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Acta Agronomica Sinica ›› 2022, Vol. 48 ›› Issue (7): 1569-1582.doi: 10.3724/SP.J.1006.2022.12044

• OCROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS •     Next Articles

Characterization and genetic mapping of a classic-abortive-type recessive genic-male-sterile mutant ap90 in rice (Oryza sativa L.)

CHEN Chi1,2(), CHEN Dai-Bo2(), SUN Zhi-Hao2, PENG Ze-Qun2, Adil Abbas2, HE Deng-Mei2, ZHANG Ying-Xin2, CHENG Hai-Tao1, YU Ping2, MA Zhao-Hui1, SONG Jian3, CAO Li-Yong2, CHENG Shi-Hua2, SUN Lian-Ping2,*(), ZHAN Xiao-Deng2,*(), LYU Wen-Yan1,*()   

  1. 1College of Agronomy, Shenyang Agricultural University, Shenyang 110866, Liaoning, China
    2China National Rice Research Institute / National Center for Rice Improvement / Key Laboratory for Zhejiang Super Rice Research / State Key Laboratory of Rice Biology, Hangzhou 311401, Zhejiang, China
    3Institute of Crop and Nuclear Technology Utilization, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, Zhejiang, China
  • Received:2021-06-29 Accepted:2021-10-19 Online:2022-07-12 Published:2021-11-03
  • Contact: SUN Lian-Ping,ZHAN Xiao-Deng,LYU Wen-Yan E-mail:595954943@qq.com;chendaibo@caas.cn;sunlianping@caas.cn;zhanxiaodeng@caas.cn;lwyzrx@syau.edu.cn
  • About author:First author contact:

    **Contributed equally to this work

  • Supported by:
    Natural Science Foundation of Zhejiang Province of China(LY17C130003);Natural Science Foundation of Zhejiang Province of China(LY21C130003);Open Project of State Key Laboratory of Rice Biology(20200105);National Natural Science Foundation of China(31801440);Chinese Academy of Agricultural Sciences Innovation Project(CAAS-ASTIP-2013-CNRRI);Liaoning Provincial Major Science and Technology Projects(2019JH1/10200001)

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Abstract:

In this study, we obtained a stable male sterile mutant abortive pollen 90 (ap90) from the mutant library of Zhonghui 8015 (wild-type, WT), an indica restorer line, induced by Ethyl Methy Sulfone (EMS). Compared to the WT, the ap90 mutant displayed no significant differences in plant height, plant type, tiller number, heading date and other agronomic traits, but the anthers were thinner, light creamy yellow and pollen grains were completely abortive. Semi-thin sections observation of anther development at different stages showed that the ap90 mutant carried an abnormal development process of anther wall cells. Namely, the tapetum cell degradation was obviously abnormal, the microspore cells could not form a normal pollen wall structure during mitosis and the starch filling process was blocked which eventually resulted in the degradation of microspores into threadlets and failure of anther dehiscence. Scanning electron microscopic (SEM) observations of the anther surface and pollen exine suggested that the anther epidermis of mutant ap90 were shrunk and covered by more compactly arranged cuticles. The shape of Ubisch distributed on the inner surface of anther locule were irregular, closely arranged and disordered. The pollen grains were shriveled and the sporopollenin on the pollen exine were abnormally arranged. Genetic analysis showed that the ap90 phenotype was controlled by a pair of recessive nuclear genes. Gene preliminary mapping located the mutation site into a 491.73 kb interval between RM21421 and RM21435 on the long arm of rice chromosome 7. Further Mut-Map sequencing analysis confirmed that there was a 37 bp deletion and a following single base substitution in the second exon region of LOC_Os07g22850 in the ap90 mutant, which resulted in the shifted coding sequence, prematurely terminated transcription and translation, leading to the entire abortive pollen and sterile spikelet in the ap90 mutant. Expression pattern analysis results demonstrated that the OsAP90 gene was specifically expressed in anthers and the OsAP90 protein was mainly located in the endoplasmic reticulum (ER). The qPCR results suggested that the relative expression level of many male sterility-related genes in the ap90 mutant was affected by the mutation site, which further proved that OsAP90 played an important role during the formation of Ubisch and pollen wall in rice anther development.

Key words: rice, recessive genic male sterility, ap90, Mut-Map, the relative expression pattern

Fig. 1

Phenotypic comparison of wild type Zhonghui 8015 (WT) and the ap90 mutant A: single plant of WT Zhonghui 8015 and the ap90 mutant at heading stage; bars: 10 cm. B: spikelets of WT and the ap90 mutant; C: flowers of WT and the ap90 mutant, the lemma and palea have been removed for clarity; D: anther of WT and the ap90 mutant; bars: 2 nm. E: I2-KI staining of pollen grains of WT; F: anther tablet of the ap90 mutant; bar: 0.1 mm."

Fig. 2

Transverse section analysis of anthers at different developmental stages in WT andap90 Locules from the anther section of WT (A to E) and ap90 (F to J) were shown as above, respectively. A and F are cross-section of anthers at the stage 8a; B and G are cross-section of anthers at the stage 9; C and H are cross-section of anthers at the stage 10; D and I are cross-section of anthers at the stage 11; E and J are cross-section of anthers at the stage 12. Ep: epidermis; En: endothecium; ML: middle layer; T: tapetum; Dy: dyad cell; Tds: tetrads; Msp: microspore; Bp: biceullar pollen; Mp: mature pollen."

Fig. 3

SEM observation of the anther surface and pollen grains in WT and the ap90 mutant A, B: the anther morphologies of WT and ap90 at stage 12 are shown as above respectively; C-F: anther epidermis; G, H: anthers inner; I, J: pollen cell; K-N: pollen exine. aUb: abnormal Ubisch; Ub: Ubisch; dSp: deformed Sporopollenin; Sp: sporopollenin. Magnifications are labeled in the figures."

Table 1

Genetic analysis of the ap90 locus"

组合
Combination		 F1单株结实率
Seed-setting rate of F1		 F2 χ(3:1) χ20.05
野生型 No. of wild type plants 突变体 No. of mutant plants
ap90/ZH8015 82.32 365 115 0.28 3.84
ap90/02428 86.59 3322 1078 0.59

Fig. 4

Mapping cloning of the male sterile gene OsAP90 A: genetic linkage and primary mapping of the ap90 locus; B: fine mapping of the AP90 locus using Mut-Map method; C: gene structure of the target gene and the ap90 mutation site; D: predicted protein change of the ap90 mutation; E: identification of separation ratio in the BC2F2 population using new-designed functional markers. M: DNA marker, 2000 bp."

Fig. 5

Tissue-specific expression pattern of the male infertility gene OsAP90 Ubiquitin was used as an internal control gene and error bars was the SDs (n = 3). MMCS, MS, YMS, PMS, and MPS indicate microspore mother cell stage, meiosis stage, young microspore cell stage, pollen mitosis stage, and mature pollen stage during anther development, respectively. WT: wild type; ap90: mutant. All data are shown as means ± SDs (n = 3). ** significant at P=0.01; * significant at P = 0.05."

Fig. 6

Subcellular localization of the AP90 protein"

Fig. 7

Relative expression profile of the genes regulating anther development in WT and the ap90 anthers Ubiquitin was used as an internal control gene and error bars show the SD (n = 3). MMCS, MS, YMS, PMS, and MPS indicate microspore mother cell stage, meiosis stage, young microspore cell stage, pollen mitosis stage, and mature pollen stage during anther development, respectively. WT: wild type; ap90: mutant. All data are shown as means ± SDs (n = 3). ** significant at P = 0.01, * significant at P = 0.05."

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