Cytological Observation of a Female and Male Sterile Osfma2 Mutant in Rice and Its Map-based Cloning

doi:10.16819/j.1001-7216.2022.210312

Abstract

Abstract:

【Objective】 The work aims to locate and clone the rice female and male sterile gene OsFMA2 by constructing a segregated population and explore its function in the regulation of rice fertility. 【Method】 Stable sterile mutants were obtained by EMS mutagenesis of japonica rice Ningjing 4, and the phenotypic and cytological observation of the mutant was conducted. Using map-based cloning and Mut-map methods, the female and male sterile gene OsFMA2 was fine-mapped and cloned. The expression pattern of OsFMA2 in various tissues was analyzed by the real-time quantitative PCR technique. Subcellular localization of OsFMA2 protein was performed with the rice protoplast expression system. 【Result】 The cytological observation revealed that the Osfma2 mutant was male- and female-sterile, and the extreme individuals were used to locate the gene in a 448-kb interval on the long arm of chromosome 6. The gene OsFMA2 is predicted to encode a replication protein that is highly conserved in monocots. The gene OsFMA2 was tissue-specific and was highly expressed in young panicles. Subcellular localization analysis showed that the OsFMA2 protein was localized on the nucleus. 【Conclusion】The gene OsFMA2 is highly expressed in young panicles, and it may participate in the homologous recombination of the first meiotic division of male gamete. At the same time, the expression of OsFMA2 also has an adverse effect on the development of female gametes.

Key words: rice, male and female sterility, gene cloning, tissue analysis, subcellular localization

摘要：

【目的】通过构建分离群体定位并克隆水稻雌雄不育基因OsFMA2,并对其在水稻育性调控中的功能进行探究。【方法】通过EMS诱变水稻宁粳4号得到稳定的不育突变体,对突变体进行表型及细胞学观察,利用精细定位和Mut-map相结合的方法克隆了水稻雌雄不育基因OsFMA2,通过实时荧光定量PCR检测其在各组织表达水平差异,利用水稻原生质体表达系统进行OsFMA2蛋白的亚细胞定位。【结果】细胞学观察发现突变体为雌雄不育。利用极端个体将其精细定位于水稻第6染色体长臂448 kb的区间内。结合全基因组重测序结果,最终确定OsFMA2为目标基因。该基因编码一个复制蛋白,在单子叶植物中高度保守。OsFMA2具有组织特异性,在幼穗中高表达。亚细胞定位结果显示OsFMA2蛋白定位于细胞核中。【结论】OsFMA2在幼穗中高表达,可能参与了水稻雄配子第一次减数分裂前期同源重组过程,同时,OsFMA2的表达对雌配子发育也产生了不利影响。

关键词: 水稻, 雌雄不育, 基因克隆, 组织表达, 亚细胞定位

ZHANG Taohui, WANG Haiyu, WAN Hua, ZHANG Liping, XIE Zhenwei, CHEN Keyi, HE Xiaodong, ZHAO Zhigang, WAN Jianmin. Cytological Observation of a Female and Male Sterile Osfma2 Mutant in Rice and Its Map-based Cloning[J]. Chinese Journal OF Rice Science, 2022, 36(1): 13-26.

张涛荟, 王海宇, 万华, 张莉萍, 谢振威, 陈可毅, 何晓栋, 赵志刚, 万建民. 水稻雌雄不育突变体Osfma2的细胞学观察及基因图位克隆[J]. 中国水稻科学, 2022, 36(1): 13-26.

Figures/Tables 13

Table 1 Fine-mapping and quantitative primer sequences used in the study.

引物名称	正向引物序列	反向引物序列	实验目的
Name	Forward sequence(5’-3’)	Reverse sequence(5’-3’)	Purpose
RM162	GCCAGCAAAACCAGGGATCCGG	CCGCAAGCCGCACAAGACCTTG	精细定位 Fine mapping
H971-5	CCGCCCGCGTTTTATTTACT	GGCAACATTCTTCGGCCTC	精细定位 Fine mapping
HF-2	CGCCGCCAAGAGCTAATTAA	TGAGAGATCTCGATCGACTTCTC	精细定位 Fine mapping
HF-3	GCCTATGTTTAGCCGCGAAA	GGAAGGAGAAGTTGAGGGGG	精细定位 Fine mapping
Hi-1	CCGGACCGTGATTTCGTTAG	TCAATACTAAAATCTTCGCCCCT	精细定位 Fine mapping
Hh-4	CTTTCTCCCCGTCGATCCTT	GCCCCACGGAGCATATCTAG	精细定位 Fine mapping
Hh-9	GGCAGGAAGTCCAAAAAGCT	TCCATAAAGCAAGCTGATGCA	精细定位 Fine mapping
He-6	CGTAGGCGAGGTGGTACAAT	GAGAGGTCACTGGTCAGCTT	精细定位 Fine mapping
RM5463	ACCCTTGCAGACAACGTACC	GCATGCAGCTGCTGGTATAT	精细定位 Fine mapping
QHG	AGCTCTGCAGCAACCAGATA	TGCTTTGGGCATTCCAGTTC	qRT-PCR

Fig. 1. Phenotypes and sterility of the wild type and the mutant Osfma2. A, Plant morphology of the wild type (Ningjing 4) and the Osfma2 mutant, bar=15 cm. B, Panicle of the wild type and the Osfma2 mutant, bar=4 cm. C, Anther of wild type, bar=600 μm. D, Anther of the mutant Osfma2, bar=600 μm. E, Pollen of Ningjing 4 stained with I2-KI, bar= 50 μm. F, Pollen of Osfma2 stained with I2-KI, bar= 50 μm.

Fig. 2. Developmental process of pollen in the wild type and the mutant Osfma2. A-H, Wild type; I-P, Mutant Osfma2. A and I, Archesporial cell; B and J, Dyads; C and K, Tetrads; D and L, Early microspore; E and M, Late microspore; F and N, Early bicellular pollen; G and O, Late bicellular pollen; H and P, Mature pollen. Bar=10 μm.

Fig. 3. Transverse section of anthers of the wild type and the mutant Osfma2. A-F, Wild type; G-L, Mutant. A and G, Before the stage of meiosis; B and H, 8b stage of anther development; C and I, The 9th stage of anther development; D and J, The 10th stage of anther development; E and K, The 11th stage of anther development; F and L, Mature stages of anther development. E, Epidermis; En, Endothecium; ML, Middle layer; T, Tapetum; MMC, Microspore mother cell; Tds, Tetrads; Msp, Microspore parietal cell; BP, Bicellular pollen; Mp, Mature pollen. Bar=10 μm.

Fig. 4. Chromosome observation of archesporial cell of the wild type and the mutant Osfma2. A~F, Wild type; G~L, Mutant Osfma2. A and G, Zygotene; B and H, Pachytene; C and I, Diplotene; D and J, Diakinesis; E and K, Telophase I; F and L, Telophase II. The white arrows point to univalents. Bar=5μm.

Fig. 5. Observation of embryo sac development of the wild type and the mutant Osfma2. A~E, Embryo sac development of wild type; F~J, Embryo sac development of mutant Osfma2. A and F, Archesporial cell stage; B and G, Dyad stage; C and H, Tetrad stage; D and I, Functional megaspore stage; E and J, Mature embryo stage. Bar=60 μm in E and J. Bar=30 μm in others.

Fig. 6. OsFMA2 gene mapping on chromosome 6. The preliminary and fine mapping of OsFMA2. The OsFMA2 gene was ultimately located in the 448-kb interval between the molecular markers RM340 and Hh-4.

Fig. 7. Whole genome re-sequencing defines the physical location of OsFMA2 gene.

Fig. 8. Cloning and sequence analysis of the sterility gene OsFMA2. A, Sequencing differences between the wild type and the mutant; B, Differential splicing of the OsFMA2 gene in the wild type and the mutant; C, Gene structure of OsFMA2. The blue box represents the exon, the line represents the intron, and the dotted box represents the RPA_C conserved domain, which is calculated from the first base of the first amino acid to the last base of the last amino acid. The solid red line represents the mutation. The white boxes represent 5′ UTR and 3′ UTR.

Fig. 9. Bio-information analysis of OsFMA2. A, Protein-structure prediction of OsFMA2. The numbers indicate the position of amino acid residue at the OsFMA2; B, Multiple sequence alignment between OsFMA2 and RPA protein of other species.

Fig. 10. Molecular Phylogenetic analysis of OsFMA2 and other RPA protein by Maximum Likelihood method.

Fig. 11. Relative expression levels of OsFMA2 in different rice tissues. R, Root; ST, Stem; L, Leaf; PA, Panicle; LS, Leaf sheath; S1-S6, Microspore mother cell stage; S7-S9, Meiotic division of male gamete; S10, Mononucleate stage; S11, Binuclear stage; S12, Tri-nuclear stage. Osubiquitin was used as an internal control. Error bars show the SD (n=3).

Fig. 12. Subcellular localization of OsFMA2 in rice protoplast. A, Subcellular localization of OsFMA2-GFP in rice protoplast; B, Localization of nuclear marker; C, Bright field of rice protoplast status; D, Merged image of GFP, nuclear marker and bright field. Bar=10 μm.

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