水稻半显性矮秆基因Si-dd1的表型分析和精细定位

doi:10.16819/j.1001-7216.2016.5113

摘要/Abstract

摘要：

以粳稻品种日本晴在组织培养过程中分离出来的一个半显性矮秆突变体Si-dd1为研究对象,通过形态学分析,发现与野生型日本晴相比,矮秆Si-dd1(AA)和半矮秆Si-dd1(Aa)植株的株高降低。结实率下降,生育期延长,一次枝梗和二次枝梗增加。激素处理结果表明突变体Si-dd1(AA)与野生型对油菜素内酯反应基本相同,而在高浓度赤霉素处理下,突变体Si-dd1(AA)表现为一定程度上的钝感。Western blot对GID2表达量分析也确定这一结果。组织切片实验表明,突变体Si-dd1(AA)相对于野生型叶片主脉气孔变小,叶肉细胞增多,茎维管束数目增加。遗传分析结果表明该突变体基因受一对核基因控制。进一步利用分子标记将该基因定位在水稻第6染色体约244 kb区间内,目前该区段并未发现已报道的矮秆相关基因。

关键词: 水稻, 半显性, 矮秆, 遗传分析, 图位克隆

Abstract:

A semi-dominant dwarf rice mutant (termed as Si-dd1) was obtained from japonica rice variety Nipponbare by tissue culture mutagenesis. Morphological analysis showed that Si-dd1(AA) and Si-dd1(Aa) exhibited shorter plant height, decreased seed-setting rate, delayed heading, increased number of primary rachis branch and secondary rachis branch compared to the wild type (WT,Nipponbare). Physiological test results showed Si-dd1 had similar response to 24-Epibrassinosteroid to the WT, while it was insensitive to Gibberellin at 10^-4μmol/L. Moreover, Western blot results also confirmed GID2 expression level in Si-dd1 differed greatly from WT. The cytological analysis showed that Si-dd1 had smaller stomas in the leaf midrib, increased mesophyll cells and stem vascular bundles compared to WT. Genetic analysis and map-based clone showed that the Si-dd1 is controlled by a single semi-dominant gene, and locates in a 244 kb region on chromosome 6.

Key words: rice, semi-dominant, dwarf, genetic analysis, map-based clone

中图分类号:

Q755
S511.01

崔永涛, 吴立文, 胡时开, 任德勇, 葛常伟, 叶卫军, 董国军, 郭龙彪, 胡兴明. 水稻半显性矮秆基因Si-dd1的表型分析和精细定位[J]. 中国水稻科学, 2016, 30(2): 152-160.

Yong-tao CUI, Li-wen WU, Shi-kai HU, De-yong REN, Chang-wei GE, Wei-jun YE, Guo-jun DONG, Long-biao GUO, Xing-ming HU. Phenotypical Analysis and Fine Mapping of a Semi-dominant Dwarfism Gene Si-dd1 in Rice[J]. Chinese Journal OF Rice Science, 2016, 30(2): 152-160.

图/表 10

表1 野生型, Si-dd1(Aa) 和Si-dd1(AA)分蘖、株高及穗部性状比较

Table 1 Comparison of tiller, plant height and panicle traits among wild type, Si-dd1(Aa) and Si-dd1(AA).

材料 Material	株高 Plant height /cm	分蘖数 Tiller number	一次枝梗 Number of primary rachis branches	二次枝梗 Number of secondary rachis branches	结实率 Seed-setting rate/%	总粒数 Total grain number
野生型WT	83.8 a	18.7	8.0	9.0 a	88.8 a	72.7 a
半矮秆Si-dd1(Aa)	60.6 b	16.7	8.0	16.3 b	61.3 b	90.7 b
矮秆Si-dd1(AA)	43.7 c	16.3	9.6	16.3 b	1.1 c	97.3 b

图 1 Si-dd1突变体成熟期表型 A-整株表型; B-穗表型; C-茎节间长; D-各节间对比。从左至右依次为野生型、中间型和突变型。

Fig. 1. Phenotypes of Si-dd1 at maturation stage. A, Whole plant; B, Panicle; C, Internode; D, Comparison of every internode. From left to right,wild type, Si-dd1(Aa) and Si-dd1(AA) in turn.

表 2 Si-dd1(AA)与NJ06、9311组合F2的遗传分析

Table 2 Genetice analysis of F2 population of Si-dd1(AA) / NJ06(9311).

组合 Cross	F₁	F₂				χ²(1:2:1)
组合 Cross	F₁	总株数 Total of plants		正常高秆 Normal plants	半矮秆 Semi-dwarf	矮秆 Dwarf
Si-dd1(AA)/9311	半矮秆 Semi-dwarf	184	48	94	42	0.478
Si-dd1(AA)/NJ06	半矮秆 Semi-dwarf	243	62	122	59	0.078
NJ06/Si-dd1(AA)	半矮秆 Semi-dwarf	193	47	100	46	0.264
9311/Si-dd1(AA)	半矮秆 Semi-dwarf	290	72	150	68	0.455

表3 杂合Si-dd1(Aa)与NJ06、9311组合F1遗传分析

Table 3 Genetice analysis of F1 population of Si-dd1(Aa) /NJ06 and Si-dd1(Aa) /9311.

组合 Cross	野生型 Wild type	半矮秆 Semi-dwarf	总株数 Total	χ²(1:1)
Si-dd1(Aa)/ 9311	54	48	102	0.353
Si-dd1(Aa)/ NJ06	59	54	113	0.220
9311 /Si-dd1(Aa)	52	45	97	0.505
NJ06 /Si-dd1(Aa)	52	48	100	0.160

表4 Si-DD1基因初定位的标记

Table 4 Makers for primary mapping of Si-DD1.

引物名称 Primer name	引物序列(5'-3') Forward primer(5'-3')	引物序列(5'-3') Reverse primer(5'-3')
M1	CAGTCTTGCTCCGTTTGTTG	CTGTGACTGACTTGGTCATAGG
M2	ATCGCAGCAATGCCTCGTG	TGCGTTTGTGTTTGGCTCG
M3	CTCAACGTTGACACCTCGTG	TCCTCCATCGAGCAGTATCA

表5 Si-DD1基因精细定位的部分标记

Table 5 Makers for fine mapping of Si-DD1.

引物名称 Primer name	引物序列(5'-3') Forward primer(5'-3')	引物序列(5'-3') Reverse primer(5'-3')
P1	GGTCGCAGCTTGAATTAATGA	GCAATCTCATTTGTTGAGAACC
P2	TGATGTTTGGCACATACTTGC	GCAAACTTTCTGATAAGGAATAG
P3	CTCCAAAGCTGACAATGGTG	TGAGAAGGAGTAGGAAGCATAACA
P4	GGTACTAACCATGTGATTGAG	CACCTGAATTACCGTATATG
P5	CGTAGGAGTCGACGCTGTC	CCCAATCCGCTGTGGTTTT
P6	GCAGGTTGTAATGGAGGTGAA	CGGCGAGCCATATTGTTTAT

图2 Si-DD1的精细定位 A-Si-DD1与标记M2和M3连锁; B-利用4500个分离单株将Si-DD1定位到244 kb区段内。

Fig. 2. Fine mapping of Si-DD1. A,Si-DD1 was linkaged with M2 and M3; B,Si-DD1 was mapped to 244 kb genome region based on 4500 separated plants.

图3 Si-dd1突变体的GA, BR激素处理结果 A-不同浓度赤霉素(GA)处理后表型; B-不同浓度GA处理后株高; C-不同浓度油菜素内酯(BR)处理后叶夹角。

Fig. 3. Si-dd1 exposed to GA and BR. WT,Wild type; GA, Gibberellic acid; BR,Brassinolide. A, Plant phenotype after GA treatment at different concentrations; B, Plant height after GA treatment at different concentrations; C, Leaf angle after BR treatment at different concentrations.

图4 GID2蛋白表达量分析

Fig. 4. Expression analysis of GID2.

图5 野生型, Si-dd1(Aa) 和Si-dd1(AA)茎和叶的显微结构比较 A,B,C-野生型(WT), Si-dd1(Aa) 和Si-dd1(AA)叶主脉横切; D,E,F- 野生型, Si-dd1(Aa) 和Si-dd1(AA)倒2节间纵切; G,H,I- 野生型, Si-dd1(Aa) 和Si-dd1(AA)倒2节间横切; J,K-维管束(1/4)和茎纵切细胞数统计。

Fig. 5. Comparison of microscopic structure among WT, Si-dd1(Aa) and Si-dd1(AA). A,B,C, Cross sections of leaf main vein of WT, Si-dd1(Aa) and Si-dd1(AA); D,E,F, Longitudinal section of the second internode from the top of WT, Si-dd1(Aa) and Si-dd1(AA); G,H,I, Cross sections of the second internode from the top of WT, Si-dd1(Aa) and Si-dd1(AA). J,K, Numbers of vascular bundles and longitudinal stem cells.

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