Identification and Gene Mapping of Male Sterile Mutant gamyb5 in Rice

doi:10.16819/j.1001-7216.2016.5154

Chinese Journal OF Rice Science ›› 2016, Vol. 30 ›› Issue (2): 143-151.DOI: 10.16819/j.1001-7216.2016.5154

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Identification and Gene Mapping of Male Sterile Mutant gamyb5 in Rice

Zheng-fu YANG¹, Ying-xin ZHANG¹, Lian-ping SUN^1,², Pei-pei ZHANG¹, Dan-dan XUAN¹, Ling LIU^1,³, Xia HU¹, Zi-he LI¹, Xiao-deng ZHAN¹, Wei-xun WU¹, Li-yong CAO^1,^*(), Shi-hua CHENG^1,^*()

¹National Center for Rice Improvement/ Key Laboratory for Zhejiang Super Rice Research, China National Rice Research Institute, Hangzhou 311401, China
²College of Agronomy,Shenyang Agricultural University, Shenyang 110866, China
³College of Agronomy, He'nan Agricultural University, Zhengzhou 450002, China

Received:2015-10-19 Revised:2015-12-11 Online:2016-03-10 Published:2016-03-10
Contact: Li-yong CAO, Shi-hua CHENG
About author:
# These authors contributed equally to this work;

水稻雄性不育突变体gamyb5的鉴定与基因定位

杨正福¹, 张迎信¹, 孙廉平^1,², 张沛沛¹, 轩丹丹¹, 刘嶺^1,³, 胡霞¹, 李紫荷¹, 占小登¹, 吴玮勋¹, 曹立勇^1,^*(), 程式华^1,^*()

¹中国水稻研究所/国家水稻改良中心/浙江省超级稻研究重点实验室, 杭州 311401
²沈阳农业大学农学院, 沈阳 110866
³河南农业大学农学院, 郑州 450002

通讯作者: 曹立勇,程式华
作者简介:
# 共同第一作者;
基金资助:
农业部农业公益性行业科技专项(20140302, 20150308);国家转基因重大专项(2014ZX08001-002);浙江省自然科学基金资助项目(Q13C130009);中国农业科学院创新工程超级稻育种创新团队、水稻杂种优势机理研究创新团队项目(CAAS-ASTIP-2013-CNRRI);国家自然科学基金资助项目(31501290)

Abstract

Abstract:

The gamyb5, a pollen-free male sterile mutant was identified from the mutant library of ⁶⁰Co-γ-treated indica cultivar Zhonghui 8015. There was no significant difference in agronomic traits such as plant type, plant height and tiller number between gamyb5 and the wild-type. But gamyb5 exhibited slender and white anthers without mature pollen grains. Observation results of anther cross-sections exhibited that the microspore mother cells failed to form functional tetrads and microspores in gamyb5. Moreover, tapetal cells of gamyb5 abnormally enlarged and the tapetum programmed cell death (PCD) was delayed. gamyb5, as the pollen acceptor, was crossed with the wild type Zhonghui 8015 and a japonica cultivar 02428, respectively. Genetic analysis of all hybridization populations indicated that gamyb5 was controlled by a single recessive nuclear gene which was mapped on the long arm of chromosome 1. With developed SSR, Indel markers and F₂ mapping population of gamyb5/02428, the gene was fine mapped to a region of 16-kb on the long arm of chromosome 1 between markers ZF-29 and ZF-31. Sequence analysis of the two open reading frames in this region revealed that the LOC_Os01g0812000, which encodes a gibberellin-induced MYB transcription factor, had an 8 nucleotides bases deletion in the second extron probably responsible for the male sterility phenotype. Additionally, real-time fluorescent quantitative PCR analysis showed that the expression level of the important regulators UDT1, TDR, CYP703A3 and CYP704B2 in anther decreased significantly in gamyb5. Together, these results suggest GAMYB plays key roles in anther meiosis and tapetum PCD.

Key words: rice, male sterility, gene mapping, gamyb5

摘要：

在⁶⁰Co-γ射线辐射诱变籼稻中恢8015的突变体库内发现了一个无花粉型雄性不育突变体gamyb5。gamyb5 的株型、株高、分蘖数等农艺性状与野生型中恢8015无差异,而其花药细长且呈白色半透明状,花药中无花粉粒。花药半薄切片观察结果表明,gamyb5的小孢子母细胞减数分裂异常,没有形成正常的四分体和小孢子,并且绒毡层异常伸长,细胞程序性死亡延迟。对以gamyb5 为母本,与野生型中恢8015 和广亲和粳稻品种02428分别配制的杂交组合遗传分析表明,gamyb5 突变性状受一个隐性核基因控制。利用gamyb5 和02428 杂交的F₂定位群体,最终将突变基因精细定位于第1染色体长臂的ZF-29和ZF-31两个标记之间,物理距离约16.9 kb。对该区域内2个完整的开放阅读框测序分析发现,编码受赤霉素诱导的MYB转录因子基因LOC_Os01g0812000的第2外显子存在8个碱基的缺失,导致翻译提前终止。qRT-PCR检测到影响花药发育的调控因子UDT1、TDR、CYP703A3和CYP704B2的表达量在突变体中比野生型中极显著降低。进一步证明GAMYB在花药减数分裂和绒毡层细胞程序性死亡过程中起关键作用。

关键词: 水稻, 雄性不育, 基因定位, gamyb5

CLC Number:

Zheng-fu YANG, Ying-xin ZHANG, Lian-ping SUN, Pei-pei ZHANG, Dan-dan XUAN, Ling LIU, Xia HU, Zi-he LI, Xiao-deng ZHAN, Wei-xun WU, Li-yong CAO, Shi-hua CHENG. Identification and Gene Mapping of Male Sterile Mutant gamyb5 in Rice[J]. Chinese Journal OF Rice Science, 2016, 30(2): 143-151.

杨正福, 张迎信, 孙廉平, 张沛沛, 轩丹丹, 刘嶺, 胡霞, 李紫荷, 占小登, 吴玮勋, 曹立勇, 程式华. 水稻雄性不育突变体gamyb5的鉴定与基因定位[J]. 中国水稻科学, 2016, 30(2): 143-151.

Figures/Tables 6

Table 1 Makers used for fine mapping and sequencing.

引物名称 Primer name	上游引物 Forward primer (5'-3')	下游引物 Reverse primer (5'-3')	实验目的 Purpose
RD0114	GCTTGTGGCAATTGGG	CGCCTGATGGATGTCG	精细定位Fine mapping
ZF-26	GATCATACCATCATCAGAAGCAGT	CAAGCAGCCCACATCACAGC	精细定位Fine mapping
ZF-28	TCTCGCTCCTCTTCGCCCAAAC	ACCCGCACCAGTACGTGACCC	精细定位Fine mapping
ZF-29	GGCAGCAGCAATCACAACAC	TCGGCACTTTCTTTACTCAACT	精细定位Fine mapping
ZF-31	CAGCCAGGCACCGCAGCAAA	AAGAAAGGCAGAGCAAGAAGG	精细定位Fine mapping
ZF-32	GCACGCTTCTCGCTACGCTAT	GCTCCAGGAACACCACCAACTC	精细定位Fine mapping
ZF-38	TGGGTGCGAGGTTTCTGTGA	AATGAATGCGTGTTTCCTGT	精细定位Fine mapping
InDel15	CAACCCCTCCAAATACCTGA	ACCGTGTTCATGCCTTTCAC	精细定位Fine mapping
GAMYB5	ACTGGAGCACTGCTGATCTTC	GCACATCTTCTCAGTTGCCAG	gamyb5位点检测gamyb5 deletion site detection
cDNA	CCCCTGCGAGTCCAATCTAC	ACCGGCTTATCTCCATGCAC	cDNA测序cDNA sequencing

Fig. 1. Phenotypic comparison of wild type Zhonghui 8015(WT)and gamyb5 . A, Plant of Zhonghui 8015 and the gamyb5 mutant at the heading stage; B, Panicle of Zhonghui 8015 and the gamyb5 mutant; C, Spikelet of the wild type Zhonghui 8015 and gamyb5 ; D, Anther of Zhonghui 8015 and gamyb5 mutant; E, Pollen fertility of Zhonghui 8015 and gamyb5.

Table 2 Genetic analysis of the gamyb5 mutant.

组合 Cross	$F 1$ 结实率 Seed-setting rate of F₁ /%	F₂		$χ 3 ∶ 1 2$	$χ 0.05 2$
组合 Cross	$F 1$ 结实率 Seed-setting rate of F₁ /%	野生型植株数 Number of wild type plants		$χ 3 ∶ 1 2$	突变体植株数 Number of mutant plants
gamyb5/02428	86.8	1895	642	0.14	3.84
gamyb5/中恢8015 gamyb5/Zhonghui 8015	87.2	234	70	0.63
中恢8015/02428 Zhonghui 8015/02428	87.6	960	0	-

Table 2 Genetic analysis of the gamyb5 mutant.

组合 Cross	$F 1$ 结实率 Seed-setting rate of F₁ /%	F₂		$χ 3 ∶ 1 2$	$χ 0.05 2$
组合 Cross	$F 1$ 结实率 Seed-setting rate of F₁ /%	野生型植株数 Number of wild type plants		$χ 3 ∶ 1 2$	突变体植株数 Number of mutant plants
gamyb5/02428	86.8	1895	642	0.14	3.84
gamyb5/中恢8015 gamyb5/Zhonghui 8015	87.2	234	70	0.63
中恢8015/02428 Zhonghui 8015/02428	87.6	960	0	-

Fig.2. Positional cloning of the gamyb5 mutated gene. A, Preliminary mapping of gamyb5; B, Fine mapping of gamyb5; C, Prediction of the candidate gene within the fine-mapped region; D, The structure of candidate gene LOC_Os01g0812000 and the mutation site in gamyb5 mutant (8 bases deleted).

Fig. 3. Transverse section analyses of the wild type and gamyb5 anthers in various of developmental stages. A to E, Wild type; G to L, The gamyb5 mutant. A and G, Cross-section of anthers at the stage 7; B and H, Cross-section of anthers at the stage 8; C and I, Cross-section of anthers at the stage 9; D and J, Cross-section of anthers at the stage 10; E and K, Cross-section of anthers at the stage 11; F and L, Cross-section of anthers at the stage 12. Ep, Epidermis; En, Endothecium; ML, Middle layer; T, Tapetum; Ms, Microsporocyte; Dy, Dyad cell; Msp, Microspore; BP, Biceullar pollen; MP, Mature pollen. Bars = 20 μm.

Fig. 4. Expression analysis of male sterility-associated genes of the wild type and the mutant. **significant at P=0.01.

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Identification and Gene Mapping of Male Sterile Mutant gamyb5 in Rice

水稻雄性不育突变体gamyb5的鉴定与基因定位

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