Identification and Mapping of a Rice Male Sterility Mutant ms102

doi:10.11983/CBB21158

Abstract

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

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

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URL: https://www.chinbullbotany.com/EN/10.11983/CBB21158

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

Figures/Tables 13

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

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

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)

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

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

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

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

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

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

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

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).

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

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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