Chinese Journal OF Rice Science ›› 2022, Vol. 36 ›› Issue (1): 13-26.DOI: 10.16819/j.1001-7216.2022.210312
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ZHANG Taohui1,#, WANG Haiyu1,#, WAN Hua1, ZHANG Liping1, XIE Zhenwei1, CHEN Keyi1, HE Xiaodong1, ZHAO Zhigang1,*(), WAN Jianmin1,2
Received:
2021-03-23
Revised:
2021-04-22
Online:
2022-01-10
Published:
2022-01-10
Contact:
ZHAO Zhigang
About author:
First author contact:#These authors contributed equally to this work;
张涛荟1,#, 王海宇1,#, 万华1, 张莉萍1, 谢振威1, 陈可毅1, 何晓栋1, 赵志刚1,*(), 万建民1,2
通讯作者:
赵志刚
作者简介:
第一联系人:#共同第一作者;
基金资助:
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.
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URL: http://www.ricesci.cn/EN/10.16819/j.1001-7216.2022.210312
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 |
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. 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. 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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