水稻雄性不育突变体ms102的鉴定和基因定位

doi:10.11983/CBB21158

摘要/Abstract

摘要： 水稻(Oryza sativa)隐性核雄性不育突变体是第三代杂交水稻技术的核心。为了挖掘优质雄性不育突变体, 该研究通过筛选优质籼稻黄华占(HHZ)的甲基磺酸乙酯(EMS)诱变突变体库, 获得1个雄性不育突变体ms102 (male sterility mutant 102)。该突变体营养生长正常, 但花药不开裂, 花粉败育。细胞学分析表明, 突变体花药绒毡层不能正常降解, 导致小孢子发育异常; 遗传分析表明, 该突变体的不育表型由1个已报道编码酰基转移酶的DPW2基因突变造成。研究获得了1个隐性核雄性不育突变体, 进一步证实了DPW2基因在水稻花药发育中的功能。

关键词: 水稻, 雄性不育, 基因克隆

Abstract: The recessive nuclear male sterility line is the base of the third-generation hybrid rice breeding technology. To explore new male sterility lines suitable for the breeding technology, we screened an ethyl methylsulfonate (EMS) mutant library derived from the elite xian variety Huanghuazhan (HHZ) and identified male sterility mutant 102 (ms102). The ms102 showed a normal vegetative development, but exhibited defective anther dehiscence and pollen abortion. Cytological analyses revealed that the degradation of the tapetum was abnormal in ms102, resulting in the abortion of microspores. Genetic analyses showed that the male sterile phenotype of ms102 was resulted from the mutation of a known male sterility gene Defective Pollen Wall 2 (DPW2), which encodes an acyl transferase. Thus, this study identified a recessive nuclear male sterility mutant, and further confirmed the function of DPW2 in rice pollen development.

Key words: rice, male sterility, gene cloning

王霞, 严维, 周志勤, 常振仪, 郑敏婷, 唐晓艳, 吴建新. 水稻雄性不育突变体ms102的鉴定和基因定位. 植物学报, 2022, 57(1): 42-55.

Xia Wang, Wei Yan, Zhiqin Zhou, Zhenyi Chang, Minting Zheng, Xiaoyan Tang, Jianxin Wu. Identification and Mapping of a Rice Male Sterility Mutant ms102. Chinese Bulletin of Botany, 2022, 57(1): 42-55.

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链接本文: https://www.chinbullbotany.com/CN/10.11983/CBB21158

https://www.chinbullbotany.com/CN/Y2022/V57/I1/42

图/表 13

表1 qRT-PCR引物序列

Table 1 Sequences of primers for qRT-PCR

Primers	Sequences (5′-3′)
OsACTIN-qPCR-F	GCTATGTACGTCGCCATCCA
OsACTIN-qPCR-R	GGACAGTGTGGCTGACACCAT
RTS-qPCR-F	ACATGTGGACTCGCTTGACT
RTS-qPCR-R	CATGGCTGCATGCAGATTCAT
TDR-qPCR-F	TGCTCTGGGAGCACAAGCC
TDR-qPCR-R	CTCGCTGTCCCTCACCATG
UDT1-qPCR-F	GAAGCACTCTGCAGCTAC
UDT1-qPCR-R	CTGCGTAGCCGTAGAAGG
DTM1-qPCR-F	AATTTAAGCCCTATCCCCTAAG
DTM1-qPCR-R	GAAGGTGGATTCCACTAGCTA
PTC1-qPCR-F	CACCAGATCATGGACCTCTG
PTC1-qPCR-R	AGCAGCCTCAGCTCCATGTG
DPW-qPCR-F	ACCACAGGAGACACGGTGATG
DPW-qPCR-R	CTTGGCCTGATGGTGACAA
PAIR1-qPCR-F	GGATGGACCCAGATTAACC
PAIR1-qPCR-R	CTGTTTAGGTGCCACCCTGT
PAIR2-qPCR-F	TGCCAGAGGAGAGGACCATTC
PAIR2-qPCR-R	CACGAGATGCTTGCTATTGAC
DTD-qPCR-F	TCATGAGTCTTTGAGCCGCA
DTD-qPCR-R	CACCTTCGCTCCAAATCTGT
OsCP1-qPCR-F	TGCAAGGATCACTCCACCTG
OsCP1-qPCR-R	GGCTGTTCTTGCTCTTGGAG
OsMSP1-qPCR-F	AGCATCGCGAGTAAGATGC
OsMSP1-qPCR-R	GTAGCCTTGTAACTTCAAGTAGGA
CYP703A3-qPCR-F	TGTACTGCTTTCTTTGCCCG
CYP703A3-qPCR-R	GCAGCAATCATGTCCTGCAT
CYP704B2-qPCR-F	GCTGGTTGATGACTTCACCT
CYP704B2-qPCR-R	CGACAGTATGTCGTGCTTGAT
OsGAMYB-qPCR-F	GCGACGGTATCATGTTCAAT
OsGAMYB-qPCR-R	GTCGCATAAGAGAACATCTG

表2 高分辨率溶解曲线(HRM)引物序列

Table 2 Sequences of primers for high resolution melting (HRM)

Primers	Sequences (5'-3')
S1-HRM-F	GACAGGCAAGCAGAAAACCTG
S1-HRM-R	GTGCAGGTAGCACATCATCCT
S2-HRM-F	CTACAGATATATACAGGACGGTG
S2-HRM-R	GCTAAGGTAGCAACGTACACAG
S3-HRM-F	GATGGAGACTCACGTCCAGA
S3-HRM-R	CCACAGCCCTCATTTTCGT

图1 水稻ms102突变体表型分析 (A) 成熟期植株的表型(bar=9 cm); (B) 成熟期野生型(WT)和ms102突变体分蘖数统计(n=23); (C) 扬花期野生型和ms102突变体叶色(bar=1 cm); (D) 去掉颖壳的成熟小花(bar=1 mm); (E) 扬花期的小花, 箭头指示开裂的花药(bar=1 cm); (F), (G) 野生型和ms102突变体的成熟花粉I2-KI染色(bars=50 μm); (H) 成熟期稻穗(bar=2.4 cm)

Figure 1 Phenotype analysis of the rice ms102 (A) Phenotype of mature plants (bar=9 cm); (B) Tiller number of mature wild type (WT) and ms102 plant (n=23); (C) Leaf color of WT and ms102 plant at flowering (bar=1 cm); (D) Mature spikelet without palea and lemma (bar=1 mm); (E) Flowering spikelet, the arrow indicates a dehisced anther (bar=1 cm); (F), (G) I2-KI-stained pollen of WT and ms102 mutant (bars=50 μm); (H) Mature panicles (bar=2.4 cm)

表3 水稻WT × ms102杂交F2代表型分离比

Table 3 Segregation of the F2 progeny of rice WT × ms102

Fertile plants	Male sterility plants	χ²(3:1)	χ²_0.05
359	102	2.034	3.841

图2 水稻野生型(WT)和ms102突变体7-12时期花药切片 BMs: 二核小孢子; DMs: 降解小孢子; E: 外皮层; En: 内皮层; M: 中层; Mp: 成熟花粉; Ms: 小孢子; PMC: 花粉母细胞; ST: 膨大绒毡层; T: 绒毡层; Tds: 四分体。Bars=10 μm

Figure 2 Transverse sections images of WT and ms102 anthers at the developmental stages 7-12 BMs: Binuclear microspores; DMs: Degenerated microspores; E: Epidermis; En: Endothecium; M: Middle layer; Mp: Mature pollen; Ms: Microspores; PMC: Pollen mother cell; ST: Swollen tapetum; T: Tapetum; Tds: Tetrads. Bars=10 μm

图3 水稻野生型(WT)和突变体(ms102)花药第9-12时期透射电镜超微结构 (A), (D) 第9时期; (B), (E) 第10时期(箭头指示小孢子外壁); (C), (F) 第12时期; (G), (H) 分别为B和E图中虚线框的放大部分(箭头指示乌氏体); T: 绒毡层; Ms: 小孢子; C: 角质层。(A), (B), (D), (E) Bars=2 μm; (C), (F), (G), (H) Bars =1 μm

Figure 3 Transmission electron microscope images of WT and ms102 anthers at the developmental stages 9-12 (A), (D) Stage 9; (B), (E) Stage 10 (the arrows indicate the microspore wall); (C), (F) Stage 12; (G), (H) The magnified region of dashed box in B and E (the arrows indicate the Ubisch bodies); T: Tapetum; Ms: Microspores; C: Cuticle. (A), (B), (D), (E) Bars=2 μm; (C), (F), (G), (H) Bars=1 μm

图4 水稻野生型(WT)和ms102突变体花药发育第12时期扫描电子显微镜超微结构 (A), (C), (E), (G) 花药外表面; (B), (F) 花粉粒; (D), (H) 乌氏体。(A), (E) Bars=100 μm; (B), (C), (F), (G) Bars=10 μm; (D), (H) Bars=2 μm

Figure 4 Scanning electron microscope images of WT and ms102 anthers at developmental stage 12 (A), (C), (E), (G) The external surfaces of anther wall; (B), (F) Pollen grains; (D), (H) Ubisch bodies. (A), (E) Bars =100 μm; (B), (C), (F), (G) Bars=10 μm; (D), (H) Bars=2 μm

图5 花药发育相关基因在野生型和ms102花药中的表达通过qRT-PCR检测野生型与ms102突变体中花药发育7-12时期相关基因的表达量。以OsACTIN1作为内参, 数据为平均值±标准差(n=3)。* P<0.05, ** P<0.01, *** P<0.001

Figure 5 Expression of genes related to pollen development in WT and ms102 anthers Anthers at stages 7-12 were used for qRT-PCR. OsACTIN1 served as an internal control. Data are shown as means±SD (n=3). * P<0.05, ** P<0.01, *** P<0.001

表4 候选基因及注释信息

Table 4 Candidate genes and annotation information

Chr. locus	Physical location	Genotype	SNP index	Gene
Chr. 1	39,147,186 (S1)	C→T	0.8966	Intergenic
Chr. 1	39,167,971 (S2)	G→A	0.8966	Intergenic
Chr. 1	40,599,831 (S3)	C→T	0.9474	LOC_Os01g7-0140-LOC_O-s01g70150

表5 3个SNP候选位点基因型-表型连锁分析

Table 5 Genotype-phenotype correlation analyses of the three candidate SNP sites

	The number of male sterile plants			The number of fertile plants
Physical location	S1	S2	S3	S1	S2	S3
Wild type	1	1	0	25	25	23
Heterozygote	8	8	0	19	19	21
Mutant	44	44	53	0	0	0

图6 水稻LOC_Os01g70140和LOC_Os01g70150基因结构及其突变体表型 (A) LOC_Os01g70140和LOC_Os01g70150基因结构及CRISPR突变体突变位点; (B)-(D) 花粉成熟期花药和雌蕊形态(bars=1 mm); (E)-(G) 成熟花粉粒I2-KI染色(bars=100 μm)。

Figure 6 Gene structures of LOC_Os01g70140 and LOC_Os01g70150 and mutant phenotypes (A) The gene structures and mutation sites of LOC_Os01g70140 and LOC_Os01g70150; (B)-(D) Morphology of mature anther and pistil (bars=1 mm); (E)-(G) Mature pollen grains stained with I2-KI (bars=100 μm).

图7 水稻ms102突变体中DPW2基因突变位点分析黑色三角形指示碱基替换突变位点, 虚线框指示缺失的核苷酸序列。

Figure 7 The mutation sites of DPW2 in ms102 The black arrow indicates the replaced nucleotide, the dashed box indicates the deleted nucleotides.

图8 水稻突变体dpw2/+ × ms102 F1的表型 (A)-(D) 花粉成熟期花药和雌蕊形态(bars=1 mm); (E)-(H) 成熟花粉粒I2-KI染色(bars=100 μm)。

Figure 8 The phenotype of the F1 progeny of dpw2/+ × ms102 The phenotype of the F1 progeny of dpw2/+ × ms102 (A)-(D) Morphology of mature anther and pistil (bars=1 mm); (E)-(H) Mature pollen grains stained with I2-KI (bars=100 μm).

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