18和野生型细胞壁组分含量 A-茎秆纤维素、半纤维素和木质素含量; B-叶片纤维素、半纤维素和木质素含量。3个测定数据的平均值;**表示在0.01水平上差异显著;横栏表示标准误。

Fig. 3. Measurement of cell wall components of Bc18 and its wild type. A and B, Cellulose, hemicellulose and lignin contents of total cell wall residues of the culm ( A ) and leaf ( B ) segments from Bc18 and its wild type plants; The average values are means of 3 measurements; ** significant at 0.01 level; The bars refer standard errors.

表3 突变体Bc18 F2分离群体

Table 3 Genetic analyses of Bc18 F2 populations.

组合 Combination	F₁	F₂群体 F₂ population			$χ 3 : 1 2$	P值 P value
组合 Combination	F₁	脆秆单株 Brittle culm			野生型 Wild type	总数 Total
中9B/Bc18 Zhong 9B/Bc18	脆秆 Brittle culm	1278	419	1697	0.04	0.769
Ⅱ-32B/Bc18	脆秆Brittle culm	1754	573	2327	0.09	0.675
Bc18/协青早B Bc18/Xieqingzao B	脆秆 Brittle culm	1455	477	1932	0.05	0.753
Bc18/明恢63 Bc18/Minghui 63	脆秆 Brittle culm	1817	595	2412	0.07	0.707
密阳46/Bc18 Milyang 46/Bc18	脆秆Brittle culm	1045	358	1403	0.10	0.655
Bc18/中恢218 Bc18/Zhonghui 218	脆秆Brittle culm	2089	703	2792	0.02	0.827

表3 突变体Bc18 F2分离群体

Table 3 Genetic analyses of Bc18 F2 populations.

组合 Combination	F₁	F₂群体 F₂ population			$χ 3 : 1 2$	P值 P value
组合 Combination	F₁	脆秆单株 Brittle culm			野生型 Wild type	总数 Total
中9B/Bc18 Zhong 9B/Bc18	脆秆 Brittle culm	1278	419	1697	0.04	0.769
Ⅱ-32B/Bc18	脆秆Brittle culm	1754	573	2327	0.09	0.675
Bc18/协青早B Bc18/Xieqingzao B	脆秆 Brittle culm	1455	477	1932	0.05	0.753
Bc18/明恢63 Bc18/Minghui 63	脆秆 Brittle culm	1817	595	2412	0.07	0.707
密阳46/Bc18 Milyang 46/Bc18	脆秆Brittle culm	1045	358	1403	0.10	0.655
Bc18/中恢218 Bc18/Zhonghui 218	脆秆Brittle culm	2089	703	2792	0.02	0.827

图4 与Bc18基因连锁的SSR标记RM7419的扩增结果 1-Bc18; 2-02428; 3-F1(02428×Bc18); 4~24-F2 不脆单株。

Fig. 4. Electrophoresis of the SSR marker RM7419 linked with the Bc18 locus. Lane 1, Bc18; Lane 2, 02428; Lane 3,F1 (02428/Bc18); Lanes 4 to 24, Non-brittle culm individuals in F2 population.

表4 用于Bc18基因精细定位的分子标记

Table 4 Markers used for fine mapping of the Bc18 gene.

标记 Marker	引物序列(5'-3') Primer sequence (5'-3')	产物大小 Product size in Nipponbare/bp
PBC4	F:GAACGTTTACAAGACTCT	124
	R:ACACAACTACTTCACTGC
PBC21	F:TCCCACCACCTCAATCTC	116
	R:CCCTACGACTCCAGCTAAT
PBC22	F:TCGCATATTCAGATAAGCATC	108
	R:TAATCGGAAAGAAGGCAAT
PBC33	F:GCTTGTTGCTGTTGCCTCTT	151
	R:TCACGGCAGAATCGCACA
PBC31	F:CTACAAAGGCACGATGATGA	185
	R:AACGCAACCTAAAAGACACTAA
PBC25	F:CTTGAGCCATTATGTGCA	166
	R:GACTCACGGACGCTATCA

图5 Bc18基因在第1染色体上的位置

Fig. 5. Location of the Bc18 gene on the chromosome 1 of rice.

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