植物学报 ›› 2019, Vol. 54 ›› Issue (4): 522-530.doi: 10.11983/CBB18229

• 技术方法 • 上一篇    下一篇

利用荧光标记高通量鉴定减数分裂重组抑制突变体

李帆,阮继伟()   

  1. 云南省农业科学院花卉研究所, 国家观赏园艺工程技术研究中心, 云南省花卉育种重点实验室, 昆明 650200
  • 收稿日期:2018-10-30 接受日期:2019-02-11 出版日期:2019-07-01 发布日期:2020-01-08
  • 通讯作者: 阮继伟 E-mail:snruanjiwei@126.com
  • 基金资助:
    云南省农业联合青年项目(2018FG001-075);云南省基础研究计划

High-throughput Identification of Meiotic Anti-CO Mutants by Fluorescent Reporters

Li Fan,Ruan Jiwei()   

  1. Yunnan Key Laboratory of Flower Breeding, National Engineering Research Center for Ornamental Horticulture, Flower Research Institute, Yunnan Academy of Agricultural Sciences, Kunming 650200, China
  • Received:2018-10-30 Accepted:2019-02-11 Online:2019-07-01 Published:2020-01-08
  • Contact: Ruan Jiwei E-mail:snruanjiwei@126.com

摘要:

正向遗传学突变体筛选被广泛用于揭示减数分裂中涉及的遗传基因, 如调控减数分裂II型交叉形成途径的重组抑制基因。该研究利用拟南芥(Arabidopsis thaliana)花粉荧光标记系进行EMS突变体的正向遗传学筛选, 鉴定拟南芥野生型Col遗传背景下的重组抑制突变体, 共获得18个重组率显著提高3倍以上的重组抑制突变体, 其中包括显性和隐性遗传突变。研究表明, 基于荧光标记高通量鉴定重组抑制突变体是可行的, 可为植物减数分裂重组调控分子机制研究提供新方法和突变材料。

关键词: 减数分裂, 重组抑制突变体, 荧光标记, 高通量可视分析法

Abstract:

The forward genetic approach has become a widespread methodology to reveal genetic factors involved in meiosis, such as the crossover negative regulators, which limit the class II crossover formation. Here we developed a forward genetics mutant screen to identify anti-CO mutants under the Col wild-type background of Arabidopsis thaliana. We isolated 18 mutant lines showing more than three-fold increase in male meiotic recombination frequency as compared with the wild type, including dominant and recessive mutants. Thus, the EMS screen based on fluorescent reporters allows for high-throughput identification of meiotic anti-CO mutants and provides a novel approach and genetic materials to study the molecular mechanism of meiotic recombination regulation.

Key words: meiosis, anti-CO mutants, fluorescent marker, high-throughput visual assay

图1

拟南芥花粉荧光标记系FTL-I2b示意图 (A) 拟南芥花粉荧光标记系FTL-I2b四分体花粉荧光显微图, 其包含1个DsRed和1个eYFP标记, 能在不同的荧光激发下分别表达红荧光和绿荧光(Bars=50 μm); (B) 拟南芥花粉荧光标记系FTL-I2b在染色体上的位置示意图(Bar=1 Mb), 红色(下)和绿色(上)椭圆分别代表对应的荧光标记DsRed和eYFP, 并构建出1个I2b标记区间(1.45 Mb)。BF: 明场"

图2

拟南芥FTL-I2b半合子荧光标记(I2b/++) (A) FTL-I2b与Col杂交示意图, F1代经过杂交后具有半合子荧光标记, 通过减数分裂形成重组和未重组的配子; (B) 杂交F1代花粉半合子荧光标记显微图, 图中黄色花粉(GR)为未重组配子, 单红色(R)和单绿色(G)花粉为重组配子(Bars=50 μm); (C) 利用流式细胞仪统计花粉中荧光标记可计算该标记区间I2b的重组率, 计算公式为重组率(RF)=(G+R)/(2GR+G+R)×100。"

图3

不同时间的EMS (75 mmol∙L-1)处理下M1代拟南芥种子的萌发率 种子萌发率为3次重复的平均发芽率, 不同小写字母表示差异显著(P<0.05)。"

图4

拟南芥减数分裂重组抑制突变体正向遗传学筛选示意图 突变体正向遗传学的筛选建立在拟南芥野生型(WT)遗传背景基础上, 利用花粉荧光标记系FTL-I2b半合子荧光标记(I2b/++)进行高通量重组率检测, 筛选获得重组率提高的突变体。图中FTL-I2b中的DsRed和eYFP荧光标记分别由R和G表示。m代表基因的显性或隐性突变。RF: 重组率"

图5

拟南芥野生型与大花粉突变体花粉显微图 (A) 拟南芥野生型(WT)花粉大小为(21.82±0.98) µm (n=10); (B) 大花粉突变体1 (big pollen 1)的大花粉率为100%, 花粉大小为(38.36±1.37) µm (n=10); (C) 大花粉突变体2 (big pollen 2)的大花粉率为50%, 花粉大小为(36.00±2.70) µm (n=10)。Bars=50 µm"

图6

拟南芥野生型与重组抑制突变体 (A) 拟南芥野生型(WT)、显性重组抑制突变体(drs)和隐性重组抑制突变体(rrs1)植株表型(Bars=1 cm); (B) 拟南芥野生型(WT)、显性重组抑制突变体(drs)和隐性重组抑制突变体(rrs1)花粉荧光标记显微图(Bars=50 μm)。"

[1] Berchowitz LE, Copenhaver GP ( 2008). Fluorescent Arabidopsis tetrads: a visual assay for quickly developing large crossover and crossover interference data sets. Nat Protoc 3, 41-50.
[2] Berchowitz LE, Copenhaver GP ( 2010). Genetic interference: don't stand so close to me. Curr Genomics 11, 91-102.
[3] Berchowitz LE, Francis KE, Bey AL, Copenhaver GP ( 2007). The role of AtMUS81 in interference-insensitive crossovers in A. thaliana. PLoS Genet 3, e132.
[4] Crismani W, Girard C, Froger N, Pradillo M, Santos JL, Chelysheva L, Copenhaver GP, Horlow C, Mercier R ( 2012). FANCM limits meiotic crossovers. Science 336, 1588-1590.
[5] Fernandes JB, Duhamel M, Seguéla-Arnaud M, Froger N, Girard C, Choinard S, Solier V, De Winne N, De Jaeger G, Gevaert K, Andrey P, Grelon M, Guerois R, Kumar R, Mercier R ( 2017). FIGL1 and its novel partner FLIP form a conserved complex that regulates homologous recombination. PLoS Genet 14, e1007317.
[6] Fernandes JB, Séguéla-Arnaud M, Larchevêque C, Lloyd AH, Mercier R ( 2018). Unleashing meiotic crossovers in hybrid plants. Proc Natl Acad Sci USA 115, 2431-2436.
[7] Francis KE, Lam SY, Harrison BD, Bey AL, Berchowitz LE, Copenhaver GP ( 2007). Pollen tetrad-based visual assay for meiotic recombination in Arabidopsis. Proc Natl Acad Sci USA 104, 3913-3918.
[8] Girard C, Chelysheva L, Choinard S, Froger N, Macaisne N, Lehmemdi A, Mazel J, Crismani W, Mercier R ( 2015). AAA-ATPase FIDGETIN-LIKE 1 and helicase FANCM antagonize meiotic crossovers by distinct mechanisms. PLoS Genet 11, e1005369.
[9] Girard C, Crismani W, Froger N, Mazel J, Lemhemdi A, Horlow C, Mercier R ( 2014). FANCM-associated proteins MHF1 and MHF2, but not the other Fanconi anemia factors, limit meiotic crossovers. Nucleic Acids Res 42, 9087-9095.
[10] Giraut L, Falque M, Drouaud J, Pereira L, Martin OC, Mézard C ( 2011). Genome-wide crossover distribution in Arabidopsis thaliana meiosis reveals sex-specific patterns along chromosomes. PLoS Genet 7, e1002354.
[11] Hatkevich T, Kohl KP, McMahan S, Hartmann MA, Williams AM, Sekelsky J ( 2017). Bloom syndrome helicase promotes meiotic crossover patterning and homolog disjunction. Curr Biol 27, 96-102.
[12] Higgins JD, Buckling EF, Franklin FC, Jones GH ( 2008). Expression and functional analysis of AtMUS81 in Arabidopsis meiosis reveals a role in the second pathway of crossing-over. Plant J 54, 152-162.
[13] Hu Q, Li YF, Wang HJ, Shen Y, Zhang C, Du GJ, Tang D, Cheng ZK ( 2017). Meiotic chromosome association 1 interacts with TOP3α and regulates meiotic recombination in rice. Plant Cell 29, 1697-1708.
[14] Huang JY, Cheng ZH, Wang C, Hong Y, Su H, Wang J, Copenhaver GP, Ma H, Wang YX ( 2015). Formation of interference-sensitive meiotic cross-overs requires sufficient DNA leading-strand elongation. Proc Natl Acad Sci USA 112, 12534-12539.
[15] Jones GH, Franklin FCH ( 2006). Meiotic crossing-over: obligation and interference. Cell 126, 246-248.
[16] Kim Y, Schumaker KS, Zhu JK ( 2006). EMS mutagenesis of Arabidopsis. In: Salinas J, Sanchez-Serrano JJ, eds. Arabidopsis Protocols. Totowa: Humana Press. pp. 101-103.
[17] Kurzbauer MT, Pradillo M, Kerzendorfer C, Sims J, Ladurner R, Oliver C, Janisiw MP, Mosiolek M, Schweizer D, Copenhaver GP, Schlögelhofer P ( 2018). Arabidopsis thaliana FANCD2 promotes meiotic crossover formation. Plant Cell 30, 415-428.
[18] Li F, De Storme N, Geelen D ( 2017). Dynamics of male meiotic recombination frequency during plant development using fluorescent tagged lines in Arabidopsis thaliana. Sci Rep 7, 42535.
[19] Lu PL, Han XW, Qi J, Yang JG, Wijeratne AJ, Li T, Ma H ( 2012). Analysis of Arabidopsis genome-wide variations before and after meiosis and meiotic recombination by resequencing Landsberg erecta and all four products of a single meiosis. Genome Res 22, 508-518.
[20] Lu PL, Wijeratne AJ, Wang ZJ, Copenhaver GP, Ma H ( 2014). Arabidopsis PTD is required for type I crossover formation and affects recombination frequency in two different chromosomal regions. J Genet Genomics 41, 165-175.
[21] Lukowitz W, Gillmor CS, Scheible WR ( 2000). Positional cloning in Arabidopsis. Why it feels good to have a genome initiative working for you. Plant Physiol 123, 795-806.
[22] Macaisne N, Novatchkova M, Peirera L, Vezon D, Jolivet S, Froger N, Chelysheva L, Grelon M, Mercier R ( 2008). SHOC1, an XPF endonuclease-related protein, is essential for the formation of class I meiotic crossovers. Curr Biol 18, 1432-1437.
[23] Macaisne N, Vignard J, Mercier R ( 2011). SHOC1 and PTD form an XPF-ERCC1-like complex that is required for formation of class I crossovers. J Cell Sci 124, 2687-2691.
[24] Mercier R, Mézard C, Jenczewski E, Macaisne N, Grelon M ( 2015). The molecular biology of meiosis in plants. Annu Rev Plant Biol 66, 297-327.
[25] Mieulet D, Aubert G, Bres C, Klein A, Droc G, Vieille E, Rond-Coissieux C, Sanchez M, Dalmais M, Mauxion JP, Rothan C, Guiderdoni E, Mercier R ( 2018). Unleashing meiotic crossovers in crops. Nat Plants 4, 1010-1016.
[26] Qi J, Chen YM, Copenhaver GP, Ma H ( 2014). Detection of genomic variations and DNA polymorphisms and impact on analysis of meiotic recombination and genetic mapping. Proc Natl Acad Sci USA 111, 10007-10012.
[27] Qu LJ, Qin GJ ( 2014). Generation and identification of Arabidopsis EMS mutants. In: Sanchez-Serrano JJ, Salinas J, eds. Arabidopsis Protocols. Totowa: Humana Press. pp. 225-239.
[28] Séguéla-Arnaud M, Choinard S, Larchevêque C, Girard C, Froger N, Crismani W, Mercier R ( 2017). RMI1 and TOP3α limit meiotic CO formation through their C-terminal domains. Nucleic Acids Res 4, 1860-1871.
[29] Séguéla-Arnaud M, Crismani W, Larchevêque C, Mazel J, Froger N, Choinard S, Lemhemdi A, Macaisne N, Van Leene J, Gevaert K, De Jaeger G, Chelysheva L, Mercier R ( 2015). Multiple mechanisms limit meiotic crossovers: TOP3α and two BLM homologs antagonize crossovers in parallel to FANCM. Proc Natl Acad Sci USA 112, 4713-4718.
[30] Wang YX, Cheng ZH, Huang JY, Shi Q, Hong Y, Copenhaver GP, Gong ZZ, Ma H ( 2012). The DNA replication factor RFC1 is required for interference-sensitive meiotic crossovers in Arabidopsis thaliana. PLoS Genet 8, e1003039.
[31] Yelina NE, Lambing C, Hardcastle TJ, Zhao XH, Santos B, Henderson IR ( 2015). DNA methylation epigenetically silences crossover hot spots and controls chromosomal domains of meiotic recombination in Arabidopsis. Gene Dev 29, 2183-2202.
[32] Yelina NE, Ziolkowski PA, Miller N, Zhao X, Kelly KA, Muñoz DF, Mann DJ, Copenhaver GP, Henderson IR ( 2013). High-throughput analysis of meiotic crossover frequency and interference via flow cytometry of fluorescent pollen in Arabidopsis thaliana. Nat Protoc 8, 2119-2134.
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