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作物学报 ›› 2024, Vol. 50 ›› Issue (5): 1104-1114.doi: 10.3724/SP.J.1006.2024.32043

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

水稻雄性不育突变体tpa1的表型鉴定与精细定位

万应春1**(), 班义结1**(), 蒋钰东2**(), 王亚欣1, 刘晶晶1, 刘晓晴1, 程育林1, 王楠1,*(), 冯萍1,*()   

  1. 1西南大学水稻研究所 / 转基因植物与安全控制重庆市市级重点实验室 / 南方山地农业教育部工程研究中心, 重庆 400716
    2四川省农业科学院水稻高粱研究所(四川省农业科学院德阳分院), 国家水稻改良中心四川泸州分中心 / 农业农村部西南水稻生物学与遗传育种重点实验室, 四川德阳 618000
  • 收稿日期:2023-10-17 接受日期:2024-01-12 出版日期:2024-05-12 网络出版日期:2024-02-08
  • 通讯作者: 冯萍, E-mail: 554180353@qq.com; 王楠, E-mail: wangnan_xndx@126.com
  • 作者简介:万应春, E-mail: wanyc1221@126.com;
    班义结, E-mail: 2263889500@qq.com;
    蒋钰东, E-mail: 289482100@qq.com第一联系人：

    **同等贡献

  • 基金资助:
    重庆市研究生科研创新项目(CYS22224);重庆市技术创新与应用发展专项重点项目(CSTB2022TIAD-KPX0014)

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 Published:2024-05-12 Published online:2024-02-08
  • Contact: E-mail: 554180353@qq.com; E-mail: wangnan_xndx@126.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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摘要：

雄性不育材料是杂交水稻育种的关键。本研究通过甲基磺酸乙酯(ethyl methane sulfonate, EMS)诱变优良籼稻保持系西农1B, 筛选得到一个雄性不育突变体tpa1。tpa1在营养生长阶段与野生型无差异, 生殖生长阶段表现雄配子不育, 雌配子发育正常。表型观察发现, tpa1花粉完全破碎消失, 花药外壁角质层异常, 花药内壁乌式体排列异常, 胼胝质合成异常, 绒毡层凋亡异常, 且花粉外壁缺失柱状层。遗传分析表明该突变性状受1对隐性核基因控制, 利用突变体tpa1与缙恢10号构建遗传群体, 最终将TPA1基因定位于4号染色体引物标记N9和N11之间, 物理距离为74 kb, 该区间共15个预测基因, 通过重测序仅发现在LOC_Os04g53380的外显子上发生了单碱基替换, 导致了翻译的提前终止, 随后对野生型和突变体tpa1该位点进行测序证实了这一突变, 因此将该基因确定为TPA1的候选基因, TPA1是一个未被报道过的新的雄性不育基因。本研究将为TPA1基因的功能研究奠定基础。

关键词: 水稻(Oryza sativa L.), 雄性隐性核不育, tpa1, 精细定位

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

表1

本研究使用的部分引物"

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

图1

野生型西农1B与突变体tpa1育性观察与扫描电镜观察 A, B: 野生型和突变体tpa1植株; C, D: 野生型和突变体tpa1小穗; E: 野生型花粉碘染; F: 野生型去壳后小花; G: 突变体tpa1去壳后小花; H: 突变体tpa1花粉碘染; I~P: 扫描电镜观察; I, J: 野生型和突变体tpa1花药; K: 野生型花药外壁; L: 野生型花药内壁; M: 野生型花药剥开露出花粉; N: 突变体tpa1花药外壁; O: 突变体tpa1花药内壁; P: 突变体tpa1花药剥开露出花粉。标尺: 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)。"

图2

野生型与突变体tpa1半薄切片与透射电镜观察 A~E: 野生型st8b~st12期花药半薄切片横切; F~J: 突变体tpa1 st8a~st12期花药半薄切片横切; K~M: 野生型st10期花药超薄切片透射电镜观察; N~P: 突变体tpa1 st10期花药超薄切片透射电镜观察。Ba: 柱状层; BP: 双核花粉; dMsp: 异常小孢子母细胞; Dy: 二分体细胞; E: 表皮; En: 内皮; ML: 中间层; MP: 成熟花粉; Msp: 小孢子母细胞; N: 细胞核; Ne: 外壁内层; Nm: 核膜; T: 绒毡层; Tds: 四分体; Ub: 乌式体。标尺: 200 μm (A~J); 2 μm (K, N); 1 μm (L, O); 0.5 μm (M, P)。"

图3

野生型与突变体tpa1胼胝质染色 A~D: 野生型st7~st9期胼胝质染色; E~H: tpa1突变体st7~st9期胼胝质染色。标尺: 200 μm (A~H)。"

表2

突变体tpa1的遗传分析"

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

表3

精细定位区间预测基因"

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

图4

TPA1基因的精细定位 A: tpa1初步定位与精细定位; B: 野生型与突变体tpa1重测序结果分析; C: 野生型与突变体tpa1突变位点测序结果。"

图5

RT-qPCR分析 A: TPA1在野生型水稻抽穗期各部位以及第6~12期的小穗中的表达水平; B: 绒毡层发育相关基因在野生型与突变体tpa1第6~9期的表达水平。"

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