玉米大斑病菌有性杂交后代的遗传多态性与原生质体制备
|
摘要
本文以01-12、01-15为亲本菌株进行有性杂交，共分离得到79个单子囊孢子后代。将79个后代菌株分别与亲本菌株01-12和01-15回交进行交配型测定，与亲本菌株01-12同属一个交配型的后代菌株有36个，与亲本菌株01-15同属一个交配型的后代菌株有20个，分离比例明显偏离1:1，表明交配型性状可能由多对基因控制。此外有3个后代菌株为两性菌株，20个为中性菌株。
试验发现，新分离菌株的交配能力明显高于经继代培养3年以上的菌株，复壮后再分离的继代菌株其交配能力有所增强。此外，将基本Sach培养基中的FeCl_3定量为每1000mL水中加入0.02mg后，可促进子囊孢子的成熟，使子囊壳的产生趋于稳定。本试验针对玉米大斑病菌子囊孢子的情况建立了一种简单易行的单孢分离手段。
以36个子囊孢子后代为研究对象，进行了玉米大斑病菌子囊孢子后代与其亲本基于RAPD的遗传多态性分析。结果发现，子囊孢子后代的遗传图谱较亲本出现明显变异。21个随机引物共扩增出144个RAPD标记，其中126个为特异性标记，占87.5％。菌株间的相似系数在0.6450～0.7914之间。此外，后代与亲本的相似性并无一定规则，甚至部分出现双亲的相似性较亲代与子代相似性高的现象，可见玉米大斑病菌遗传机制的复杂性。在双亲遗传物质重组的过程中，可能亲本菌株01-12的遗传物质更占优势，而01-15的遗传力相对较弱。
本试验对玉米大斑病菌的RAPD扩增程序做了进一步摸索，建立了适宜的、较为稳定的反应体系和扩增程序，即：在25μL体系中，加入Taq酶1U，dNTP200μM，10×PCR buffer 2.5μL，随机引物0.8μM，模板DNA 50ng，用灭菌超纯水补足至25μL。扩增程序为：95℃预变性3min；之后94℃变性1min，45℃退火50sec，72℃延伸2min，进行40个循环；接下来72℃延伸5min；最后在4℃下保存。
对亲本及子囊孢子后代的酯酶同工酶分析表明，亲本与后代菌株在同工酶谱上存在显著差异，其中Rf=0.042的酶带为区分两亲本的特异性酶带。个别后代菌株出现较大变异。
本文还对玉米大斑病菌原生质体的制备条件进行了初步摸索，研究了酶系统、渗透压稳定剂、酶解时间、酶解温度对原生质体制备的影响。结果表明，玉
米大斑病菌原生质体制备的适宜条件为:取PD液体培养基振荡培养4d后的玉
米大斑病菌菌丝，用试管形玻璃组织研磨器将其磨碎，以0.8:nol几甘露醇做渗
透压稳定剂，加入10 mg/mL纤维素酶+l 0 mg/mL蜗牛酶的混合酶，28℃卜静置
酶解72h。
Two isolates belonging to opposite mating type, 01-12 and 01-15, were selected as parent isolates to induce ascospore production of Setosphaeria turcica, and 79 single ascospores were isolated. 79 crossing progenies belonging to which mating type were identified by sexual cross with parental isolates. 36 isolates belonged to 'a' mating type, and were the same as parent isolate 01-12, and 20 isolates belonged to 'A' mating type and were the same as parent isolate 01-15. But the ratio of two mating type deviated 1:1 apparently, as deduced that the mating character was decided by polygene site. Otherwise, 3 isolates were bisexual and 20 isolates were neuter.
The experimental results had found that new isolates collected had greater mating ability than those isolates which subcultured for 3 years. In the favorite conditions that the quantity of FeCl3 was 0.02 mg per 1000 mL medium, the number of mature ascospores and the stability of asci occurrence were greatly increased. A simplified method for isolating monosacospores was also set up in this test.
Progenies of 36 ascospores selected were used to study the genetic diversity of parent isolates and sexual crossing progenies of S. turcicum based on RAPD analysis. The results indicated that there were evident variation on genetic map between parent isolates and progenies isolates. There were total of 144 RAPD markers with 21 arbitrary primers, and 126 markers presented the diversity and the percentage was 87.5%. The similarity coefficient of diferrent strains was 0.6450~0.7914. Moreover, there were no definite rules as to whether markers in progenies were more similar to parents by similarity analysis, even though that within parents were more similar than between parent and progeny. It showed that the genetics of Setosphaeria turcica were very complex. Parent isolate 01-12 maybe had stonger genetic ability than parent isolate 01-15 in the course of sexual cross.
The stabilized RAPD reaction system was established in this paper. In 25 L
reactions, there were 1 units of Taq DNA polymerase, 200 M of mixed dNTP, 2.5 L of 10 PCR buffer, 0.8 M of arbitrary primer and 50ng of genomic DNA, the rest volume filled with aseptic ddH2O. Amplification was performed by the following program: initial denaturation at 95 for 3 min followed by 40 cycles of denaturation at 94 for 1 min, annealing at 45 for 50 sec, extension at 72 for 2 min, and final extension at 72 for 5 min, kept at 4 at last.
There were great diversity in the esterase isozyme map among parent isolates and progenies. The specific band in Rf=0.042 location was a distinctive marker for parents. A progeny occurred apparent mutation.
Mycelia of S. turcica could be used to prepare the protoplast. The yield of protoplasts was significantly correlated with enzymes, osmotic stabilizer, temperature and treating time. The proper condition shown in this paper was that the ground mycelium of S. turcica was treated by mixed enzymes (10 mg/mL Cellulase and 10 mg/mL Snailase) for 72 hours at 28癈 after shocking cultrued in PD medium for 4 days, and the mixed enzymes had a better effect when they were dissolved with 0.8 mol / L mannitol osmotic stabilizer.
试验发现，新分离菌株的交配能力明显高于经继代培养3年以上的菌株，复壮后再分离的继代菌株其交配能力有所增强。此外，将基本Sach培养基中的FeCl_3定量为每1000mL水中加入0.02mg后，可促进子囊孢子的成熟，使子囊壳的产生趋于稳定。本试验针对玉米大斑病菌子囊孢子的情况建立了一种简单易行的单孢分离手段。
以36个子囊孢子后代为研究对象，进行了玉米大斑病菌子囊孢子后代与其亲本基于RAPD的遗传多态性分析。结果发现，子囊孢子后代的遗传图谱较亲本出现明显变异。21个随机引物共扩增出144个RAPD标记，其中126个为特异性标记，占87.5％。菌株间的相似系数在0.6450～0.7914之间。此外，后代与亲本的相似性并无一定规则，甚至部分出现双亲的相似性较亲代与子代相似性高的现象，可见玉米大斑病菌遗传机制的复杂性。在双亲遗传物质重组的过程中，可能亲本菌株01-12的遗传物质更占优势，而01-15的遗传力相对较弱。
本试验对玉米大斑病菌的RAPD扩增程序做了进一步摸索，建立了适宜的、较为稳定的反应体系和扩增程序，即：在25μL体系中，加入Taq酶1U，dNTP200μM，10×PCR buffer 2.5μL，随机引物0.8μM，模板DNA 50ng，用灭菌超纯水补足至25μL。扩增程序为：95℃预变性3min；之后94℃变性1min，45℃退火50sec，72℃延伸2min，进行40个循环；接下来72℃延伸5min；最后在4℃下保存。
对亲本及子囊孢子后代的酯酶同工酶分析表明，亲本与后代菌株在同工酶谱上存在显著差异，其中Rf=0.042的酶带为区分两亲本的特异性酶带。个别后代菌株出现较大变异。
本文还对玉米大斑病菌原生质体的制备条件进行了初步摸索，研究了酶系统、渗透压稳定剂、酶解时间、酶解温度对原生质体制备的影响。结果表明，玉
米大斑病菌原生质体制备的适宜条件为:取PD液体培养基振荡培养4d后的玉
米大斑病菌菌丝，用试管形玻璃组织研磨器将其磨碎，以0.8:nol几甘露醇做渗
透压稳定剂，加入10 mg/mL纤维素酶+l 0 mg/mL蜗牛酶的混合酶，28℃卜静置
酶解72h。
Two isolates belonging to opposite mating type, 01-12 and 01-15, were selected as parent isolates to induce ascospore production of Setosphaeria turcica, and 79 single ascospores were isolated. 79 crossing progenies belonging to which mating type were identified by sexual cross with parental isolates. 36 isolates belonged to 'a' mating type, and were the same as parent isolate 01-12, and 20 isolates belonged to 'A' mating type and were the same as parent isolate 01-15. But the ratio of two mating type deviated 1:1 apparently, as deduced that the mating character was decided by polygene site. Otherwise, 3 isolates were bisexual and 20 isolates were neuter.
The experimental results had found that new isolates collected had greater mating ability than those isolates which subcultured for 3 years. In the favorite conditions that the quantity of FeCl3 was 0.02 mg per 1000 mL medium, the number of mature ascospores and the stability of asci occurrence were greatly increased. A simplified method for isolating monosacospores was also set up in this test.
Progenies of 36 ascospores selected were used to study the genetic diversity of parent isolates and sexual crossing progenies of S. turcicum based on RAPD analysis. The results indicated that there were evident variation on genetic map between parent isolates and progenies isolates. There were total of 144 RAPD markers with 21 arbitrary primers, and 126 markers presented the diversity and the percentage was 87.5%. The similarity coefficient of diferrent strains was 0.6450~0.7914. Moreover, there were no definite rules as to whether markers in progenies were more similar to parents by similarity analysis, even though that within parents were more similar than between parent and progeny. It showed that the genetics of Setosphaeria turcica were very complex. Parent isolate 01-12 maybe had stonger genetic ability than parent isolate 01-15 in the course of sexual cross.
The stabilized RAPD reaction system was established in this paper. In 25 L
reactions, there were 1 units of Taq DNA polymerase, 200 M of mixed dNTP, 2.5 L of 10 PCR buffer, 0.8 M of arbitrary primer and 50ng of genomic DNA, the rest volume filled with aseptic ddH2O. Amplification was performed by the following program: initial denaturation at 95 for 3 min followed by 40 cycles of denaturation at 94 for 1 min, annealing at 45 for 50 sec, extension at 72 for 2 min, and final extension at 72 for 5 min, kept at 4 at last.
There were great diversity in the esterase isozyme map among parent isolates and progenies. The specific band in Rf=0.042 location was a distinctive marker for parents. A progeny occurred apparent mutation.
Mycelia of S. turcica could be used to prepare the protoplast. The yield of protoplasts was significantly correlated with enzymes, osmotic stabilizer, temperature and treating time. The proper condition shown in this paper was that the ground mycelium of S. turcica was treated by mixed enzymes (10 mg/mL Cellulase and 10 mg/mL Snailase) for 72 hours at 28癈 after shocking cultrued in PD medium for 4 days, and the mixed enzymes had a better effect when they were dissolved with 0.8 mol / L mannitol osmotic stabilizer.
引文
[1]董金皋．农业植物病理学(北方本)[M]．北京：中国农业出版社，2001，84～85
[2]Luttrell E S. Leptosphaeria perfect stages for Helminthosporium turcieum and H. rostratum[J]. Phytopathology, 1957,47(5): 313
[3]Luttrell E S. The perfect stage of Helminthosporium turcicum[J]. Phytopathology, 1958,48(5): 281～287
[4]任金平．玉米大斑、小斑、圆斑病菌的有性态[J]．吉林农业科学，1990，3：47～62
[5]Leonard k J, Suggs E G. Setosphaeria prolata, the ascigerous state of Exserohilum prolatum[J]. Mycologia, 1974,66(20:281～297
[6]Nelson R R. A major gene locus for compatibility in Trichometasphaeria turciea[J]. Phytopathology, 1959, 49(3): 159～160
[7]Nelson R R. Heterothallism in Helminthosporium maydis[J]. Phytopathology, 1957,47(4): 191～192
[8]Nelson R R. A major gene locus for compatibility in Cochliobolus heterostrophus [J]. Phytopathology, 1957,47(12): 18～23
[9]Nelson R R. Genetics of Cochliobolus heterostrophus I. variability in degree of compatibility[J]. Mycologia, 1959,5(l): 18～23
[10]Nelson R R. The genetics of compatibility in Cochliobolus carbonum[J]. Phytopathology, 1960,50(2): 158～160
[11]Pedersen W L, Brndenburg L J. Mating types,virulence and cultural characteristics of Exserohilum turcicum race 2[J]. Plant Disease, 1986,70(94): 290～292
[12]Nelson R R, Huisingh D, Webster R K. Sexual differentiation in Cochliobolus carbonum as influenced by inhibition and repair of steroid biosynthesis[J]. Phytopathology, 1967,57(10): 1081～1085
[13]桂秀梅．玉米大斑病菌遗传多态性与有性态诱导[D]．保定：河北农业大学硕士学位论文，2002
[14]Lim S M, Kinsey J K, Hooker A L. Inheritance of virulence in Helminthosporium turcicum to monogenic resistant corn[J]. Phytopathology, 1974,64(8): 1150～
1151
[15]Fallah M P, Pataky J K. Reactions of isolates from matings of races 1 and 23N of Exserohilum turcicum[J]. Plant Disease, 1994,78(8): 767～771
[16]Nelson R R. Evaluating genetic protentials in Helminthosporium turcicum[J]. Phytopathology, 1965,55(8): 418～420
[17]Rodriguez A E, Ullstrup A J. Pathogenicity of monoascosporic progenies of Trichometasphaeria turcica[J]. Phytopathology, 1962,52(5): 599～601
[18]安鑫龙．玉米大斑病菌生理小种的鉴定和有性态的诱导[D]．保定：河北农业大学硕士学位论文，2001
[19]孟翠勉．玉米大斑病菌毒素研究[D]．保定：河北农业大学硕士学位论文，1990
[20]Knox-davies P S, Dickson J C. Cytology of Helminthosporium turcicum and its ascigerous stage, Trichometasphaeria turcica[J]. American Journal of Botany, 1960,47(5): 328～339
[21]Williams J G, Kubelik A R, Livak K J. DNA polymorphisms amplified by arbitrary primers useful as genetic markers[J]. Nucleic Acids Research, 1990, 18(22): 6531～6536
[22]Welsh J, Mcclelland M. Fingerprinting genoues using PCR with arbitrary primers[J]. Nucleic Acids Research, 1990,18(24): 7213～7218
[23]Abadi R, Perl-Treves R, Levy Y. Molecular variability among Exserohilum turcicum isolates using RAPD[J]. Candian Journal of Plant Pathology, 1996,18(1): 29～34
[24]Borchardt D S, Welt H G, Geiger H H. Genetic stricture of Setosphaeria turcica population in tropical and temperates[J]. Phytopathology, 1998,88(4):322～329
[25]Borchardt D S, Welt H G, Geiger H H. Molecular marker analysis of European Setosphaeria turcica population[J]. European Journal of Plant Pathology, 1998,104: 611～617
[26]Nicholson P．用RAPD分析和DNA指纹鉴别玉米小斑病菌的O、C、T三个小种[J]．植物病理学报，1993，23(2)：114～119
[27]单卫星，陈受宜，吴立人等．中国小麦条锈菌流行小种的RAPD分析[J]．中
国农业科学，1995，28(5)：1～7
[28]Shi Y L. Analysis of the genetic relationships among the wheat bunt fungi using RAPD and ribosomal DNA markers[J]. Phytopathology, 1996,86:311～318
[29]刘敏学．大豆灰斑病菌生理小种的RAPD标记[J]．菌物系统，1997，16(2)：128～132
[30]朱衡．水稻白叶枯病菌的RAPD分析[J]．生物工程学报，1995，11(3)：281～284
[31]冯洁，孙文姬，石磊岩等．中国棉花枯萎菌生理小种的RAPD分析[J]．菌物系统，2000，19(4)：45～50
[32]朱有勇，王云月，Multani D S等．棉花黄萎病菌RAPD指纹谱型研究[A]．刘仪主编．中国病害研究与防治．中国植物病理学会第六届代表大会暨学术年会论文选编[C]．北京：中国农业科技出版社，1998，451～456
[33]秦敏，何忠全，何明等．RAPD鉴别稻瘟病菌生理小种的研究[J]．西南农业大学学报，1998，10(5)：493～496
[34]惠东威，陈受宜．RAPD技术及其应用[J]．生物工程进展，1992，12(6)：1～5
[35]徐大庆．小麦叶锈菌毒性及RAPD多态性分析[D]．保定：河北农业大学硕士学位论文，2000
[36]朴春根，唐文华，曾士迈等．RAPD技术和聚类分析在小麦条锈病菌生理小种研究中的应用[J]．植物病理学报，1996，26(3)：205～210
[37]鲁东国，王宝华，赵志颖等．福建稻瘟菌群体遗传多样性RAPD分析[J]．福建农业大学学报，2000，28(1)：54～59
[38]胡能书，万国贤．同工酶技术及其应用[M]．长沙：湖南科学技术出版社，1985
[39]Market C L, Moller F. Multiple forms of enzymes: tissue, ontogenetic and species specific patterus[J]. Proceedings of the National Academy of Sciences USA, 1959, 45:753～763
[40]王中仁，植物等位酶分析[M]．北京：科学出版社，1998
[41]董金皋，唐蕊，赵光耀等．玉米大斑病菌的酯酶同工酶与HT-毒素组分关系研究[J]．河北省科学院学报，1999，16(3)：262～266
[42]董金皋，贾建航，李正平．玉米大斑病菌生理小种同工酶的薄层扫描图谱[J]．
玉米科学，1995，3：67～70
[43]吕金殿．我国棉花枯黄萎病菌酯酶同工酶鉴定初报[J]．植物病理学报，1982，12(20)：41～45
[44]魏艳敏，刘大群，张汀．棉花黄萎病菌同工酶电泳研究[J]．植物病理学报，2000，30(1)：87～88
[45]袁凤杰，徐金星，杨庆凯．大豆灰斑病菌生理小种的同工酶鉴定[J]．大豆科学，1998，17(3)：119～224
[46]朱培良，沈瑛，袁筱萍．稻瘟菌不同小种间可溶性蛋白及酯酶同工酶的多态性[J]．植物保护，1995，6：15～16
[47]刘曙照．稻瘟病菌生理小种酯酶同工酶特性的研究[J]．植物病理学报，1992，212(2)：179～184
[48]张修军，胡传炯，周启．利用菌体酯酶同工酶和可溶性蛋白SDS-PAGE鉴定链霉菌融合子[J]．微生物学杂志，1999，19(2)：40～42
[49]Liu Z L. Genetic diversity of Rhizoctonia solani anastomosis group 2[J]. Phytopathology, 1992,82:778～787
[50]陈宏宇，文景芝，于鑫．大豆疫霉菌与其它几种疫霉菌同工酶电泳比较研究[J]．大豆科学，2001，20(1)：1～4
[51]邹庆道，朱勇，刘丽等．运用同工酶标记对玉米穗、茎腐病串珠镰孢菌亲缘关系的研究[J]．杂粮作物，2000，20(1)：46～49
[52]喻子牛，孔晓瑜．同工酶电泳技术与遗传变异研究[J]．海洋湖沼通报，1997，2：32～42
[53]刘伟成，吕国忠，周永力．球壳孢目真菌同工酶电泳研究Ⅰ．分类学意义的评价[J]．沈阳农业大学学报，2001，32(2)：102～106
[54]Hunter R L, Market CL. Histochemical demonstration of enzymes separated by zone electrophoresis in starch gels[J]. Science, 1957,125:1294～1259
[55]电泳技术——分子生物学技术[EB／OL]．http://www.cx.yn.cninfo.net/edu/science/specil-1.htm，2002-10-8
[56]王中仁，张方．复合淀粉凝胶电泳同工酶分析[J]．植物学通报，1995，12(2)：57～62
[57]石玉玲，陈彦红，刘讯．AST同工酶的琼脂糖凝胶电泳NBT显色法[J]．生物
化学与生物物理进展，1994，21(3)：273～276
[58]黄宏文．超薄平板微型聚丙烯酰胺凝胶的等电聚焦电泳方法——介绍一种高效、快速的生物同工酶分析方法[J]．武汉植物学研究，2000，18(3)：224～228
[59]林清华，李常健，张楚富．水稻谷氨酰胺合成酶同工酶免疫学性质比较研究[J]．植物学报，2000，42(5)：471～475
[60]徐庆毅．我国生物技术发展的回顾与展望[J]．生物工程进展，1995，15(1)：3～7
[61]柴容明．生物技术在未来农业中的角色[J]．世界农业，2000，4：29～30
[62]郭丽琼，陈守才，林俊芳，食用菌遗传转化研究进展[J]．食用菌学报，2001，8(4)：47～53
[63]林良斌，刘彦中，杨志新．植物基因工程在应用中存在的问题及对策[J]．云南农业大学学报，2001，16(2)：144～148
[64]赵永昌，张树庭．食用菌原生质体研究的发展史[J]．中国食用菌，1995，14(4)：3～7
[65]陈波．两种微型绿藻营养生理及原生质体制备于杂交育种的研究[D]．广州：暨南大学硕士学位论文．，2000
[66]陈晓琳．两种虫草无性型原生质体融合及蜂头虫草发酵条件的研究[D]．合肥：安徽农业大学硕士学位论文，2001
[67]辛明秀，马玉娥．微生物的原生质体融合及应用[J]．微生物学通报，1995，22(6)：365～370
[68]曹文芩，郭顺星，徐锦堂．大型真菌原生质体融合技术研究进展[J]．生物学通报，1998，33(10)：2～4
[69]甘志波．微生物原生质体融合：问题与对策[J]．微生物学杂志，1996，1：46～51
[70]上官舟建，食用菌原生质体技术研究进展[J]．生物学通报，1996，31(1)：4～6
[71]何强泰，张李杨．大型真菌原生质体技术的研究与进展[J]．南京晓庄学院学报，2001，17(4)：47～50
[72]宋庆涛．玉米大斑病菌原生质体制备、再生及根癌农杆菌介导的转化研究[D]
北京：中国农业大学硕士学位论文，2002
[73]张志光，李东屏，方芳等．丝状真菌原生质体技术研究——培养条件对原生质体的影响(Ⅶ)[J]．湖南师范大学自然科学学报，1998，21(1)：60～65
[74]孙剑秋，周东坡．微生物原生质体技术[J]．生物学通报，2002，37(7)：9～11
[75]张志光，李东屏，邹寿长等．丝状真菌原生质体技术的研究(Ⅷ)——渗透压稳定剂对原生质体的影响[J]．湖南师范大学自然科学学报，1998，21(2)：67～71
[76]徐敬友，胡广淦，童蕴慧等．禾谷镰孢原生质体的形成与再生[J]．江苏农学院学报，1997，18(2)：71～73．
[77]Schwartz D C, Cantor C R. Separation of yearst chromosome-sized DNAs by Pulsed Field Gradient Gel Electrophoresis[J]. Ce11,1984(37): 67～73
[78]Chu G, Vollrath D, Davis R W. Separation of large DNA molecules by contour-clamped homogeneous electric fields[J]. Science, 1986, 234: 1582～1585
[79]Cooley R N, Canten C E. Variation in electrophoretic karyitype between strains of Septoria nodorum[J]. Mol. Gen. Genet, 1991, 228:17～23
[80]孙广宇，张天宇．一种检疫单孢子分离技术[J]．植物检疫，1996，10(1)：40～41
[81]高玉亮，李葵花．病原真菌单孢子显微镜下分离技巧[J]．植物保护，1999，25(6)：48
[82]Stewart N C Jr, Via L E. A Rapid CTAB DNA isolation technique useful for RAPD fingerprinting and other PCR applications[J]. BioTechniques, 1993, 14(5): 748～749
[83]安鑫龙，董金皋，韩建民．玉米大斑病菌的RAPD分析Ⅰ．应用CTAB法提取玉米大斑病菌DNA[J]．河北农业大学学报，2001，24(1)：38～41
[84]李培金，朱苏文，程备久．玉米RAPD分析影响因素的研究[J]．安徽农业大学学报，2000，27(3)：254～257
[85]Zou K H, Wells W M, Kaus M R, Statistical validation of automated probabilistic segmentation against composite latent expert ground truth in MR imaging of Brain Tumors[EB/OL]. http://splweb.bwh.harvard.edu:8OOO/pages/papers/pubs/../zou/zou-miccai2OO2.pdf,
2003-3-30
[86]Nei M, Li W H. Mathematical model for studying genetic variation in terms of restriction endonucleases[J]. Proceedings of the National Academy of Sciences USA, 1979, 76: 5269～5273.
[87]Nei M. Analysis of gene diversity in subdivided populations[J]. Proceedings of the National Academy of Sciences USA, 1973, 70(12): 3321～3323
[88]Nei M. Estimation of average heterozygosity and genetic distance from a small number of individuals[J]. Genetics, 1978, 89: 583～590.
[89]黄胜忠，蔡奇助，许谦信．利用RAPD分子标志进行菊花杂交后代遗传性之研究[J]．台中区农业改良场研究简报，2001，67：27～36
[90]黄学跃，肖炳光，赵立红等．云南省地方晒烟品种酯酶同工酶分析[RB／OL]．http://www.tobaccoinfo.com.cn/yuzhong/nanfangxb/wen/13564.htm，2003-4-1
[91]李东屏，张志光，陈作红．丝状真菌原生质体技术研究(Ⅵ)——高脚小伞原生质体的制备与再生[J]．湖南师范大学自然科学学报，1997，20(2)：82-85．
[92]周益军，范永坚，王金生等，稻瘟病菌原生质体的制备和再生菌株的致病性[J]．江苏农业学报，2000，16(2)：83-87．
[93]Macedo M B, Azevedo J L. Techniques for colonial growth and protoplast production in Humicola grisea var. thermoidea[EB/OL]. http://www. fgsc.net/fgn38/macedo.html, 2002-7-1
[94]张国珍．稻瘟病菌对三个水稻品种无毒性的遗传分析及分子标记[D]．北京：中国农业大学博士学位论文，2002
[95]刘春林，官春云，李梅．植物RAPD标记的可靠性研究[J]．生物技术通报，1992，(2)：32
[96]曹庆辉．应用同工酶标记与RAPD标记对葡萄亲缘关系的比较研究[D]．南宁：广西大学硕士学位论文，2002．
[97]赵素珍．旱稻品种遗传差异的RAPD分析[J]．呼和浩特：内蒙古农业大学硕士学位论文，2001
[98]李刚，李宝健．灵芝原生质体分离与再生研究[J]．菌物系统，1999，18(1)：79～88．
[2]Luttrell E S. Leptosphaeria perfect stages for Helminthosporium turcieum and H. rostratum[J]. Phytopathology, 1957,47(5): 313
[3]Luttrell E S. The perfect stage of Helminthosporium turcicum[J]. Phytopathology, 1958,48(5): 281～287
[4]任金平．玉米大斑、小斑、圆斑病菌的有性态[J]．吉林农业科学，1990，3：47～62
[5]Leonard k J, Suggs E G. Setosphaeria prolata, the ascigerous state of Exserohilum prolatum[J]. Mycologia, 1974,66(20:281～297
[6]Nelson R R. A major gene locus for compatibility in Trichometasphaeria turciea[J]. Phytopathology, 1959, 49(3): 159～160
[7]Nelson R R. Heterothallism in Helminthosporium maydis[J]. Phytopathology, 1957,47(4): 191～192
[8]Nelson R R. A major gene locus for compatibility in Cochliobolus heterostrophus [J]. Phytopathology, 1957,47(12): 18～23
[9]Nelson R R. Genetics of Cochliobolus heterostrophus I. variability in degree of compatibility[J]. Mycologia, 1959,5(l): 18～23
[10]Nelson R R. The genetics of compatibility in Cochliobolus carbonum[J]. Phytopathology, 1960,50(2): 158～160
[11]Pedersen W L, Brndenburg L J. Mating types,virulence and cultural characteristics of Exserohilum turcicum race 2[J]. Plant Disease, 1986,70(94): 290～292
[12]Nelson R R, Huisingh D, Webster R K. Sexual differentiation in Cochliobolus carbonum as influenced by inhibition and repair of steroid biosynthesis[J]. Phytopathology, 1967,57(10): 1081～1085
[13]桂秀梅．玉米大斑病菌遗传多态性与有性态诱导[D]．保定：河北农业大学硕士学位论文，2002
[14]Lim S M, Kinsey J K, Hooker A L. Inheritance of virulence in Helminthosporium turcicum to monogenic resistant corn[J]. Phytopathology, 1974,64(8): 1150～
1151
[15]Fallah M P, Pataky J K. Reactions of isolates from matings of races 1 and 23N of Exserohilum turcicum[J]. Plant Disease, 1994,78(8): 767～771
[16]Nelson R R. Evaluating genetic protentials in Helminthosporium turcicum[J]. Phytopathology, 1965,55(8): 418～420
[17]Rodriguez A E, Ullstrup A J. Pathogenicity of monoascosporic progenies of Trichometasphaeria turcica[J]. Phytopathology, 1962,52(5): 599～601
[18]安鑫龙．玉米大斑病菌生理小种的鉴定和有性态的诱导[D]．保定：河北农业大学硕士学位论文，2001
[19]孟翠勉．玉米大斑病菌毒素研究[D]．保定：河北农业大学硕士学位论文，1990
[20]Knox-davies P S, Dickson J C. Cytology of Helminthosporium turcicum and its ascigerous stage, Trichometasphaeria turcica[J]. American Journal of Botany, 1960,47(5): 328～339
[21]Williams J G, Kubelik A R, Livak K J. DNA polymorphisms amplified by arbitrary primers useful as genetic markers[J]. Nucleic Acids Research, 1990, 18(22): 6531～6536
[22]Welsh J, Mcclelland M. Fingerprinting genoues using PCR with arbitrary primers[J]. Nucleic Acids Research, 1990,18(24): 7213～7218
[23]Abadi R, Perl-Treves R, Levy Y. Molecular variability among Exserohilum turcicum isolates using RAPD[J]. Candian Journal of Plant Pathology, 1996,18(1): 29～34
[24]Borchardt D S, Welt H G, Geiger H H. Genetic stricture of Setosphaeria turcica population in tropical and temperates[J]. Phytopathology, 1998,88(4):322～329
[25]Borchardt D S, Welt H G, Geiger H H. Molecular marker analysis of European Setosphaeria turcica population[J]. European Journal of Plant Pathology, 1998,104: 611～617
[26]Nicholson P．用RAPD分析和DNA指纹鉴别玉米小斑病菌的O、C、T三个小种[J]．植物病理学报，1993，23(2)：114～119
[27]单卫星，陈受宜，吴立人等．中国小麦条锈菌流行小种的RAPD分析[J]．中
国农业科学，1995，28(5)：1～7
[28]Shi Y L. Analysis of the genetic relationships among the wheat bunt fungi using RAPD and ribosomal DNA markers[J]. Phytopathology, 1996,86:311～318
[29]刘敏学．大豆灰斑病菌生理小种的RAPD标记[J]．菌物系统，1997，16(2)：128～132
[30]朱衡．水稻白叶枯病菌的RAPD分析[J]．生物工程学报，1995，11(3)：281～284
[31]冯洁，孙文姬，石磊岩等．中国棉花枯萎菌生理小种的RAPD分析[J]．菌物系统，2000，19(4)：45～50
[32]朱有勇，王云月，Multani D S等．棉花黄萎病菌RAPD指纹谱型研究[A]．刘仪主编．中国病害研究与防治．中国植物病理学会第六届代表大会暨学术年会论文选编[C]．北京：中国农业科技出版社，1998，451～456
[33]秦敏，何忠全，何明等．RAPD鉴别稻瘟病菌生理小种的研究[J]．西南农业大学学报，1998，10(5)：493～496
[34]惠东威，陈受宜．RAPD技术及其应用[J]．生物工程进展，1992，12(6)：1～5
[35]徐大庆．小麦叶锈菌毒性及RAPD多态性分析[D]．保定：河北农业大学硕士学位论文，2000
[36]朴春根，唐文华，曾士迈等．RAPD技术和聚类分析在小麦条锈病菌生理小种研究中的应用[J]．植物病理学报，1996，26(3)：205～210
[37]鲁东国，王宝华，赵志颖等．福建稻瘟菌群体遗传多样性RAPD分析[J]．福建农业大学学报，2000，28(1)：54～59
[38]胡能书，万国贤．同工酶技术及其应用[M]．长沙：湖南科学技术出版社，1985
[39]Market C L, Moller F. Multiple forms of enzymes: tissue, ontogenetic and species specific patterus[J]. Proceedings of the National Academy of Sciences USA, 1959, 45:753～763
[40]王中仁，植物等位酶分析[M]．北京：科学出版社，1998
[41]董金皋，唐蕊，赵光耀等．玉米大斑病菌的酯酶同工酶与HT-毒素组分关系研究[J]．河北省科学院学报，1999，16(3)：262～266
[42]董金皋，贾建航，李正平．玉米大斑病菌生理小种同工酶的薄层扫描图谱[J]．
玉米科学，1995，3：67～70
[43]吕金殿．我国棉花枯黄萎病菌酯酶同工酶鉴定初报[J]．植物病理学报，1982，12(20)：41～45
[44]魏艳敏，刘大群，张汀．棉花黄萎病菌同工酶电泳研究[J]．植物病理学报，2000，30(1)：87～88
[45]袁凤杰，徐金星，杨庆凯．大豆灰斑病菌生理小种的同工酶鉴定[J]．大豆科学，1998，17(3)：119～224
[46]朱培良，沈瑛，袁筱萍．稻瘟菌不同小种间可溶性蛋白及酯酶同工酶的多态性[J]．植物保护，1995，6：15～16
[47]刘曙照．稻瘟病菌生理小种酯酶同工酶特性的研究[J]．植物病理学报，1992，212(2)：179～184
[48]张修军，胡传炯，周启．利用菌体酯酶同工酶和可溶性蛋白SDS-PAGE鉴定链霉菌融合子[J]．微生物学杂志，1999，19(2)：40～42
[49]Liu Z L. Genetic diversity of Rhizoctonia solani anastomosis group 2[J]. Phytopathology, 1992,82:778～787
[50]陈宏宇，文景芝，于鑫．大豆疫霉菌与其它几种疫霉菌同工酶电泳比较研究[J]．大豆科学，2001，20(1)：1～4
[51]邹庆道，朱勇，刘丽等．运用同工酶标记对玉米穗、茎腐病串珠镰孢菌亲缘关系的研究[J]．杂粮作物，2000，20(1)：46～49
[52]喻子牛，孔晓瑜．同工酶电泳技术与遗传变异研究[J]．海洋湖沼通报，1997，2：32～42
[53]刘伟成，吕国忠，周永力．球壳孢目真菌同工酶电泳研究Ⅰ．分类学意义的评价[J]．沈阳农业大学学报，2001，32(2)：102～106
[54]Hunter R L, Market CL. Histochemical demonstration of enzymes separated by zone electrophoresis in starch gels[J]. Science, 1957,125:1294～1259
[55]电泳技术——分子生物学技术[EB／OL]．http://www.cx.yn.cninfo.net/edu/science/specil-1.htm，2002-10-8
[56]王中仁，张方．复合淀粉凝胶电泳同工酶分析[J]．植物学通报，1995，12(2)：57～62
[57]石玉玲，陈彦红，刘讯．AST同工酶的琼脂糖凝胶电泳NBT显色法[J]．生物
化学与生物物理进展，1994，21(3)：273～276
[58]黄宏文．超薄平板微型聚丙烯酰胺凝胶的等电聚焦电泳方法——介绍一种高效、快速的生物同工酶分析方法[J]．武汉植物学研究，2000，18(3)：224～228
[59]林清华，李常健，张楚富．水稻谷氨酰胺合成酶同工酶免疫学性质比较研究[J]．植物学报，2000，42(5)：471～475
[60]徐庆毅．我国生物技术发展的回顾与展望[J]．生物工程进展，1995，15(1)：3～7
[61]柴容明．生物技术在未来农业中的角色[J]．世界农业，2000，4：29～30
[62]郭丽琼，陈守才，林俊芳，食用菌遗传转化研究进展[J]．食用菌学报，2001，8(4)：47～53
[63]林良斌，刘彦中，杨志新．植物基因工程在应用中存在的问题及对策[J]．云南农业大学学报，2001，16(2)：144～148
[64]赵永昌，张树庭．食用菌原生质体研究的发展史[J]．中国食用菌，1995，14(4)：3～7
[65]陈波．两种微型绿藻营养生理及原生质体制备于杂交育种的研究[D]．广州：暨南大学硕士学位论文．，2000
[66]陈晓琳．两种虫草无性型原生质体融合及蜂头虫草发酵条件的研究[D]．合肥：安徽农业大学硕士学位论文，2001
[67]辛明秀，马玉娥．微生物的原生质体融合及应用[J]．微生物学通报，1995，22(6)：365～370
[68]曹文芩，郭顺星，徐锦堂．大型真菌原生质体融合技术研究进展[J]．生物学通报，1998，33(10)：2～4
[69]甘志波．微生物原生质体融合：问题与对策[J]．微生物学杂志，1996，1：46～51
[70]上官舟建，食用菌原生质体技术研究进展[J]．生物学通报，1996，31(1)：4～6
[71]何强泰，张李杨．大型真菌原生质体技术的研究与进展[J]．南京晓庄学院学报，2001，17(4)：47～50
[72]宋庆涛．玉米大斑病菌原生质体制备、再生及根癌农杆菌介导的转化研究[D]
北京：中国农业大学硕士学位论文，2002
[73]张志光，李东屏，方芳等．丝状真菌原生质体技术研究——培养条件对原生质体的影响(Ⅶ)[J]．湖南师范大学自然科学学报，1998，21(1)：60～65
[74]孙剑秋，周东坡．微生物原生质体技术[J]．生物学通报，2002，37(7)：9～11
[75]张志光，李东屏，邹寿长等．丝状真菌原生质体技术的研究(Ⅷ)——渗透压稳定剂对原生质体的影响[J]．湖南师范大学自然科学学报，1998，21(2)：67～71
[76]徐敬友，胡广淦，童蕴慧等．禾谷镰孢原生质体的形成与再生[J]．江苏农学院学报，1997，18(2)：71～73．
[77]Schwartz D C, Cantor C R. Separation of yearst chromosome-sized DNAs by Pulsed Field Gradient Gel Electrophoresis[J]. Ce11,1984(37): 67～73
[78]Chu G, Vollrath D, Davis R W. Separation of large DNA molecules by contour-clamped homogeneous electric fields[J]. Science, 1986, 234: 1582～1585
[79]Cooley R N, Canten C E. Variation in electrophoretic karyitype between strains of Septoria nodorum[J]. Mol. Gen. Genet, 1991, 228:17～23
[80]孙广宇，张天宇．一种检疫单孢子分离技术[J]．植物检疫，1996，10(1)：40～41
[81]高玉亮，李葵花．病原真菌单孢子显微镜下分离技巧[J]．植物保护，1999，25(6)：48
[82]Stewart N C Jr, Via L E. A Rapid CTAB DNA isolation technique useful for RAPD fingerprinting and other PCR applications[J]. BioTechniques, 1993, 14(5): 748～749
[83]安鑫龙，董金皋，韩建民．玉米大斑病菌的RAPD分析Ⅰ．应用CTAB法提取玉米大斑病菌DNA[J]．河北农业大学学报，2001，24(1)：38～41
[84]李培金，朱苏文，程备久．玉米RAPD分析影响因素的研究[J]．安徽农业大学学报，2000，27(3)：254～257
[85]Zou K H, Wells W M, Kaus M R, Statistical validation of automated probabilistic segmentation against composite latent expert ground truth in MR imaging of Brain Tumors[EB/OL]. http://splweb.bwh.harvard.edu:8OOO/pages/papers/pubs/../zou/zou-miccai2OO2.pdf,
2003-3-30
[86]Nei M, Li W H. Mathematical model for studying genetic variation in terms of restriction endonucleases[J]. Proceedings of the National Academy of Sciences USA, 1979, 76: 5269～5273.
[87]Nei M. Analysis of gene diversity in subdivided populations[J]. Proceedings of the National Academy of Sciences USA, 1973, 70(12): 3321～3323
[88]Nei M. Estimation of average heterozygosity and genetic distance from a small number of individuals[J]. Genetics, 1978, 89: 583～590.
[89]黄胜忠，蔡奇助，许谦信．利用RAPD分子标志进行菊花杂交后代遗传性之研究[J]．台中区农业改良场研究简报，2001，67：27～36
[90]黄学跃，肖炳光，赵立红等．云南省地方晒烟品种酯酶同工酶分析[RB／OL]．http://www.tobaccoinfo.com.cn/yuzhong/nanfangxb/wen/13564.htm，2003-4-1
[91]李东屏，张志光，陈作红．丝状真菌原生质体技术研究(Ⅵ)——高脚小伞原生质体的制备与再生[J]．湖南师范大学自然科学学报，1997，20(2)：82-85．
[92]周益军，范永坚，王金生等，稻瘟病菌原生质体的制备和再生菌株的致病性[J]．江苏农业学报，2000，16(2)：83-87．
[93]Macedo M B, Azevedo J L. Techniques for colonial growth and protoplast production in Humicola grisea var. thermoidea[EB/OL]. http://www. fgsc.net/fgn38/macedo.html, 2002-7-1
[94]张国珍．稻瘟病菌对三个水稻品种无毒性的遗传分析及分子标记[D]．北京：中国农业大学博士学位论文，2002
[95]刘春林，官春云，李梅．植物RAPD标记的可靠性研究[J]．生物技术通报，1992，(2)：32
[96]曹庆辉．应用同工酶标记与RAPD标记对葡萄亲缘关系的比较研究[D]．南宁：广西大学硕士学位论文，2002．
[97]赵素珍．旱稻品种遗传差异的RAPD分析[J]．呼和浩特：内蒙古农业大学硕士学位论文，2001
[98]李刚，李宝健．灵芝原生质体分离与再生研究[J]．菌物系统，1999，18(1)：79～88．