栝楼性别转化的RAPD-SCAR标记研究
摘要
栝楼(Trichosanthes kirilowii Maxim)是葫芦科栝楼属的一种药食两用植物,多年生草质藤本。栝楼种植历史悠久,栝楼根、果皮、果实均可入药。随着近年栝楼籽休闲食品的开发,栝楼的根与果实的主要用途发生很大变化,市场价格相差悬殊,从综合效益考虑,有效控制雌、雄株比例在生产上是非常必要的。栝楼雌、雄异株,在栝楼未开花前从形态上很难辨别雌雄株,所以,所以在苗期利用分子生物学的方法鉴别未成熟栝楼的性别,可为栝楼生产上合理的雌、雄性植株田间配置以及育种工作中的早期选择等提供科学依据,对栝楼产业持续、快速、稳定发展奠定了基础。
本文利用RAPD和SCAR分子标记技术对同一来源栝楼块根经组织培养后分化成的不同性别的二年生栝楼进行研究,发现了在雄性栝楼中,可以稳定的得到一条569bp的条带,而雌性栝楼中没有。该结果为栝楼苗期性别鉴定筛选提供了新的途径,为进一步的栝楼性别分化的机理研究提供了参考和依据。
主要试验研究内容与结果如下所述：
1.栝楼叶片含有多糖和多酚,通过改良的CTAB法,所提取的栝楼基因组DNA效果较好,其OD260/OD280的值均在1.7～1.9之间,通过1.5%琼脂糖凝胶电泳后,谱带明亮清晰,没有拖尾现象,能够满足PCR扩增需要。
2.采用L16 (44) RAPD反应体系正交实验设计,对RAPD-PCR影响反应较大的Mg2+浓度、TaqDNA聚合酶、dNTPs、引物浓度进行4因素4水平正交实验设计。优化后的反应体系为：在25μL反应体系中,含10×buffer 2.5μL,Mg2+2.0mmol·L-1, TaqDNA聚合酶1U,引物0.8μmol·L-1,dNTPs 0.1mmol·L-1。反应程序为94℃预变性2mim；94℃变性0.5mim,37℃退火40s,72℃延伸1.5mim,36次循环；72℃延伸10mim,4℃保存。
3.利用92个随机引物对雌、雄栝楼DNA分别进行PCR扩增,共产生大约600多条带,经过多次重复,发现在所选择雌、雄性栝楼样品DNA之间出现的差异性的引物只有1条。
4.采取上述优化体系,找到1条引物S1200(序列5'-GTGAACGCTC-3')能在雄性栝楼DNA中产生1条600bp左右的特异性的扩增带,将这一特异条带命名为S1200-600。经过多次重复试验,结果表明其重复性较好。
5.对S1200-600进行回收、克隆和测序,得到569bp的序列,根据测得的序列,利用Primer Premier5.0软件设计了一对特异性引物,通过退火温度的筛选,最终选择55℃为最适合的退火温度,得到适合于SCAR标记的体系为：在25μL反应体系中,含10×buffer 2.5μL,Mg2+2μL,TaqDNA聚合酶0.2μL,引物Ⅰ2μL,引物Ⅱ2μL,dNTPs0.25μL。反应程序为94℃预变性2mim；94℃变性50s,55℃退火温度40s,72℃延伸1.5mim,36次循环；72℃延伸10mim,10℃保存。经过多次验证,最终成功实现RAPD转化为SCAR标记。可作为栝楼品种早期性别鉴定的遗传标记。
Snakegourd, or Trichosanthes (Trichosanthes kirilowii Maxim) is Perennial Herb liana of medicinal plant in family Cucurbitaceae. Trichosanthes have a long history of cultivation in china. With the development of Trichosanthes as a leisure food, great changes had been taken place in the main Purpose of the root and fruit and there is also have great difference in their price. From the consideration of Comprehensive Benefit, it is very necessary to control the rate of male and female plant economically. Nowadays it is very difficult to distinguish the male and female plants from their Morphology prior to flowering. This article is to determinate the sex of premature Trichosanthes seedlings by the way of molecular biology, which will give scientific basis to the earlier selection of Trichosanthes seedlings. This research will not only lay a foundation for reasonable configuration of Trichosanthes seedlings of different sex in the production but also promote the industry development of Trichosanthes.
In this article the DNA of male and female Trichosanthes seedlings that originate from same clone by the way of tissue culture is amplified by RAPD primers. A primer named S1200 can amplify a specific band of 600bp in DNA of the male, but the DNA of female did not. This result will not only provide a reference for earlier sex selection of Trichosanthes seedlings but also give a basis for Trichosanthes breeding as well as the study of mechanism of Sexual Development.
The main contents and results studied are as follows:
1.In order to get high quality DNA from Trichosanthes's leaves enriching polysaccharides and polyphenols, the genomic DNA obtained by improvement method of CTAB extraction showed to be more integral and pure. The value of OD260/OD280 was between 1.7 and 1.9. The extracted DNA by this method showed a clear band when electrophoresed in 1.5% agrarose gel, and produced clear polymorphic patterns when amplified by PCR.
2. Using L16(44) orthogonal experiment to get the optimized RAPD-PCR reaction system of Trichosanthes. The result shows that the optimal concentration was 10×buffer 2.5μL,Mg2+ 2.0mmol·L-1,Taq polymerase 1U,primer 0.8μmol·L-1,dNTPs 0.1mmol·L-1 per 25μL and the most suitable program was one cycle at 94℃pre-denaturation for 2 min,36cycles of 94℃for 30s,37℃for 40s,72℃for 1.5 min, and 10 min 72℃extension. Store at 4℃.
3. The DNA of male and female Trichosanthes seedlings by using 92 random RAPD primers, more than 600 bands was produced. After repeated experiments, the result revealed that only one primers can amplified polymorphism between the DNA of male and female. The level of polymorphism is very low, about 1%.
4. Through the mentioned optimized system, a RAPD primer named S1200 (5'-GTGAACGCTC-3') can amplified a specific band of 600bp in the male after many repeated experiments. We called this band S1200-600.
5. The fragment S1200-600 was recycled, cloned and sequenced. According to the sequence, a specific primer was designed by the Primer Premier 5.0 software. Through the screening of annealing temperature,55℃was proved to be the suitable annealing temperature. The optimized reaction system for specific primer is as follow:10×buffer 2.5μL, Mg2+2μL, Taq polymerase 0.2μL, primerⅠ2μL, primerⅡ2μL, dNTPs 0.25μL per 25μL reaction system and the most suitable program was one cycle at 94℃pre-denaturation for 2min,36cycles of 94℃for 50s,55℃for 40s,72℃for 1.5 min, and 72℃10 min for extension, Store at 10℃. Through many repeated experiments, RAPD marker was proved to be successfully converted into SCAR marker. This will provide a new way to determinate the sex of Trichosanthes seedlings.
本文利用RAPD和SCAR分子标记技术对同一来源栝楼块根经组织培养后分化成的不同性别的二年生栝楼进行研究,发现了在雄性栝楼中,可以稳定的得到一条569bp的条带,而雌性栝楼中没有。该结果为栝楼苗期性别鉴定筛选提供了新的途径,为进一步的栝楼性别分化的机理研究提供了参考和依据。
主要试验研究内容与结果如下所述：
1.栝楼叶片含有多糖和多酚,通过改良的CTAB法,所提取的栝楼基因组DNA效果较好,其OD260/OD280的值均在1.7～1.9之间,通过1.5%琼脂糖凝胶电泳后,谱带明亮清晰,没有拖尾现象,能够满足PCR扩增需要。
2.采用L16 (44) RAPD反应体系正交实验设计,对RAPD-PCR影响反应较大的Mg2+浓度、TaqDNA聚合酶、dNTPs、引物浓度进行4因素4水平正交实验设计。优化后的反应体系为：在25μL反应体系中,含10×buffer 2.5μL,Mg2+2.0mmol·L-1, TaqDNA聚合酶1U,引物0.8μmol·L-1,dNTPs 0.1mmol·L-1。反应程序为94℃预变性2mim；94℃变性0.5mim,37℃退火40s,72℃延伸1.5mim,36次循环；72℃延伸10mim,4℃保存。
3.利用92个随机引物对雌、雄栝楼DNA分别进行PCR扩增,共产生大约600多条带,经过多次重复,发现在所选择雌、雄性栝楼样品DNA之间出现的差异性的引物只有1条。
4.采取上述优化体系,找到1条引物S1200(序列5'-GTGAACGCTC-3')能在雄性栝楼DNA中产生1条600bp左右的特异性的扩增带,将这一特异条带命名为S1200-600。经过多次重复试验,结果表明其重复性较好。
5.对S1200-600进行回收、克隆和测序,得到569bp的序列,根据测得的序列,利用Primer Premier5.0软件设计了一对特异性引物,通过退火温度的筛选,最终选择55℃为最适合的退火温度,得到适合于SCAR标记的体系为：在25μL反应体系中,含10×buffer 2.5μL,Mg2+2μL,TaqDNA聚合酶0.2μL,引物Ⅰ2μL,引物Ⅱ2μL,dNTPs0.25μL。反应程序为94℃预变性2mim；94℃变性50s,55℃退火温度40s,72℃延伸1.5mim,36次循环；72℃延伸10mim,10℃保存。经过多次验证,最终成功实现RAPD转化为SCAR标记。可作为栝楼品种早期性别鉴定的遗传标记。
Snakegourd, or Trichosanthes (Trichosanthes kirilowii Maxim) is Perennial Herb liana of medicinal plant in family Cucurbitaceae. Trichosanthes have a long history of cultivation in china. With the development of Trichosanthes as a leisure food, great changes had been taken place in the main Purpose of the root and fruit and there is also have great difference in their price. From the consideration of Comprehensive Benefit, it is very necessary to control the rate of male and female plant economically. Nowadays it is very difficult to distinguish the male and female plants from their Morphology prior to flowering. This article is to determinate the sex of premature Trichosanthes seedlings by the way of molecular biology, which will give scientific basis to the earlier selection of Trichosanthes seedlings. This research will not only lay a foundation for reasonable configuration of Trichosanthes seedlings of different sex in the production but also promote the industry development of Trichosanthes.
In this article the DNA of male and female Trichosanthes seedlings that originate from same clone by the way of tissue culture is amplified by RAPD primers. A primer named S1200 can amplify a specific band of 600bp in DNA of the male, but the DNA of female did not. This result will not only provide a reference for earlier sex selection of Trichosanthes seedlings but also give a basis for Trichosanthes breeding as well as the study of mechanism of Sexual Development.
The main contents and results studied are as follows:
1.In order to get high quality DNA from Trichosanthes's leaves enriching polysaccharides and polyphenols, the genomic DNA obtained by improvement method of CTAB extraction showed to be more integral and pure. The value of OD260/OD280 was between 1.7 and 1.9. The extracted DNA by this method showed a clear band when electrophoresed in 1.5% agrarose gel, and produced clear polymorphic patterns when amplified by PCR.
2. Using L16(44) orthogonal experiment to get the optimized RAPD-PCR reaction system of Trichosanthes. The result shows that the optimal concentration was 10×buffer 2.5μL,Mg2+ 2.0mmol·L-1,Taq polymerase 1U,primer 0.8μmol·L-1,dNTPs 0.1mmol·L-1 per 25μL and the most suitable program was one cycle at 94℃pre-denaturation for 2 min,36cycles of 94℃for 30s,37℃for 40s,72℃for 1.5 min, and 10 min 72℃extension. Store at 4℃.
3. The DNA of male and female Trichosanthes seedlings by using 92 random RAPD primers, more than 600 bands was produced. After repeated experiments, the result revealed that only one primers can amplified polymorphism between the DNA of male and female. The level of polymorphism is very low, about 1%.
4. Through the mentioned optimized system, a RAPD primer named S1200 (5'-GTGAACGCTC-3') can amplified a specific band of 600bp in the male after many repeated experiments. We called this band S1200-600.
5. The fragment S1200-600 was recycled, cloned and sequenced. According to the sequence, a specific primer was designed by the Primer Premier 5.0 software. Through the screening of annealing temperature,55℃was proved to be the suitable annealing temperature. The optimized reaction system for specific primer is as follow:10×buffer 2.5μL, Mg2+2μL, Taq polymerase 0.2μL, primerⅠ2μL, primerⅡ2μL, dNTPs 0.25μL per 25μL reaction system and the most suitable program was one cycle at 94℃pre-denaturation for 2min,36cycles of 94℃for 50s,55℃for 40s,72℃for 1.5 min, and 72℃10 min for extension, Store at 10℃. Through many repeated experiments, RAPD marker was proved to be successfully converted into SCAR marker. This will provide a new way to determinate the sex of Trichosanthes seedlings.
引文
[1]YAMPOLSKY C, YAMPOLSKY H. Distribution of the sex forms in the phanerogamic flora[J]. Bibliotheca Genetica,1922,3:1-62.
[2]李明珠,陈清华,程志学等.葫芦科瓜类蔬菜性别分化控制研究进展[J].广东农业科学2008年第8期41-45.
[3]寿森炎,汪俏梅.高等植物性别分化研究进展[J].植物学通报,2000,17(6)：528-535.
[4]任吉君,王艳,刘洪家等.高等植物性别分化研究概述[J].生物学杂志,1993,53(3)：4-7.
[5]娄群峰,余纪柱,陈劲枫等.植物性别分化的遗传基础与标记物研究[J].植物学通报,2002,19(6)：684-691.
[6]夏仁学.园艺植物性别分化的研究进展[J].植物学通报,1996,13：12-19.
[7]Ren J J,Wang Y,Liu H J.General survey on sex differentiation of higher plants[J]. Journal of Biology, 1993,3:4-7.
[8]邵宏波,初立业.高等植物的性别研究及其特点[J].生命科学,1994,6(1)：11-14.
[9]George W L. Influence of genetic background on sex conversion by 2-chloroethyphosphonic acid in monoecious cucumbers. Hort. Sci,1971,96:152-154.
[10]CHARLESWORTH D. Plant sex determination and sex chromosomes[J]. Heredity,2002, 88(2):94-101.
[11]张双全.天花粉蛋白的结构、功能及其临床应用的分子基础[J].中草药,1991,22(5)：228-232.
[12]范怡梅,王勇,张祖暄等.银杏叶中萜类内酯含量与生长季节及植株性别的相关性研究(简报)[J].中国中药杂志,1999,24(2)：82-83.
[13]国家药典委员会.中华人民共和国药典(2000年版一部)[M].北京：化学工业出版社,2000,82-84.
[14]PARKER J S. Sex chromosomes and sexual differentiation in flowering plants[J]. Chromosomes Today,1990,10:187-198.
[15]汪俏梅,曾广文.苦瓜性别分化的特异蛋白质研究[J].植物学报,1998,40(3)：241-246.
[16]Wang Q M, Zeng G W. Studies on expression of sex differentiation program in Momrdica charantia L.[J]. Journal of Zhejiang University (Agricl & Life Sci.),2002,28(3):243-248.
[17]Friedlander M, Atsrmon D, Galan E. Sexual differentiation in cucumber:Abscisic acid and gibberellic acid contents of various sex genotypes[J]. Plant and Cell Physial,1997(18):681-691.
[18]邓俭英,张玲玲,方锋学等.园艺植物性别鉴定的研究概述[J].长江蔬菜,2005,12：31-33.
[19]李瑞丽,卢龙斗,高武军等.雌、雄异株植物性别鉴定的研究进展[J].广西植物,2006,26(4)：387-391.
[202]汪俏梅,曾广文.苦瓜性别分化的形态与组织化学研究.浙江农业大学学报[J].1997,23(2)：149-153.
[21]董莉娜,苏雪,孙坤.DNA分子标记在雌、雄异株植物性别鉴定中的应用[J]广西植物,26(1)：63-68.
[22]宋振巧,王洪刚,王建华.栝楼的研究进展[J].山东农业科学,2005,5：27-29.
[23]李珊,程舟,李彦等.雌、雄栝楼植株内含物比较研究[J].中草药,2008,39(2)：260-263.
[24]何长征,艾辛,匡逢春.不同性型黄瓜植株保护酶类活性的差异[J].湖南农业大学学报,2001,27(4)：289-291.
[25]孙颖,黄瓜性别表达研究[J].吉林师范大学学报(自然科学版)[J].2004,2：118-119.
[26]高武军,姬艳克,肖理会等.雌、雄异株植物性别决定相关功能基因研究进展[J].植物遗传资源学报2008,9(1)：125-129.
[27]BUZEK J, KOUTNIKOVA H, HOUBEN A, et al. Isolation and characterization of X chromosome-derived DNA sequences from a dioecious plant Melandrium album [J]. Chromosome Research,1997,5:57-65.
[28]MATSUNAGA S, KAWANO S, MICHIMOTO T, et al. Semi-automatic laser beam microdissection of the Y chromosome and analysis of Y chromosome DNA in a dioecious plant, Silene latifolia[J]. Plant and Cell Physiology,1999,40:60-68.
[29]LAN T Y,CHEN R Y,L I X L, et al. Microdissection and painting of the W chromosome in Ginkgo biloba showed different labeling patterns[J]. Botanical Studies,2008,49:33-37.
[30]王敏,黄璐琦,付桂芳.栝楼3个农家品种间亲缘近似关系的RAPD分析[J],中国中药杂志,1999,24(6)：336-337.
[3 1]黄璐琦,王敏,杨滨.栝楼农家品种苗期的分子标识鉴别[J].中国药学杂志,1999,34(9)：642.
[32]于凤池,孙耘子等.栝楼性别分化特异大分子标记物研究[J].浙江农业学报,2003,15(6)：332-335.
[33]李彦农,栝楼性别的分子标记研究[J].南京农业大学.2005.
[34]盖树鹏,孟祥栋.分子标记技术及其在作物育种中的应用.中国农学通报,2003,19(6)：12-15.
[35]涂振东,叶凯.生物技术在农作物遗传育种中应用的进展[J].新疆农业科学,2003,40(6)：381-383.
[36]徐小勇,刘继红,邓秀新.柑橘生物技术研究进展[J].中国生物工程杂志,2003,23(8)：35-38.
[37]韦晓霞,熊月明等.果树品种鉴定方法概述[J].福建果树,2002(4)：11-13.
[38]尹逸,李斌.DNA分子标记技术在茶树遗传多样性研究上的应用[J].广东茶叶,2000(3)11-14.
[39]彭建营,束怀瑞等.分子标记技术及其在果树种质资源研究上的应用[J].山东农业大学学报,2001,27(6)：41-45.
[40]F.Bartolozzi, M.L.Warburton, S. Arulsekar et al. Genetic characterization and relatedness among califonia almond cultivars and breeding lines detected by Randomly Amplified Polymorphic DNA Analysis. J. Amer. Soc. Hort. Sci,1998,123(3):381-387.
[41]邓九生,赖炽吕,彭民璋.柑橘RAPD技术与体细胞杂种鉴定.园艺学报,1998,25(2)：105-110.
[42]Roberto Botia, Aziz Akkak. Identification of pear cultivars by molecular marker. Acta Horticultural,1998,457：63-70.
[43]史永忠,郭文武,邓秀新.柑橘RAPD技术建立与体细胞杂种鉴定.园艺学报,1998,25(2)：105-110.
[44]罗正荣,米森敬三,杉浦明.应用RAPD技术研究柿品种间的亲缘关系果树科学[J].1998,15(4)：311-316.
[45]Williams JG, Kubelik AR, Livak KJ, Rafalski JA, Tingey SV. DNA polymorphisms amplified by arbitrary primers are useful as genetic markers.Nucleic Acids Res,1990,18:6531-6535.
[46]刘继红,胡春根.RAPD技术在果树研究中的应用[J].生命的化学,1998,18(1)：33-35.
[47]刘广林,李杨瑞,彭宏祥.RAPD技术应用于果树遗传育种的研究进展[J].广西农业科学,2006,37(6)：632-636.
[48]Harvey C F, Gil] G P, Fraser L G, et al. Sex determination in Actinidia 1. Sex-linked markers and progeny sex ratio in diploid A.chinensis[J]. Sex Plant Reprod,1997,10:149-154.
[49]陈劲枫,娄群峰,余纪柱等.黄瓜性别基因连锁的分子标记筛选[J].上海农业学报,2003,19(4)：11-14.
[50]Jiang C X, Sink K C. RAPD and SCAR markers linked to the sex expression locusM in Asparagus[J]. Euphytica,1997,94:329-333.
[51]Mulcahy D L, Weeden N F, Kessel R, et al. DNA p robes for the Y-chromosome of Silene latifolia, a dioecious angiosperm [J]. Sex Plant Reprod,1992,5:86-88.
[52]Paran I, Michelmore R W. Development of reliable PCR-based markers linked to downy mildew resistance genes in lettuce [J]. Theor App 1 Genet,1993,85:985-993.
[53]OttoT, N andor B, Erzsebet K, et al. Novel male-specific molecular markers (MADC5, MADC6) in hemp [J]. Euphytica,2002,127:209-218.
[54]Urasaki N, Tokumoto M, Tarora K, et al. A male and hermaphrodite specific RAPD marker for papaya (Carica papaya L.)[J]. TheorApp 1 Genet,2002,104:281-285.
[55]王筱璐,杭悦宇,周义峰等.与黄独(Dioscorea bulbifera)性别相关的RAPD-SCAR标记研究[J],植物资源与环境学报,2007,16(2)：1-5.
[56]谭冬梅,罗淑萍,李疆等.阿月浑子性别鉴定的RAPD分析[J].北方果树,2003,20(2)124-126.
[57]周国辉,李华平,张曙光等.番木瓜两性基因的RAPD标记[J].热带亚热带植物学报,2001, 9(3)：190-193.
[58]陈其军,韩玉珍,傅永福等.大麻性别的RAPD和SCAR分子标记[J].植物生理学报,2001,27(2)：173-178.
[59]王关林,方宏筠.植物基因工程(第二版)[M].北京：科学出版社,2002：533-535.
[60]Pich U, Schubert I. Method for isolation of DNA from Plants with a high content of polyphenolics. Nucleic Acids Research,1993,2(14):3328-3332.
[61]杨英军,李学强.落叶果树韧皮部提取DNA的方法研究[J].河南农业大学学报,2001,35(1)：97-98.
[62]奥斯伯F M,金斯顿R E,塞德曼J G等.马学军,舒跃龙等译校.精编分子生物学实验指南(第四版)[M].北京：科学出版社,2005：54-55.
[63]Paterson A H, Brubaber C L, Wendel J F. A rap id method for extraction of cotton (Gossypium spp.) genomic DNA suitable for RFLP of PCR analysis[J]. PlantMol Biol Rep,1993,11 (3):122-127.
[64]蒋立科,杨婉身.现代生物化学实验技术[M].北京：中国农业出版社,2003：253-256.
[65]朱英,陶刚,刘作易,等.普遍适用于农作物基因DNA的提取方法及值得注意的几个问题[J].贵州农业科学,2005,33(5)：20～21.
[66]付燕,罗楠,杨芩等,枇杷属植物ISSR反应体系的建立和优化[J].果树学报,2009,26(2)：180-185.
[67]张今今,王跃进.苹果PAPD反应体系的研究[J].西北农业大学学报,2000,28(5)：65-59.
[68]王得元,张长远等.辣椒RAPD反应体系研究.广东农业科学,2001,(1)：21-22.
[69]曾莉娟,郑成木.硅胶干燥旱稻叶片制备DNA样品及其RAPD反应体系的建立[J].热带农业科学,2002,22(5)：9-12.
[70]蒋曹德,邓昌彦.随机扩增多态DNA规范化反应技系探讨[J].生物技术通报,2002,158(3)：40-43.
[71]何正文,刘运生,陈立华等.正交设计直观分析法优化条件PCR[J].湖南医科大学学报,1998,23(4)：403-404.
[72]汪结明,项艳,吴大强等.杨树ISSR反应体系的建立及正交设计优化[J].核农学报,2007,21(5)：470-473.
[73]许红星,刘红彦,李锁平等.小麦抗白粉病基因pm6的RAPD标记和SCAR标记[J].河南农业科学.2004,4：25-28.
[74]盖树鹏,孟祥栋.大葱胞质雄性不育位点RAPD标记转化为SCAR标记的研究[J].莱阳农学院学报,2004,21(3)：189-192.
[75]柳波,孙彬,金德敏等.紫菜自由丝状体SCAR标记的获得[J].高技术通讯,2004,12：88-92.
[2]李明珠,陈清华,程志学等.葫芦科瓜类蔬菜性别分化控制研究进展[J].广东农业科学2008年第8期41-45.
[3]寿森炎,汪俏梅.高等植物性别分化研究进展[J].植物学通报,2000,17(6)：528-535.
[4]任吉君,王艳,刘洪家等.高等植物性别分化研究概述[J].生物学杂志,1993,53(3)：4-7.
[5]娄群峰,余纪柱,陈劲枫等.植物性别分化的遗传基础与标记物研究[J].植物学通报,2002,19(6)：684-691.
[6]夏仁学.园艺植物性别分化的研究进展[J].植物学通报,1996,13：12-19.
[7]Ren J J,Wang Y,Liu H J.General survey on sex differentiation of higher plants[J]. Journal of Biology, 1993,3:4-7.
[8]邵宏波,初立业.高等植物的性别研究及其特点[J].生命科学,1994,6(1)：11-14.
[9]George W L. Influence of genetic background on sex conversion by 2-chloroethyphosphonic acid in monoecious cucumbers. Hort. Sci,1971,96:152-154.
[10]CHARLESWORTH D. Plant sex determination and sex chromosomes[J]. Heredity,2002, 88(2):94-101.
[11]张双全.天花粉蛋白的结构、功能及其临床应用的分子基础[J].中草药,1991,22(5)：228-232.
[12]范怡梅,王勇,张祖暄等.银杏叶中萜类内酯含量与生长季节及植株性别的相关性研究(简报)[J].中国中药杂志,1999,24(2)：82-83.
[13]国家药典委员会.中华人民共和国药典(2000年版一部)[M].北京：化学工业出版社,2000,82-84.
[14]PARKER J S. Sex chromosomes and sexual differentiation in flowering plants[J]. Chromosomes Today,1990,10:187-198.
[15]汪俏梅,曾广文.苦瓜性别分化的特异蛋白质研究[J].植物学报,1998,40(3)：241-246.
[16]Wang Q M, Zeng G W. Studies on expression of sex differentiation program in Momrdica charantia L.[J]. Journal of Zhejiang University (Agricl & Life Sci.),2002,28(3):243-248.
[17]Friedlander M, Atsrmon D, Galan E. Sexual differentiation in cucumber:Abscisic acid and gibberellic acid contents of various sex genotypes[J]. Plant and Cell Physial,1997(18):681-691.
[18]邓俭英,张玲玲,方锋学等.园艺植物性别鉴定的研究概述[J].长江蔬菜,2005,12：31-33.
[19]李瑞丽,卢龙斗,高武军等.雌、雄异株植物性别鉴定的研究进展[J].广西植物,2006,26(4)：387-391.
[202]汪俏梅,曾广文.苦瓜性别分化的形态与组织化学研究.浙江农业大学学报[J].1997,23(2)：149-153.
[21]董莉娜,苏雪,孙坤.DNA分子标记在雌、雄异株植物性别鉴定中的应用[J]广西植物,26(1)：63-68.
[22]宋振巧,王洪刚,王建华.栝楼的研究进展[J].山东农业科学,2005,5：27-29.
[23]李珊,程舟,李彦等.雌、雄栝楼植株内含物比较研究[J].中草药,2008,39(2)：260-263.
[24]何长征,艾辛,匡逢春.不同性型黄瓜植株保护酶类活性的差异[J].湖南农业大学学报,2001,27(4)：289-291.
[25]孙颖,黄瓜性别表达研究[J].吉林师范大学学报(自然科学版)[J].2004,2：118-119.
[26]高武军,姬艳克,肖理会等.雌、雄异株植物性别决定相关功能基因研究进展[J].植物遗传资源学报2008,9(1)：125-129.
[27]BUZEK J, KOUTNIKOVA H, HOUBEN A, et al. Isolation and characterization of X chromosome-derived DNA sequences from a dioecious plant Melandrium album [J]. Chromosome Research,1997,5:57-65.
[28]MATSUNAGA S, KAWANO S, MICHIMOTO T, et al. Semi-automatic laser beam microdissection of the Y chromosome and analysis of Y chromosome DNA in a dioecious plant, Silene latifolia[J]. Plant and Cell Physiology,1999,40:60-68.
[29]LAN T Y,CHEN R Y,L I X L, et al. Microdissection and painting of the W chromosome in Ginkgo biloba showed different labeling patterns[J]. Botanical Studies,2008,49:33-37.
[30]王敏,黄璐琦,付桂芳.栝楼3个农家品种间亲缘近似关系的RAPD分析[J],中国中药杂志,1999,24(6)：336-337.
[3 1]黄璐琦,王敏,杨滨.栝楼农家品种苗期的分子标识鉴别[J].中国药学杂志,1999,34(9)：642.
[32]于凤池,孙耘子等.栝楼性别分化特异大分子标记物研究[J].浙江农业学报,2003,15(6)：332-335.
[33]李彦农,栝楼性别的分子标记研究[J].南京农业大学.2005.
[34]盖树鹏,孟祥栋.分子标记技术及其在作物育种中的应用.中国农学通报,2003,19(6)：12-15.
[35]涂振东,叶凯.生物技术在农作物遗传育种中应用的进展[J].新疆农业科学,2003,40(6)：381-383.
[36]徐小勇,刘继红,邓秀新.柑橘生物技术研究进展[J].中国生物工程杂志,2003,23(8)：35-38.
[37]韦晓霞,熊月明等.果树品种鉴定方法概述[J].福建果树,2002(4)：11-13.
[38]尹逸,李斌.DNA分子标记技术在茶树遗传多样性研究上的应用[J].广东茶叶,2000(3)11-14.
[39]彭建营,束怀瑞等.分子标记技术及其在果树种质资源研究上的应用[J].山东农业大学学报,2001,27(6)：41-45.
[40]F.Bartolozzi, M.L.Warburton, S. Arulsekar et al. Genetic characterization and relatedness among califonia almond cultivars and breeding lines detected by Randomly Amplified Polymorphic DNA Analysis. J. Amer. Soc. Hort. Sci,1998,123(3):381-387.
[41]邓九生,赖炽吕,彭民璋.柑橘RAPD技术与体细胞杂种鉴定.园艺学报,1998,25(2)：105-110.
[42]Roberto Botia, Aziz Akkak. Identification of pear cultivars by molecular marker. Acta Horticultural,1998,457：63-70.
[43]史永忠,郭文武,邓秀新.柑橘RAPD技术建立与体细胞杂种鉴定.园艺学报,1998,25(2)：105-110.
[44]罗正荣,米森敬三,杉浦明.应用RAPD技术研究柿品种间的亲缘关系果树科学[J].1998,15(4)：311-316.
[45]Williams JG, Kubelik AR, Livak KJ, Rafalski JA, Tingey SV. DNA polymorphisms amplified by arbitrary primers are useful as genetic markers.Nucleic Acids Res,1990,18:6531-6535.
[46]刘继红,胡春根.RAPD技术在果树研究中的应用[J].生命的化学,1998,18(1)：33-35.
[47]刘广林,李杨瑞,彭宏祥.RAPD技术应用于果树遗传育种的研究进展[J].广西农业科学,2006,37(6)：632-636.
[48]Harvey C F, Gil] G P, Fraser L G, et al. Sex determination in Actinidia 1. Sex-linked markers and progeny sex ratio in diploid A.chinensis[J]. Sex Plant Reprod,1997,10:149-154.
[49]陈劲枫,娄群峰,余纪柱等.黄瓜性别基因连锁的分子标记筛选[J].上海农业学报,2003,19(4)：11-14.
[50]Jiang C X, Sink K C. RAPD and SCAR markers linked to the sex expression locusM in Asparagus[J]. Euphytica,1997,94:329-333.
[51]Mulcahy D L, Weeden N F, Kessel R, et al. DNA p robes for the Y-chromosome of Silene latifolia, a dioecious angiosperm [J]. Sex Plant Reprod,1992,5:86-88.
[52]Paran I, Michelmore R W. Development of reliable PCR-based markers linked to downy mildew resistance genes in lettuce [J]. Theor App 1 Genet,1993,85:985-993.
[53]OttoT, N andor B, Erzsebet K, et al. Novel male-specific molecular markers (MADC5, MADC6) in hemp [J]. Euphytica,2002,127:209-218.
[54]Urasaki N, Tokumoto M, Tarora K, et al. A male and hermaphrodite specific RAPD marker for papaya (Carica papaya L.)[J]. TheorApp 1 Genet,2002,104:281-285.
[55]王筱璐,杭悦宇,周义峰等.与黄独(Dioscorea bulbifera)性别相关的RAPD-SCAR标记研究[J],植物资源与环境学报,2007,16(2)：1-5.
[56]谭冬梅,罗淑萍,李疆等.阿月浑子性别鉴定的RAPD分析[J].北方果树,2003,20(2)124-126.
[57]周国辉,李华平,张曙光等.番木瓜两性基因的RAPD标记[J].热带亚热带植物学报,2001, 9(3)：190-193.
[58]陈其军,韩玉珍,傅永福等.大麻性别的RAPD和SCAR分子标记[J].植物生理学报,2001,27(2)：173-178.
[59]王关林,方宏筠.植物基因工程(第二版)[M].北京：科学出版社,2002：533-535.
[60]Pich U, Schubert I. Method for isolation of DNA from Plants with a high content of polyphenolics. Nucleic Acids Research,1993,2(14):3328-3332.
[61]杨英军,李学强.落叶果树韧皮部提取DNA的方法研究[J].河南农业大学学报,2001,35(1)：97-98.
[62]奥斯伯F M,金斯顿R E,塞德曼J G等.马学军,舒跃龙等译校.精编分子生物学实验指南(第四版)[M].北京：科学出版社,2005：54-55.
[63]Paterson A H, Brubaber C L, Wendel J F. A rap id method for extraction of cotton (Gossypium spp.) genomic DNA suitable for RFLP of PCR analysis[J]. PlantMol Biol Rep,1993,11 (3):122-127.
[64]蒋立科,杨婉身.现代生物化学实验技术[M].北京：中国农业出版社,2003：253-256.
[65]朱英,陶刚,刘作易,等.普遍适用于农作物基因DNA的提取方法及值得注意的几个问题[J].贵州农业科学,2005,33(5)：20～21.
[66]付燕,罗楠,杨芩等,枇杷属植物ISSR反应体系的建立和优化[J].果树学报,2009,26(2)：180-185.
[67]张今今,王跃进.苹果PAPD反应体系的研究[J].西北农业大学学报,2000,28(5)：65-59.
[68]王得元,张长远等.辣椒RAPD反应体系研究.广东农业科学,2001,(1)：21-22.
[69]曾莉娟,郑成木.硅胶干燥旱稻叶片制备DNA样品及其RAPD反应体系的建立[J].热带农业科学,2002,22(5)：9-12.
[70]蒋曹德,邓昌彦.随机扩增多态DNA规范化反应技系探讨[J].生物技术通报,2002,158(3)：40-43.
[71]何正文,刘运生,陈立华等.正交设计直观分析法优化条件PCR[J].湖南医科大学学报,1998,23(4)：403-404.
[72]汪结明,项艳,吴大强等.杨树ISSR反应体系的建立及正交设计优化[J].核农学报,2007,21(5)：470-473.
[73]许红星,刘红彦,李锁平等.小麦抗白粉病基因pm6的RAPD标记和SCAR标记[J].河南农业科学.2004,4：25-28.
[74]盖树鹏,孟祥栋.大葱胞质雄性不育位点RAPD标记转化为SCAR标记的研究[J].莱阳农学院学报,2004,21(3)：189-192.
[75]柳波,孙彬,金德敏等.紫菜自由丝状体SCAR标记的获得[J].高技术通讯,2004,12：88-92.