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Acta Agronomica Sinica ›› 2024, Vol. 50 ›› Issue (5): 1104-1114.doi: 10.3724/SP.J.1006.2024.32043

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

Phenotypic identification and fine mapping of male sterile mutant tpa1 in rice

WAN Ying-Chun1**(), BAN Yi-Jie1**(), JIANG Yu-Dong2**(), WANG Ya-Xin1, LIU Jing-Jing1, LIU Xiao-Qing1, CHENG Yu-Lin1, WANG Nan1,*(), FENG Ping1,*()   

  1. 1Rice Research Institute of Southwest University / Chongqing Key Laboratory of Application and Safety Control of Genetically Modified Crops / Engineering Research Center of South Upland Agriculture, Ministry of Education, Chongqing 400716, China
    2Key Laboratory of Southwest Rice Biology and Genetic Breeding, Ministry of Agriculture and Rural Affairs / Luzhou Branch of National Rice Improvement Center, Rice and Sorghum Research Institute, Sichuan Academy of Agricultural Sciences (Deyang Branch of Sichuan Academy of Agricultural Sciences), Deyang 618000, Sichuan, China
  • Received:2023-10-17 Accepted:2024-01-12 Online:2024-05-12 Published:2024-02-08
  • Contact: E-mail: 554180353@qq.com; E-mail: wangnan_xndx@126.com E-mail:wanyc1221@126.com;2263889500@qq.com;289482100@qq.com;wangnan_xndx@126.com;554180353@qq.com
  • About author:First author contact:

    **Contributed equally to this work

  • Supported by:
    Chongqing Graduate Research and Innovation Project(CYS22224);Chongqing Technology Innovation and Application Development Special Key Project(CSTB2022TIAD-KPX0014)

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Abstract:

Male sterile material is the key to hybrid rice breeding. In this study, a male sterile mutant tpa1 was screened by ethyl methane sulfonate (EMS) mutagenesis of excellent indica cultivar Xinong 1B. There was no difference between tpa1 and wild type at vegetative growth stage. At reproductive stage, male gametes were sterile and female gametes developed normally. Phenotypic observation showed that the pollen of tpa1 was completely broken and disappeared, the stratum corneum of the outer wall of the anther was abnormal, the Ubisch body of the inner wall of the anther was abnormal, the callose synthesis was abnormal, the tapetum apoptosis was abnormal, and the outer wall of the pollen was missing bacula layer. Genetic analysis showed that the mutant trait was controlled by a pair of recessive nuclear genes. A genetic population was constructed using the tpa1 mutant and Jinhui 10. Finally, TPA1 gene was located between the markers N9 and N11 on chromosome 4, with a physical distance of 74 kb. There were 15 predicted genes in this interval. Resequencing identified that only a single base substitution occurred in the exon of LOC_Os04g53380, leading to an early termination of translation. Subsequent sequencing of the wild-type and mutant tpa1 confirmed this mutation, thus identified the gene as a candidate gene for TPA1, indicating that TPA1 was a new male sterile gene. This study will lay a foundation for the functional study of TPA1 gene.

Key words: rice (Oryza sativa L.), male recessive nuclear infertility, tpa1, fine mapping

Table 1

Primers used in this study"

用途
Usage		 引物名称
Primer name		 正向序列
Forward sequence (5'-3')		 反向序列
Reverse sequence (5′-3′)
Fine mapping R1 ATCCATCCTTCAGGCTTCG CCTGTGGTGTCCTAAATCCC
R2 TGCCATCATACAAGCGATTTT ATCAGCGTTAGGGTTCGGT
R3 AGGCTGGGAGATCAATGTCTT CATGCTCAAAAACTGAGCTACTG
R4 CACCGATCCTCCCACAAGT CTCTTTGAATCTGTTGTTATGCC
R5 GGGATGCAGTGTGAACTTATTC CTTTTGTGAAAAAATGGGAGG
R6 CGTTACCAGGTTGCTCTTGG CATAGCGCTTGGCCTGTTAC
R7 CTCTCTTCCACGTATGCTTATAGC CATAGTTGCATCCAACGAGC
R8 GAGATTATCCTGCGGTCTTAATC TTGATGATACGATGACCAAGTTC
R9 CACCGATCCTCCCACAAGT CTCTTTGAATCTGTTGTTATGCC
R10 GCAATCTTGTGACCTAATCTTAGTAC GACCATCTAATCAGTACTTCCTCC
N8 TCAGATCCGACCTTTGCGAC CTTTCCTCCTTCCTACCGTA
N9 TAGGTTTAAAATATTCATCT TGATTGCGTGCTGCCGTGCT
N10 AAGGTTGTCCCCTGGTAAAC TGAAGACAGCATCCGTTGGA
N11 GTACTACGGCGGCTAAACGC ACTTCTGGGTTTTTGACTCG
TPA1-check CAATTCTAGGTCTAACATGCGC ATTGCCATTGGGCTCTGGGA
RT-qPCR Actin GACCCAGATCATGTTTGAGACCT CAGTGTGGCTGACACCATCAC
TPA1 CATATCCCTAGCAGCGGACA GCTCGAGTGCACATATTGGG
TDR TGGAGGTGGCACCAGTTTGGATGCT CTGTGCTTCAAGCTCACTGCTGCA
EAT1 CAGAGGAGGTCAAAGGAATG TCCAATCCTGGTCAAATAAG
OsAP37 CGACGCGAACAGGTACGGAAT GCGGCAGCCGGATCTCCA
OsAP25 AGGACGCCATCCCGAACTA AGCGAGCCGGAGAAGTAGTA
OsCYP703B2 TGTTGTCCTCTCATGGATCTTGG GGTAATTGGTGAAGTTGGTCTTCA
PTC2 TTCCATTGGAGGACATGATGAT AACAAATTAAGTAGCAGCAGCATA
TIP2 AAGGAGGATAGAGCGACGGT GATGTTCACGTCGTCCTCCA
WDA1 CCTACTATACCTCGGCGCAC ATGTGATGGGCTCAGTGACG

Fig. 1

Fertility and SEM observation of wild-type Xinong 1B and mutant tpa1 A, B: plants of wild type and mutant tpa1; C, D: spikelet of wild type and mutant tpa1; E: pollen iodine staining of wild type; F: spikelet of wild type; G: spikelet of mutant tpa1; H: pollen iodine staining of mutant tpa1; I-P: SEM observation; I, J: anthers of wild type and mutant tpa1; K: outer wall of wild type anther; L: inner wall of wild type anther; M: wild type anthers peeled out pollen; N: anther outer wall of mutant tpa1; O: the inner wall of anther of mutant tpa1; P: the anther of mutant tpa1 peeled and exposed pollen. Bar: 10 cm (A, B), 1 mm (C, F), 0.5 mm (D, G), 0.1 mm (E, H), 100 μm (I, J), 10 μm (K-P)."

Fig. 2

Semi-thin section and TEM observation of wild type and mutant tpa1 A-E: section-thin sections of wild type anthers from st8b-st12; F-J: section-thin sections of tpa1 anthers from st8b-st12; K-M: TEM of wild type st10 anther ultra-thin sections; N-P: TEM of tpa1 st10 anther ultra-thin sections. Ba: bacula; BP: binuclear pollen; dMsp: abnormal microspore mother cells; Dy: dyad cells; E: epidermis; En: endothelium; ML: middle layer; MP: mature pollen; Msp: microspore mother cells; N: nucleus; Ne: nexine; Nm: nuclear membrane; T: tapetum; Tds: tetrad; Ub: Ubisch body. Bar: 200 μm (A-J); 2 μm (K, N); 1 μm (L, O); 0.5 μm (M, P)."

Fig. 3

Callose staining of wild type and mutant tpa1 A-D: callose staining of wild type anthers from stages st7 to st9; E-H: callose staining of mutant tpa1 anthers from stages st7 to st9. Bar: 200 μm (A-H)."

Table 2

Genetic analysis of mutant tpa1"

群体
Population		 总株数
Total plant number		 可育植株
Fertile plants		 不育植株
Sterile plans		 期望分离比
Segregation ratio		 P值
P-value		 χ2 3:1
(JH10×tpa1) F2 1624 1202 422 3∶1 0.52 0.41

Table 3

Fine mapping interval predicted genes"

序号
Serial number		 基因编号
Gene number		 基因功能注释
Gene function annotation
Gene1 LOC_Os04g53310 OsSSIIIb Soluble starch synthase III
Gene2 LOC_Os04g53320 The expressed protein
Gene3 LOC_Os04g53330 RNA recognition motif containing protein
Gene4 LOC_Os04g53340 The expressed protein
Gene5 LOC_Os04g53350 The expressed protein
Gene6 LOC_Os04g53360 The expressed protein
Gene7 LOC_Os04g53370 The acyltransferase
Gene8 LOC_Os04g53380 The expressed protein
Gene9 LOC_Os04g53390 MBTB2-Bric-a-Brac, Tramtrack, Broad Complex BTB domain with Meprin and TRAF Homology MATH-related domain
Gene10 LOC_Os04g53400 MBTB6-Bric-a-Brac, Tramtrack, Broad Complex BTB domain with Meprin and TRAF Homology MATH domain
Gene11 LOC_Os04g53410 MBTB7-Bric-a-Brac, Tramtrack, Broad Complex BTB domain with Meprin and TRAF Homology MATH domain
Gene12 LOC_Os04g53420 The expressed protein
Gene13 LOC_Os04g53430 MBTB8-Bric-a-Brac, Tramtrack, Broad Complex BTB domain with Meprin and TRAF Homology MATH domain
Gene14 LOC_Os04g53440 RNA recognition motif containing protein
Gene15 LOC_Os04g53450 The expressed protein

Fig. 4

Fine mapping of TPA1 A: preliminary positioning and fine positioning of tpa1; B: the resequencing results of wild type and mutant tpa1; C: wild type and mutant tpa1 mutation site sequencing results."

Fig. 5

RT-qPCR analysis A: the relative expression level of TPA1 genes in different parts of heading stage and spikelets of st6-st12 in wild-type rice; B: the relative expression level of tapetum development-related genes in wild type and mutant tpa1 at st6-st9 stage."

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