利用荧光原位杂交技术分析新合成异源四倍体拟南芥

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Nora Cobb
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1 作物学报 ACTA AGRONOMICA SINICA 2010, 36(7): ISSN ; CODEN TSHPA9 DOI: /SP.J 利用荧光原位杂交技术分析新合成异源四倍体拟南芥 * 位芳张改生 / /, :,,,, A. suecica, DAPI ;, : ; ; ; DAPI; FISH Analysis of Resynthesized Allotetraploid Arabidopsis WEI Fang and ZHANG Gai-Sheng * Key Laboratory of Crop Heterosis of Shaanxi Province, Wheat Breeding Engineering Research Center, Ministry of Education, Northwest A&F University, Yangling , China Abstract: Allopolyploidy breeding or introduction of preferential gene(s) via allopolyploidization is widely attempted in production of new crops. Thus at the onset of establishment of new allopolyploids, the functional gametes are reproduced conditioning that normal events such as synapsis and pairing and correct segregation of homologous chromosomes are guaranteed with the avoidance of possible interference of homoeologous chromosomes during meiosis. In the present study, we reported on meiotic synapsis and pairing of homologous chromosomes during the development of pollen mother cells in the newly resynthesized allotetraploid Arabidopsis aided with DAPI staining and FISH technique. The results showed that the correct number of nuclear chromosomes and balanced segregation were frequently observed with DAPI staining, and FISH analysis further provided the improved resolution of synapsis and pairing of homologous chromosomes, and the investigated nuclear chromosomes derived from different parental lines had no interference with each other, and the synapsis and pairing of homologous chromosomes were clearly identified. Therefore, the results indicated that the newly resynthesized allotetraploid Arabidopsis may achieve the normal meiosis and propagate the functionalized gametes for fertilization, confirming a potential vigor of intra/interspecific hybridization and a cytological basis for allopolyploid breeding. Keywords: Arabidopsis; Allotetraploid; Resynthesized; DAPI; FISH, (hybridization) (genome doubling) [1],,,, [2-3] ;,,, [4-6] ;,, DNA, [7-8],,,,,,,,,,,, (AABB) (AABBDD), 5B Ph1,, (863 ) (2009AA101102) (2007ZDKG-02) (99-1A) * (Corresponding author):, zhanggsh@public.xa.sn.cn Received( ): ; Accepted( :

2 7 : 1217, [9-10],, Ph1 (Ph1-like), (genomic shock),, (transient synapsis) (pairing) [11] (fluorescence in situ hybridization, FISH),, DNA DNA [12-15],, [16], (evolution of genome stability) 1 材料与方法 1.1 A. thaliana (2n=4x=20) A. arenosa (2n=4x=32), (, ), F 1 F 1 ( 4, 2 ), ( 22 ), A. suecica (2n=4x=26), 18 h, 70% 1.2 DNA 2~5 mg, CTAB DNA [17] dutp ( bio-dutp dig-dutp, Roche ), PCR, 10 PCR buffer (Mg 2+ ) 5 μl 2.5 mmol L 1 dntp ( datp/dctp/dgtp) Mixture 3 μl 2.5 mmol L 1 dttp bio-dutp ( dig-dutp) 1 μl 5 U μl 1 Taq (Fermentas ) 1 μl 0 μmol L 1 At-cen-F/ At-cen-R Aa-cen-1-F/Aa-cen-1-F 2.5 μl, ddh 2 O 50 μl PCR 94 2 min; s; s; s, 35 PCR, 150 ng μl 1 At-cen-F: 5 -CATGGTGGTAGCCAAA GTCCATA-3 ; At-cen-R: 5 -GCTTTGAGAAGCAAGAAG AAGG-3 ; Aa-cen-1-F: 5 -AGCTTCTTATTGCTCTCAACG G-3 ; Aa-cen-1-R: 5 -TTAGAAGCTCCAAAACCGAAAA-3 1.3,, Ross Armstrong [18-19], A. suecica, 0.5~2.0 mm,,, 2 h, 3, (ph 4.5) 2, (, Sigma ) min,,,,,,, 10 μg ml 1 DAPI (4,6-diamidino-2-phenylindole, Roche ),,, SSC 5~10 min, (70% 80% 90% 100%) ; 25 μl I ( 100% 10 μl; 50% 8 μl; 20 SSC 2.5 μl; 10% SDS 2.5 μl; 2 μl),, 95 3 min;, 37 ; SF50 (50% 2 SSC) 42 3 ; 2 SSC 42 3 ; 4T (0.05% Tween SSC) 42 5 min; min; 4T 42 5 min; 100 μl II ( Avidin~Texas Red ), min; TNT (Tris-HCl, ph % Tween-20) 42 5 min; 100 μl III ( goat-anti-avidin~biotin mouse-anti-digoxigenin, ), min; TNT 42 3 ; 100 μl IV [ Avidin (Texas Red) goat-antimouse Alexa 488, ], min; TNT 42 3 ;, DAPI, 2 结果与分析 A. suecica (2n=4x=26), 10 A. thaliana (2n=4x=20), 180 bp, (biotin) ;, 16 A. arenosa (2n=4x=32), 180 bp, PCR (digoxigenin), (Texas Red Alexa 488), A. thaliana A. arenosa, A. suecica (2n=4x=26), DAPI, ( )

1218 36 ( 1-a); ( 1-a);,, ( 1-b), ; I,,, ( 1-c), ( 1-d), I, I ( 1-e), II ( 1-f) DAPI,,,, A. suecica, S DNA, ( ),, ( ) I, ( 2-a);,,, ( 2-b), I,, ( 2-c); I, ( 2-d~e), ; I,, ( 2-f);, ( 2-g~h), A.

3 ( 1-a); ( 1-a);,, ( 1-b), ; I,,, ( 1-c), ( 1-d), I, I ( 1-e), II ( 1-f) DAPI,,,, A. suecica, S DNA, ( ),, ( ) I, ( 2-a);,,, ( 2-b), I,, ( 2-c); I, ( 2-d~e), ; I,, ( 2-f);, ( 2-g~h), A. suecica (2n=4x =26), 图 1 异源四倍体 A. suecica 减数分裂期染色体行为 DAPI 染色 Fig. 1 Chromosome (chromatin) behavior during meiosis in allotetraploid A. suecica stained with DAPI a:, ; b:,, ; c: I, ; d: I, ; e: I, ; f:, a: thread-like chromatin at prophase I; b: condensed chromatin and homologous chromosomes gradually paired at late prophase I; c: at metaphase I homologous chromosome completely paired with their partners located at the equatorial plate. d: the paired chromosomes disassociated at late metaphase I; e: at anaphase I the separated chromosomes distributed in two opposite pole and formed into dyad; f: after the meiosis II the chromosomes equally distributed in four micronuclei and formed a tetrad. 3 讨论, [20], [21-22],, DNA [8] DNA A. thaliana A. lyrata, A. lyrata,, A. lyrata (nucleolar dominance) DNA [23],, A. suecica, [24],,,,,

4 7 : 1219 图 2 以 FISH 分析异源四倍体 A. suecica 减数分裂期染色体行为 Fig. 2 FISH analysis of chromosome (chromatin) behaviors during meiosis in allotetraploid A. suecica a:,,,, At-CEN( ) Aa-CEN( ), DAPI ; b c: I,,, 5 ( ) A. thaliana, 8 ( ) A. arenosa; d e: I, ; f: I I,, ; g h: II, a: thread-like chromatin at prophase I, regularly diffused euchromatin and highly condensed heterochromatin, At-CEN(red) and Aa-CEN(green); b and c: condensed chromatin and homologous chromosomes gradually paired at late prophase I (five pairs from A. thaliana and eight pairs from A. arenosa); d and e: at metaphase I, homologous chromosomes completely paired with their partners located at the equatorial plate; f: after metaphase I the paired chromosomes separated into two opposite poles; g and h: after meiosis II, four nuclei formed with equal number of chromosomes.,,, Moore [25],, Ph1 (pairing homoeologous 1) (ABD),, Ph1,,, [1],,,, A. suecica, Ph1 ( ),,,,,,,, References [1] Levsky J M, Singer R H. Fluorescence in situ hybridization: past, present and future. J Cell Sci, 2003, 116: [2] Jiang J M, Gill B S. Current status and the future of fluorescence in situ hybridization (FISH) in plant genome research. Genome, 2006, 49: [3] Gorman P, Roylance R. Fluorescence in situ hybridization and comparative genomic hybridization. Methods Mol Med, 2006, 120: [4] Ali H B, Lysak M A, Schubert I. Genomic in situ hybridization in plants with small genomes is feasible and elucidates the chromosomal parentage in interspecific Arabidopsis hybrids. Genome, 2004, 47: [5] Doyle J J, Flagel L E, Paterson A H, Rapp R A, Soltis D E, Soltis P S, Wendel J F. Evolutionary genetics of genome merger and

5 doubling in plants. Annu Rev Genet, 2008, 42: [6] Comai L, Tyagi A P, Winter K, Holmes-Davis R, Reynolds S H, Stevens Y, Byers B. Phenotypic instability and rapid gene silencing in newly formed Arabidopsis allotetraploids. Plant Cell, 2000, 12: [7] Schranz M E, Osborn T C. Novel flowering time variation in the resynthesized polyploid Brassica napus. J Hered, 2000, 91: [8] Madlung A, Tyagi A P, Watson B, Jiang H, Kagochi T, Doerge R W, Martienssen R, Comai L. Genomic changes in synthetic Arabidopsis polyploids. Plant J, 2005, 41: [9] Song K, Lu P, Tang K, Osborn T C. Rapid genome change in synthetic polyploids of Brassica and its implications for polyploid evolution. Proc Natl Acad Sci USA, 1995, 92: [10] Pikaard C S. Genomic change and gene silencing in polyploids. Trends Genet, 2001, 17: [11] Chen Z J. Genetic and epigenetic mechanisms for gene expression and phenotypic variation in plant polyploids. Annu Rev Plant Biol, 2007, 58: [12] Liu B, Wendel J F. Epigenetic phenomena and the evolution of plant allopolyploids. Mol Phylogenet Evol, 2003, 29: [13] Roberts M A, Reader S M, Dalgliesh C, Miller T E, Foote T N, Fish L J, Snape J W, Moore G. Induction and characterization of Ph1 wheat mutants. Genetics, 1999, 153: [14] Griffiths S, Sharp R, Foote T N, Bertin I, Wanous M, Reader S, Colas I, Moore G. Molecular characterization of Ph1 as a major chromosome pairing locus in polyploid wheat. Nature, 2006, 439: [15] Comai L, Madlung A, Josefsson C, Tyagi A. Do the different parental heteromes cause genomic shock in newly formed allopolyploids. Philos Trans R Soc Lond B Biol Sci, 2003, 358: [16] Wang A-Y( ), Chen D-L( ), Cai D-T( ). Applications of wide hybridization and allopolyploidization in Rice Breeding. J Wuhan Bot Res ( ), 2005, 23(5): (in Chinese with English abstract) [17] Stewart C N Jr, Via L E. A rapid CTAB DNA isolation technique useful for RAPD fingerprinting and other PCR applications. Biotechniques, 1993, 14: [18] Ross K J, Fransz P, Jones G H. A light microscopic atlas of meiosis in Arabidopsis thaliana. Chromosome Res, 1996, 4: [19] Armstrong S J, Sanchez-Moran E, Franklin F C. Cytological analysis of Arabidopsis thaliana meiotic chromosomes. Methods Mol Biol, 2009, 558: [20] Comai L. The advantages and disadvantages of being polyploid. Nat Rev Genet, 2005, 6: [21] Zhuang Y( ), Chen L-Z( ), Yang Y-G( ), Lou Q-F( ), Chen J-F( ). Changes in gene expression in evolution of plant allopolyploids. Chin Bull Bot ( ), 2006, 23(2): (in Chinese with English abstract) [22] Xiong Z-Y( ), Gao Y( ), He G-Y( ), Gu M-G( ), Guo L-Q( ), Song Y-C( ). Distribution of the knob heterochromatin repeat sequence on chromosome in maize, perennial diploid maize and their offspring. Chin Sci Bull ( ), 2004, 12(49): (in Chinese with English abstract) [23] Wang J L, Tian L, Lee H S, Wei N E, Jiang H M, Brian W, Andreas M, Osborn T C, Doerge R W, Comai L, Chen Z J. Genomewide nonadditive gene regulation in Arabidopsis allotetraploids. Genetics, 2006, 172: [24] Beaulieu J, Jean M, Belzile F. The allotetraploid Arabidopsis thaliana-arabidopsis lyrata subsp. petraea as an alternative model system for the study of polyploidy in plants. Mol Genet Genom, 2009, 281: [25] Moore G. Meiosis in allopolyploids the importance of Teflon chromosomes. Trends Genet, 2002, 18:

小麦与玉米杂交产生小麦单倍体与双单倍体的稳定性

作物学报 ACTA AGRONOMICA SINICA 2013, 39(12): 2247 2252 http://zwxb.chinacrops.org/ ISSN 0496-3490; CODEN TSHPA9 E-mail: xbzw@chinajournal.net.cn DOI: 10.3724/SP.J.1006.2013.02247 小麦与玉米杂交产生小麦单倍体与双单倍体的稳定性 *