黄皮（Clausena lansium Lour.）种质资源的ISSR分析
|
摘要
黄皮(Clausena lansium Lour.)Skeels属芸香科黄皮属,原产于我国南部,目前世界上发现黄皮属约有30多个种,在我国约有11个种,主要分布于福建、广东、广西、海南等省,是我国南方重要特产水果之一,已有1 500多年的栽培历史。广东和广西已经开展了相关的基础性研究,主要集中在栽培、管理、生理、形态及细胞学等方面,但从分子生物学方面进行研究的相对较少,对现有的资源遗传多样性及种质鉴定工作还缺乏系统的研究。
本研究在开展黄皮栽培品种资源的调查基础上,运用ISSR分子标记,研究了40份黄皮材料的遗传多样性,为黄皮种质资源的有效保护和合理利用提供科学依据,为黄皮新品种的培育提供遗传背景。主要研究结果如下:
1.针对黄皮富含酚类化合物和多糖等物质导致DNA提取和纯化困难的问题,采用经过改良的SDS法提取的黄皮DNA,不论从数量还是纯度上均可满足ISSR分析要求,摸索提出了合适的黄皮DNA提取方法。
2.研究建立了黄皮ISSR-PCR反应的技术体系。在20μL的反应体系中,TaqDNA聚合酶的用量为1.5 U,引物浓度为0.4μmol/L,dNTP的浓度为300μmol/L,模板DNA浓度为30 ng/反应,10×Buffer 2μL。按照此体系,在94℃预变性5 min,94℃变性30 s,在最适退火温度下退火60 s,72℃延伸90 s,进行40个循环,循环结束后72℃延伸10 min。该体系扩增出的条带清晰,效果良好,能够满足ISSR分析。
3.从96个引物中筛选出15个ISSR引物,对40份黄皮材料进行了ISSR分析。共扩增出165条带,其中多态性为100条,多态性百分率为60.6%。同时研究中发现含(AG)n、(GA)n序列的ISSR引物在黄皮上扩增的多态性水平较高,说明黄皮基因组中(TC)n/(CT)n含量丰富。
4.依据ISSR分析结果,对供试的40个品种进行聚类分析,结果表明:它们之间的遗传相似系数在0.714-1.000之间,揭示了不同省(区)地方性黄皮品种之间遗传多样性、遗传相似性与亲缘关系,进一步明晰了品种间的遗传距离。为以后更有目的、有选择的引种、育种,缩短育种年限,提高育种效率奠定了一定的理论基础。
Wampee(Clausena lansium Lour. Skeels) belongs to Rutacease, consists of about 30 species all over the world. It is one of the most well-known special fruit tree in south china, where is the native land of Wampee, there are 11 species, mainly distributing in Fujian, Guangdong, Guangxi, and Hainan. Wampee has been cultivated in china for 1500 years old. Guangdong and Guangxi have carried some basic studies accordingly, including cultivation, management, morphologically, physiologically and so on, seldom from the angle of molecular biology. The research on both genetic diversity investigation and idioplasm evaluation systematically are still absent.
In this study, inter-simple sequence repeat(ISSR) method was applied to detect genetic variation of cultivated Wampee from 40 samples. The results could provide scientific basis for making right protection strategy and help to foster new cultivars. The main results were as follow:
1. Faced the fact that it is difficulty to extract DNA from Wampee which contains phenolic compound and quite a lot of sugar, the researcher used improved SDS method to extract DNA of Wampee, which meets ISSR requirements in quantity and purity. So far it is quite a proper way to extract DNA from Wampee.
2. The technological system of ISSR-PCR reaction of Wampee has been built up. In the 20μL reaction system, there is 1.5 U of TaqDNAenzyme, 0.4μmol/L of primer, 300μmol/L of dNTP, 30 ng/reaction of template DNA, 2.0μL 10×Buffer solution. According to the system, the experimental steps are as follows. 5 min predenaturation at 94℃, 30 seconds denaturation at 94℃, 60 seconds annealing, 90 seconds extension at 72℃. The cycle repeats forty times and ends in 10 min extention at 72℃.
3. A total of 15 ISSR primers were employed on 40 individuals of cultivated Wampee from 96 primers, and 165 scorable amplified polymorphic were detected, of which 100(60.6%) were polymorphic loci. And this research found that primers containing ( AG) n, (GA) n sequence resulted in higher levels of amplified polymorphism of Wampee.
4. According to the clustering analysis of ISSR results, it proved that the genetic similarity index was 0.714～1.000. From the result, genetic diversity and similarity and genetic relationships were revealed.
本研究在开展黄皮栽培品种资源的调查基础上,运用ISSR分子标记,研究了40份黄皮材料的遗传多样性,为黄皮种质资源的有效保护和合理利用提供科学依据,为黄皮新品种的培育提供遗传背景。主要研究结果如下:
1.针对黄皮富含酚类化合物和多糖等物质导致DNA提取和纯化困难的问题,采用经过改良的SDS法提取的黄皮DNA,不论从数量还是纯度上均可满足ISSR分析要求,摸索提出了合适的黄皮DNA提取方法。
2.研究建立了黄皮ISSR-PCR反应的技术体系。在20μL的反应体系中,TaqDNA聚合酶的用量为1.5 U,引物浓度为0.4μmol/L,dNTP的浓度为300μmol/L,模板DNA浓度为30 ng/反应,10×Buffer 2μL。按照此体系,在94℃预变性5 min,94℃变性30 s,在最适退火温度下退火60 s,72℃延伸90 s,进行40个循环,循环结束后72℃延伸10 min。该体系扩增出的条带清晰,效果良好,能够满足ISSR分析。
3.从96个引物中筛选出15个ISSR引物,对40份黄皮材料进行了ISSR分析。共扩增出165条带,其中多态性为100条,多态性百分率为60.6%。同时研究中发现含(AG)n、(GA)n序列的ISSR引物在黄皮上扩增的多态性水平较高,说明黄皮基因组中(TC)n/(CT)n含量丰富。
4.依据ISSR分析结果,对供试的40个品种进行聚类分析,结果表明:它们之间的遗传相似系数在0.714-1.000之间,揭示了不同省(区)地方性黄皮品种之间遗传多样性、遗传相似性与亲缘关系,进一步明晰了品种间的遗传距离。为以后更有目的、有选择的引种、育种,缩短育种年限,提高育种效率奠定了一定的理论基础。
Wampee(Clausena lansium Lour. Skeels) belongs to Rutacease, consists of about 30 species all over the world. It is one of the most well-known special fruit tree in south china, where is the native land of Wampee, there are 11 species, mainly distributing in Fujian, Guangdong, Guangxi, and Hainan. Wampee has been cultivated in china for 1500 years old. Guangdong and Guangxi have carried some basic studies accordingly, including cultivation, management, morphologically, physiologically and so on, seldom from the angle of molecular biology. The research on both genetic diversity investigation and idioplasm evaluation systematically are still absent.
In this study, inter-simple sequence repeat(ISSR) method was applied to detect genetic variation of cultivated Wampee from 40 samples. The results could provide scientific basis for making right protection strategy and help to foster new cultivars. The main results were as follow:
1. Faced the fact that it is difficulty to extract DNA from Wampee which contains phenolic compound and quite a lot of sugar, the researcher used improved SDS method to extract DNA of Wampee, which meets ISSR requirements in quantity and purity. So far it is quite a proper way to extract DNA from Wampee.
2. The technological system of ISSR-PCR reaction of Wampee has been built up. In the 20μL reaction system, there is 1.5 U of TaqDNAenzyme, 0.4μmol/L of primer, 300μmol/L of dNTP, 30 ng/reaction of template DNA, 2.0μL 10×Buffer solution. According to the system, the experimental steps are as follows. 5 min predenaturation at 94℃, 30 seconds denaturation at 94℃, 60 seconds annealing, 90 seconds extension at 72℃. The cycle repeats forty times and ends in 10 min extention at 72℃.
3. A total of 15 ISSR primers were employed on 40 individuals of cultivated Wampee from 96 primers, and 165 scorable amplified polymorphic were detected, of which 100(60.6%) were polymorphic loci. And this research found that primers containing ( AG) n, (GA) n sequence resulted in higher levels of amplified polymorphism of Wampee.
4. According to the clustering analysis of ISSR results, it proved that the genetic similarity index was 0.714～1.000. From the result, genetic diversity and similarity and genetic relationships were revealed.
引文
[1]翁树章.华南特种果树栽培技术[M].广州:广东科技出版社,1997:1-2.
[2]宋立人,洪恂,丁绪亮,等.现代中药学大词典(下册)[M].北京:人民卫生出版社,2001:1895.
[3]潘建平,袁沛元,曾杨,等.华南地区黄皮良种、生产现状与发展对策[J].2007,(1):103-104.
[4]吴健华,赵向阳.无核黄皮的形成机理及主要品种[J].广西农业科学,2004,35(2):116-117.
[5]江海林,江根玲,伍丽芳.黄皮早结丰产栽培[M].广州:广东科技出版社,2000:11-12.
[6]潘晓芳,秦彦梅.黄皮花粉萌发研究[J].广西农业生物科学,2001,20(3):183-184.
[7]冯莉,刘蕊,郭丽荣,等.郁南无核黄皮花果生长发育的生物学特性研究[J].广东农业科学,2005,(6):42-44.
[8]王长龙,杨培新.黄皮果实生长发育规律研究[J].广东农业科学,2006,(3):25-27.
[9]郭文武,庞晓明,霍合强,等.黄皮染色体数目观察[J].华中农业大学学报,2000,19(2):166-167.
[10]姜燕,郭启高,李春艳,等.黄皮属2个种的染色体分析[J].西南农业大学学报(自然科学版),2005,27(4):544-546.
[11]陈瑞阳,李秀兰,宋文芹,等.我国部分热带果树染色体研究[J].武汉植物学研究,1985,3(4):423-428.
[12]冯莉,刘蕊.郁南无核黄皮染色体倍性变异研究初报[J].中国农学通报,2007,23(9):115-117.
[13]屈红霞,蒋跃明,李月标,等.黄皮耐贮性与果皮超微结构的研究[J].果树学报,2004,21(2):153-157.
[14]陈树思,唐为萍.黄皮次生木质部导管分子观察研究[J].湖北林业科技,2006,(139):18-20.
[15]李升锋,陈卫东,徐玉娟,等.无核黄皮的营养成分[J].食品科技,2005,(6):96-98.
[16]黄亚非,张永明,黄际薇,等.黄皮果挥发油化学成分及微量元素的研究[J].中国中药杂志,2006,31(11):898-900.
[17]张永明,黄亚非,黄际薇,等.黄皮果氨基酸成分分析[J].中药材,2006,29(9):921-923.
[18]潘小平,李美茹,杨谦.黄皮愈伤组织诱导及其对不同激素的响应[J].广西植物,2004,24(2):144-145.
[19]王瑞霞,何业华,罗吉,等.黄皮胚培养研究[J].果树学报,2006,23(4):581-586.
[20]刘小梅,潘建平,姜玲.黄皮RAPD-PCR反应体系的优化[J].广东农业科学,2006,(3):28-31.
[21]刘小梅,潘建平,曾杨,等.黄皮种质资源的RAPD分析[J].广东农业科学,2007,(2):13-15.
[22]郭永泽,何新华,李杨瑞,等.黄皮LEAFY同源基因的克隆与序列分析[J].林业科学,2007,43(10):130-133.
[23]秦民坚,王衡奇.黄皮树树皮的化学成分研究[J].林产化学与工业,2003,23(4):42-46.
[24]唐闻宁,康文艺,穆淑珍,等.黄皮果挥发油成分研究[J].天然产物研究与开发,2002,14(2):26.
[25]李瑞珍,廖华卫,陈飞苑.广州黄皮果挥发油成分研究[J].广东药学院学报,2007,23(2):141-143.
[26]钟秋平,林美芳.黄皮果中总黄酮含量的测定及其黄酮种类的鉴别[J].食品科学,2007,28(8):411-413.
[27]张建和,蔡春,罗辉,等.黄皮果核挥发油成分的研究[J].中药材,1997,20(10):518-519.
[28]卢海博,万树青,周丽兴.黄皮果核有机溶剂浸提物对稗草光活化生长抑制活性[J].杂草科学,2005,(2):11-13.
[29]罗辉,蔡春,张建和,等.黄皮叶挥发油化学成分研究[J].中药材,1998,21(8):405-406.
[30]廖华卫,邓金梅,黄敏仪.黄皮果皮挥发油成分研究[J].广东药学院学报,2006,22(2):139-141.
[31]杨明河,曹延怀,李伟勋,等.黄皮叶中黄皮酰胺的分离和结构测定[J].药学学报,1987,22(1):33.
[32]戴斌,丘翠嫦.黄皮叶的生药学研究[J].中国民族民间医药杂志,1997,6(29):38-41.
[33]饶尔昌,程家宠,杨光中,等.黄皮酰胺的合成[J].药学学报,1994,(29):502.
[34]侯软玲,综述,林军,等.黄皮酰胺的研究进展[J].广西医学,2007,29(3):377-379.
[35]吴继军,肖更生,张友胜,等.黄皮浸提汁及其酿酒工艺的研究[J].广东农业科学2006,(11):73-74.
[36]钟秋平,林美芳.黄皮果酒酿造工艺研究[J].食品科学,2007,28(10):650-651.
[37]丘华.黄皮、黑芝麻冰淇淋的开发研究[J].中国乳业,2002,(6):15-17.
[38]潘建平,袁沛元,林志雄,等.黄皮生产现状与发展对策[J].中国热带农业,2006,(2):35-39.
[39]Zietkiewicz E,Rafalski A,Labuda D.Genome fingerprinting by simple sequence repeat (SSR)-anchored polymerase chain reaction amplification[J].Genomics,1994,20(2):176-183.
[40]References S,Litt M,Luty J.A hypervariable microsatellite revealed by in vitro amplification of a dinucleotide repeat within the cardiac muscle actin gene[J].Am J Hum Genet,1989,44(3):397-401.
[41]周延清.DNA分子标记技术在植物研究中的应用[M].北京:化学工业出版社,2005:13-14.
[42]Fernandez M,Figueiras A,Benito C.The use of ISSR and RAPD markers for detecting DNA polymorphism,genotype identification and genetic diversity among barley cultivars with known origin[J].Theoretical and Applied Genetics,2002,104(5):845-851.
[43]王明明,宋振巧,王建华.ISSR标记技术及其在药用植物遗传育种中的应用[J].中草药,2007,38(1):134-136.
[44]GuptaM,ChyiYS.Amplification of DNA markers from evolutionarily diverse genomes using single primers of simple sequence repeats[J].Theoretical and Applied Genetics,1994,89:998-1006.
[45]References S,Wu K,Jones R,et al.Detection of microsatellite polymorphisms without cloning[J].Nucleic Acids Res,1994,22(15):3257-3258.
[46]Blair M W,Panaud O,McCouch S R.Inter-simple sequence repeat(ISSR)amplification for analysis of-microsatellite motif frequency and fingerprinting in rice(Oryza sativa L.)[J].Theoretical and Applied Genetics,1999,98(5):780-792.
[47]Wang Z,Weber J L,Zhong G,et al.Survey of plant short tandem DNA repeats[J].Theoretical and Applied Genetics,1994,88(1):1-6.
[48]Fang D Q,Roose M L.Identification of closely related citrus cultivars with inter-simple sequence repeat markers[J].Theoretical and Applied Genetics,1997,95(3):408-417.
[49]Tsumura Y,Ohba K,Strauss S H.Diversity and inheritance of inter-simple sequence repeat polymorphisms in Douglas-fir(Pseudotsuga menziesii)and sugi(Cryptomeria japonica)[J].Theoretical and Applied Genetics,1996,92(1):40-45.
[50]张立荣,徐大庆.SSR和ISSR分子标记及其在植物遗传育种研究中的应用[J].河北农业大学学报,2002,25(1):90-94.
[51]Nagaoka T,Ogihara Y.Applicability of inter-simple sequence repeat polymorphisms in wheat for use as DNA markers in comparison to RFLP and RAPD markers[J].Theoretical and Applied Genetics,1997,94(5):597-602.
[52]邓辉,黄晓燕,乙引.ISSR-PCR技术在植物种间和种下关系中的应用[J].种子,2006,25(4):55-57.
[53]王建波.ISSR分子标记及其在植物遗传学研究中的应用[J].遗传,2002,24(005):613-616.
[54]朴红梅,李万良,穆楠,等.ISSR标记的研究与应用[J].吉林农业科学,2007,32(5):28-30.
[55]Monte-Corvo L,Goulao L,Oliveira C.ISSR Analysis of Cultivars of Pearand Suitability of Molecular Markers for Clone Discrimination[J].J Amer Soc Hort Sci,2001,126(5):517-522.
[56]Amau G,Lallemand J,Bourgoin M.Fast and reliable strawbetrry cultivar identification using inter-simple sequence repeat(ISSR)amplification[J].Euphytica,1999,129:69-79.
[57]乔玉山,章镇,房经贵,等.李种质资源ISSR反应体系的建立[J],果树学报,2003,(4):270-274.
[58]Fang G,Hammar S,Grumet R.A quick and inexpensive method for removing polysaccharides from plant genomic DNA[J].Biotechniques,1992,(13):52-57.
[59]Moreno S,Martin J P,Ortiz J M.Inter-simple sequence repeats PCR for characterization of closely related grapevine germplasm[J].Euphytica,1998,101(1):117-125.
[60]邱英雄,傅承新.杨梅不同品种的ISSR分析[Jj.农业生物技术学报,2002,10(4):343-346.
[61]Prevost A,Wilkinson M J.A new system of comparing PCR primers applied to ISSR fingerprinting of potato cultivars[J].TAG Theoretical and Applied Genetics,1999,98(1):107-112.
[62]Potter D,Gao F Y,Aiello G,et al.Intersimple sequence repeat markers for fingerprinting and determining genetic relationships of walnut(Juglans regia)cultivars[J].Journal of the American Society for Horticultural Science,2002,127:75-81.
[63]杨玉玲,马祥庆,张木清.ISSR分子标记及其在树木遗传育种研究中的应用[J].亚热带农业研究,2006,2(1):18-24.
[64]Eiadthong W,Yonemori K,Sugiura A,et al.Identification of mango cultivars of Thailand and evaluation of their genetic variation using the amplified fragments by simple sequence repeat-(SSR-)anchored primers[J].Scientia Horticulturae,1999,82(1-2):57-66.
[65]杨若林,孔俊,吴鑫.ISSR标记在辣椒资源遗传多态性分析中的初步应用[J].上海大学学报(自然科学版),2005,11(4):235-237.
[66]Lanham P G,Korycinska A,Brennan R M.Genetic diversity within a secondary gene pool for Ribes nigrum L.revealed by RAPD and ISSR markers[J].Journal of Horticultural Science and Biotechnology,2000,75:371-375.
[67]葛永奇,邱英雄,丁炳扬,等.孑遗植物银杏群体遗传多样性的ISSR分析[J].生物多样性,2003,11(4):276-287.
[68]刘万勃,刘富中.RAPD和ISSR标记对甜瓜种质遗传多样性的研究[J].农业生物技术学报,2002,(3):231-236.
[69]Rajesh P N,Sant V J,Gupta V S,et al.Genetic relationships among annual and perennial wild species of Cicer using inter simple sequence repeat(ISSR)polymorphism[J].Euphytica,2003,129(1):15-23.
[70]姚明哲,黄海涛,余继忠,等.ISSR在茶树品种分子鉴别和亲缘关系研究中的适用性分析[J].茶叶科学,2005,25(2):153-157.
[71]Fang D,Krueger R R,Roose M L.Phylogenetic relationships among selected Citrus germplasm accessions revealed by inter-simple sequence repeat(ISSR)marker[J].Journal of the American Society for Horticultural Science,1998,123(4):612-617.
[72]Gulsen O,Roose M L.Lemons:Phylogenetic relationships among selected Citrus germplasm accessions revealed by inter-simple sequence repeat(ISSR)markers[J].J Amer Soc Hort Sci,1998,123(4):612-617.
[73]Goulao L,Monte-Corvo L,Oliveira C M.Phenetic characterization of plum cultivars by high multiplex ratio markers:amplified fragment length polymorphisms and inter-simple sequence repeats[J].Journal of the American Society for Horticultural Science,2001,126(1):72-77.
[74]Huang J C,Sun M.Genetic diversity and relationships of sweetpotato and its wild relatives in Ipomoea series Batatas(Convolvulaceae)as revealed by inter-simple sequence repeat(ISSR)and restriction analysis of chloroplast DNA[J].Theoretical and Applied Genetics,2000,100(7):1050-1060.
[75]陈龙,王家良,杨贤松.ISSR分子标记及其在植物分子生物学中的应用[J].种子,2007,26(10):49-52.
[76]吴旭红.甜菜抗丛根病基因(Rzl)ISSR分子标记的初步研究[J].中国糖料,2005,(2):9-11.
[77]吴旭红.甜菜抗根腐病基因ISSR分子标记的初步研究[J].齐齐哈尔大学学报,2005,6(2):30-32.
[78]Ratnaparkhe M B,Tekeoglu M,Muehlbauer F J.Inter-simple-sequence-repeat(ISSR)polymorphisms are useful for finding markers associated with disease resistance gene clusters[J].Theoretical and Applied Genetics,1998,97(4):515-519.
[79]Levin I,Gilboa N,Yeselson E,et al.Fgr,a major locus that modulates the fructose to glucose ratio in mature tomato fruits[J].Theoretical and Applied Genetics,2000,100(2):256-262.
[80]王绪,邓俭英,方锋学.ISSR分子标记技术及其在园艺作物中的应用[J].广西农业科学,2007,38(4):371-373.
[81]张青林,罗正荣.ISSR及其在果树上的应用[J].果树学报,2004,21(1):54-58.
[82]张仁兵,易克,许勇,等.用重组自交系构建西瓜分子遗传图谱[J].分子植物育种,2003,1(4):481-489.
[83]易克,徐向利,卢向阳,等.利用SSR和ISSR标记技术构建西瓜分子遗传图谱[J].湖南农业大学学报,2003,29(4):333-337.
[84]Sankar A A,Moore G A.Evaluation of inter-simple sequence repeat analysis for mapping in Citrus and extension of the genetic linkage map[J].Theoretical and Applied Genetics,2001,102(2):206-214.
[85]Cekic C,Battey N H,Wilkinson M J.The potential of ISSR-PCR primer-pair combinations for genetic linkage analysis using the seasonal flowering locus in Fragaria as a model[J].Theoretical and Applied Genetics,2001,103(4):540-546.
[86]朱元娣,李光晨,李春雨,等.苹果柱型基因的ISSR分子标记研究[J].园艺学报,2003,30(5):505-510.
[87]Yamamoto T,Kimura T,Shoda M,et al.Genetic linkage maps constructed by using an interspeciflc cross between Japanese and European pears[J].Theoretical and Applied Genetics,2002,106(1):9-18.
[88]Serquen F C,Bather J,Staub J E.Mapping and QTL analysis of horticultural traits in a narrow cross in cucumber(Cucumis sativus L.)using random-amplified polymorphic DNA markers[J].Molecular Breeding,1997,3(4):257-268.
[89]Yu Y G,Maroof M A S,Buss G R.Divergence and allelomorphic relationship of a soybean virus resistance gene based on tightly linked DNA microsatellite and RFLP markers[J].Theoretical and Applied Genetics,1996,92(1):64-69.
[90]方宣钧,宛煜嵩.我国“应县小黑豆”对SCN4号生理小种抗性的SSR及ISSR分子标记研究[J].农业生物技术学报,2002,10(1):81-85.
[91]杨传平,魏利,姜静,等.应用ISSR-PCR对西伯利亚红松19个种源的遗传多样[J].东北林业大学学报,2005,33(1):1-3.
[92]Holden M,Shipley G.Inquiry into the problems left over in PCR[J].Biotech World,2005,10:21-26.
[93](美)萨姆布鲁克(Sambrook,J.)等.分子克隆实验指南(下册)[M].黄培堂.第3版.北京:科学出版社,2002:1566-1570.
[94]林郑和.我国39个茶树品种的RAPD与ISSR分析[D].福州:福建农林大学,2006.
[95]丁晓东,吕柳新.从顽拗植物荔枝中提取基因组DNA技术的研究[J].应用与环境生物学报,2000,6(2):142-145.
[96]刘萍,代红军.DNA提取方法与植物种类的效应比较[J].宁夏农林科技,1998,(1):15-18.
[97]Porebski S,Bailey L G,Baum B R.Modification of a CTAB DNA extraction protocol for plants containing high polysaccharide and polyphenol components[J].Plant Molecular Biology Reporter,1997,15(1):8-15.
[98]Tesniere C,Vayda M E.Method for the isolation of high-quality RNA from grape berry tissues without contaminating tannins or carbohydrates[J].Plant Molecular Biology Reporter,1991,9(3):242-251.
[99]王卓伟,余茂德.PVP在桑叶总DNA提取中的应用[J].西南农业大学学报,2001,23(1):62-62.
[100]陈桂信,吕柳新,赖钟雄,等.嫩芽总RNA的提取与纯化[J].江西农业大学学报,2004,26(3):324-328.
[101]Williams J G K,Kubelik A R,Livak K J,et al.DNA polymorphisms amplified by arbitrary primers are useful as genetic markers[J].Nucleic Acids Res,1990,18(22):6531-6535.
[102]Welsh J,McClelland M.Genomic fingerprinting using arbitrarily primed PCR and a matrix of pairwise combinations of primers[J].Nucleic Acids Res,1991,19(19):5275-5279.
[103]Smith J J,Scott-Craig J S,Leadbetter J R,et al.Characterization of random amplified polymorphic DNA(RAPD)products from Xanthomonas campestris and some comments on the use of RAPD products in phylogenetic analysis[J].Mol Phylogenet Evol,1994,3(2):135-145.
[104]Ammiraju J S S,Dholakia B B,Santra D K,et al.Identification of inter simple sequence repeat (ISSR)markers associated with seed size in wheat[J].Theoretical and Applied Genetics,2001,102(5):726-732.
[105]张恒庆,安利佳.红松RAPD实验中各组成成份含量对实验结果的影响[J].植物研究,1999,19(2):183-188.
[106]彭海,张静,张梁,等.长豇豆品种ISSR-PCR最佳退火温度的筛选[J].长江蔬菜,2007,(9):3-8.
[107]Barrett B A,Kidwell K K.AFLP-based genetic diversity assessment among wheat cultivars from the Pacific Northwest[J].Crop science,1998,38(5):1261-1271.
[108]Hamrick J L,Loveless M D.The genetic structure of tropical tree populations:associations with reproductive biology[J].The Evolutionary Ecology of Plants,1989:129-146.
[109]Godwin I D,Aitken E A,Smith L W.Application of inter simple sequence repeat(ISSR)markers to plant genetics[J].Electrophoresis,1997,18(9):1524-1528.
[110]刘小梅,潘建平,曾杨,等.黄皮种质资源的RAPD分析[J].广东农业科学,2007,(2):13-15.
[2]宋立人,洪恂,丁绪亮,等.现代中药学大词典(下册)[M].北京:人民卫生出版社,2001:1895.
[3]潘建平,袁沛元,曾杨,等.华南地区黄皮良种、生产现状与发展对策[J].2007,(1):103-104.
[4]吴健华,赵向阳.无核黄皮的形成机理及主要品种[J].广西农业科学,2004,35(2):116-117.
[5]江海林,江根玲,伍丽芳.黄皮早结丰产栽培[M].广州:广东科技出版社,2000:11-12.
[6]潘晓芳,秦彦梅.黄皮花粉萌发研究[J].广西农业生物科学,2001,20(3):183-184.
[7]冯莉,刘蕊,郭丽荣,等.郁南无核黄皮花果生长发育的生物学特性研究[J].广东农业科学,2005,(6):42-44.
[8]王长龙,杨培新.黄皮果实生长发育规律研究[J].广东农业科学,2006,(3):25-27.
[9]郭文武,庞晓明,霍合强,等.黄皮染色体数目观察[J].华中农业大学学报,2000,19(2):166-167.
[10]姜燕,郭启高,李春艳,等.黄皮属2个种的染色体分析[J].西南农业大学学报(自然科学版),2005,27(4):544-546.
[11]陈瑞阳,李秀兰,宋文芹,等.我国部分热带果树染色体研究[J].武汉植物学研究,1985,3(4):423-428.
[12]冯莉,刘蕊.郁南无核黄皮染色体倍性变异研究初报[J].中国农学通报,2007,23(9):115-117.
[13]屈红霞,蒋跃明,李月标,等.黄皮耐贮性与果皮超微结构的研究[J].果树学报,2004,21(2):153-157.
[14]陈树思,唐为萍.黄皮次生木质部导管分子观察研究[J].湖北林业科技,2006,(139):18-20.
[15]李升锋,陈卫东,徐玉娟,等.无核黄皮的营养成分[J].食品科技,2005,(6):96-98.
[16]黄亚非,张永明,黄际薇,等.黄皮果挥发油化学成分及微量元素的研究[J].中国中药杂志,2006,31(11):898-900.
[17]张永明,黄亚非,黄际薇,等.黄皮果氨基酸成分分析[J].中药材,2006,29(9):921-923.
[18]潘小平,李美茹,杨谦.黄皮愈伤组织诱导及其对不同激素的响应[J].广西植物,2004,24(2):144-145.
[19]王瑞霞,何业华,罗吉,等.黄皮胚培养研究[J].果树学报,2006,23(4):581-586.
[20]刘小梅,潘建平,姜玲.黄皮RAPD-PCR反应体系的优化[J].广东农业科学,2006,(3):28-31.
[21]刘小梅,潘建平,曾杨,等.黄皮种质资源的RAPD分析[J].广东农业科学,2007,(2):13-15.
[22]郭永泽,何新华,李杨瑞,等.黄皮LEAFY同源基因的克隆与序列分析[J].林业科学,2007,43(10):130-133.
[23]秦民坚,王衡奇.黄皮树树皮的化学成分研究[J].林产化学与工业,2003,23(4):42-46.
[24]唐闻宁,康文艺,穆淑珍,等.黄皮果挥发油成分研究[J].天然产物研究与开发,2002,14(2):26.
[25]李瑞珍,廖华卫,陈飞苑.广州黄皮果挥发油成分研究[J].广东药学院学报,2007,23(2):141-143.
[26]钟秋平,林美芳.黄皮果中总黄酮含量的测定及其黄酮种类的鉴别[J].食品科学,2007,28(8):411-413.
[27]张建和,蔡春,罗辉,等.黄皮果核挥发油成分的研究[J].中药材,1997,20(10):518-519.
[28]卢海博,万树青,周丽兴.黄皮果核有机溶剂浸提物对稗草光活化生长抑制活性[J].杂草科学,2005,(2):11-13.
[29]罗辉,蔡春,张建和,等.黄皮叶挥发油化学成分研究[J].中药材,1998,21(8):405-406.
[30]廖华卫,邓金梅,黄敏仪.黄皮果皮挥发油成分研究[J].广东药学院学报,2006,22(2):139-141.
[31]杨明河,曹延怀,李伟勋,等.黄皮叶中黄皮酰胺的分离和结构测定[J].药学学报,1987,22(1):33.
[32]戴斌,丘翠嫦.黄皮叶的生药学研究[J].中国民族民间医药杂志,1997,6(29):38-41.
[33]饶尔昌,程家宠,杨光中,等.黄皮酰胺的合成[J].药学学报,1994,(29):502.
[34]侯软玲,综述,林军,等.黄皮酰胺的研究进展[J].广西医学,2007,29(3):377-379.
[35]吴继军,肖更生,张友胜,等.黄皮浸提汁及其酿酒工艺的研究[J].广东农业科学2006,(11):73-74.
[36]钟秋平,林美芳.黄皮果酒酿造工艺研究[J].食品科学,2007,28(10):650-651.
[37]丘华.黄皮、黑芝麻冰淇淋的开发研究[J].中国乳业,2002,(6):15-17.
[38]潘建平,袁沛元,林志雄,等.黄皮生产现状与发展对策[J].中国热带农业,2006,(2):35-39.
[39]Zietkiewicz E,Rafalski A,Labuda D.Genome fingerprinting by simple sequence repeat (SSR)-anchored polymerase chain reaction amplification[J].Genomics,1994,20(2):176-183.
[40]References S,Litt M,Luty J.A hypervariable microsatellite revealed by in vitro amplification of a dinucleotide repeat within the cardiac muscle actin gene[J].Am J Hum Genet,1989,44(3):397-401.
[41]周延清.DNA分子标记技术在植物研究中的应用[M].北京:化学工业出版社,2005:13-14.
[42]Fernandez M,Figueiras A,Benito C.The use of ISSR and RAPD markers for detecting DNA polymorphism,genotype identification and genetic diversity among barley cultivars with known origin[J].Theoretical and Applied Genetics,2002,104(5):845-851.
[43]王明明,宋振巧,王建华.ISSR标记技术及其在药用植物遗传育种中的应用[J].中草药,2007,38(1):134-136.
[44]GuptaM,ChyiYS.Amplification of DNA markers from evolutionarily diverse genomes using single primers of simple sequence repeats[J].Theoretical and Applied Genetics,1994,89:998-1006.
[45]References S,Wu K,Jones R,et al.Detection of microsatellite polymorphisms without cloning[J].Nucleic Acids Res,1994,22(15):3257-3258.
[46]Blair M W,Panaud O,McCouch S R.Inter-simple sequence repeat(ISSR)amplification for analysis of-microsatellite motif frequency and fingerprinting in rice(Oryza sativa L.)[J].Theoretical and Applied Genetics,1999,98(5):780-792.
[47]Wang Z,Weber J L,Zhong G,et al.Survey of plant short tandem DNA repeats[J].Theoretical and Applied Genetics,1994,88(1):1-6.
[48]Fang D Q,Roose M L.Identification of closely related citrus cultivars with inter-simple sequence repeat markers[J].Theoretical and Applied Genetics,1997,95(3):408-417.
[49]Tsumura Y,Ohba K,Strauss S H.Diversity and inheritance of inter-simple sequence repeat polymorphisms in Douglas-fir(Pseudotsuga menziesii)and sugi(Cryptomeria japonica)[J].Theoretical and Applied Genetics,1996,92(1):40-45.
[50]张立荣,徐大庆.SSR和ISSR分子标记及其在植物遗传育种研究中的应用[J].河北农业大学学报,2002,25(1):90-94.
[51]Nagaoka T,Ogihara Y.Applicability of inter-simple sequence repeat polymorphisms in wheat for use as DNA markers in comparison to RFLP and RAPD markers[J].Theoretical and Applied Genetics,1997,94(5):597-602.
[52]邓辉,黄晓燕,乙引.ISSR-PCR技术在植物种间和种下关系中的应用[J].种子,2006,25(4):55-57.
[53]王建波.ISSR分子标记及其在植物遗传学研究中的应用[J].遗传,2002,24(005):613-616.
[54]朴红梅,李万良,穆楠,等.ISSR标记的研究与应用[J].吉林农业科学,2007,32(5):28-30.
[55]Monte-Corvo L,Goulao L,Oliveira C.ISSR Analysis of Cultivars of Pearand Suitability of Molecular Markers for Clone Discrimination[J].J Amer Soc Hort Sci,2001,126(5):517-522.
[56]Amau G,Lallemand J,Bourgoin M.Fast and reliable strawbetrry cultivar identification using inter-simple sequence repeat(ISSR)amplification[J].Euphytica,1999,129:69-79.
[57]乔玉山,章镇,房经贵,等.李种质资源ISSR反应体系的建立[J],果树学报,2003,(4):270-274.
[58]Fang G,Hammar S,Grumet R.A quick and inexpensive method for removing polysaccharides from plant genomic DNA[J].Biotechniques,1992,(13):52-57.
[59]Moreno S,Martin J P,Ortiz J M.Inter-simple sequence repeats PCR for characterization of closely related grapevine germplasm[J].Euphytica,1998,101(1):117-125.
[60]邱英雄,傅承新.杨梅不同品种的ISSR分析[Jj.农业生物技术学报,2002,10(4):343-346.
[61]Prevost A,Wilkinson M J.A new system of comparing PCR primers applied to ISSR fingerprinting of potato cultivars[J].TAG Theoretical and Applied Genetics,1999,98(1):107-112.
[62]Potter D,Gao F Y,Aiello G,et al.Intersimple sequence repeat markers for fingerprinting and determining genetic relationships of walnut(Juglans regia)cultivars[J].Journal of the American Society for Horticultural Science,2002,127:75-81.
[63]杨玉玲,马祥庆,张木清.ISSR分子标记及其在树木遗传育种研究中的应用[J].亚热带农业研究,2006,2(1):18-24.
[64]Eiadthong W,Yonemori K,Sugiura A,et al.Identification of mango cultivars of Thailand and evaluation of their genetic variation using the amplified fragments by simple sequence repeat-(SSR-)anchored primers[J].Scientia Horticulturae,1999,82(1-2):57-66.
[65]杨若林,孔俊,吴鑫.ISSR标记在辣椒资源遗传多态性分析中的初步应用[J].上海大学学报(自然科学版),2005,11(4):235-237.
[66]Lanham P G,Korycinska A,Brennan R M.Genetic diversity within a secondary gene pool for Ribes nigrum L.revealed by RAPD and ISSR markers[J].Journal of Horticultural Science and Biotechnology,2000,75:371-375.
[67]葛永奇,邱英雄,丁炳扬,等.孑遗植物银杏群体遗传多样性的ISSR分析[J].生物多样性,2003,11(4):276-287.
[68]刘万勃,刘富中.RAPD和ISSR标记对甜瓜种质遗传多样性的研究[J].农业生物技术学报,2002,(3):231-236.
[69]Rajesh P N,Sant V J,Gupta V S,et al.Genetic relationships among annual and perennial wild species of Cicer using inter simple sequence repeat(ISSR)polymorphism[J].Euphytica,2003,129(1):15-23.
[70]姚明哲,黄海涛,余继忠,等.ISSR在茶树品种分子鉴别和亲缘关系研究中的适用性分析[J].茶叶科学,2005,25(2):153-157.
[71]Fang D,Krueger R R,Roose M L.Phylogenetic relationships among selected Citrus germplasm accessions revealed by inter-simple sequence repeat(ISSR)marker[J].Journal of the American Society for Horticultural Science,1998,123(4):612-617.
[72]Gulsen O,Roose M L.Lemons:Phylogenetic relationships among selected Citrus germplasm accessions revealed by inter-simple sequence repeat(ISSR)markers[J].J Amer Soc Hort Sci,1998,123(4):612-617.
[73]Goulao L,Monte-Corvo L,Oliveira C M.Phenetic characterization of plum cultivars by high multiplex ratio markers:amplified fragment length polymorphisms and inter-simple sequence repeats[J].Journal of the American Society for Horticultural Science,2001,126(1):72-77.
[74]Huang J C,Sun M.Genetic diversity and relationships of sweetpotato and its wild relatives in Ipomoea series Batatas(Convolvulaceae)as revealed by inter-simple sequence repeat(ISSR)and restriction analysis of chloroplast DNA[J].Theoretical and Applied Genetics,2000,100(7):1050-1060.
[75]陈龙,王家良,杨贤松.ISSR分子标记及其在植物分子生物学中的应用[J].种子,2007,26(10):49-52.
[76]吴旭红.甜菜抗丛根病基因(Rzl)ISSR分子标记的初步研究[J].中国糖料,2005,(2):9-11.
[77]吴旭红.甜菜抗根腐病基因ISSR分子标记的初步研究[J].齐齐哈尔大学学报,2005,6(2):30-32.
[78]Ratnaparkhe M B,Tekeoglu M,Muehlbauer F J.Inter-simple-sequence-repeat(ISSR)polymorphisms are useful for finding markers associated with disease resistance gene clusters[J].Theoretical and Applied Genetics,1998,97(4):515-519.
[79]Levin I,Gilboa N,Yeselson E,et al.Fgr,a major locus that modulates the fructose to glucose ratio in mature tomato fruits[J].Theoretical and Applied Genetics,2000,100(2):256-262.
[80]王绪,邓俭英,方锋学.ISSR分子标记技术及其在园艺作物中的应用[J].广西农业科学,2007,38(4):371-373.
[81]张青林,罗正荣.ISSR及其在果树上的应用[J].果树学报,2004,21(1):54-58.
[82]张仁兵,易克,许勇,等.用重组自交系构建西瓜分子遗传图谱[J].分子植物育种,2003,1(4):481-489.
[83]易克,徐向利,卢向阳,等.利用SSR和ISSR标记技术构建西瓜分子遗传图谱[J].湖南农业大学学报,2003,29(4):333-337.
[84]Sankar A A,Moore G A.Evaluation of inter-simple sequence repeat analysis for mapping in Citrus and extension of the genetic linkage map[J].Theoretical and Applied Genetics,2001,102(2):206-214.
[85]Cekic C,Battey N H,Wilkinson M J.The potential of ISSR-PCR primer-pair combinations for genetic linkage analysis using the seasonal flowering locus in Fragaria as a model[J].Theoretical and Applied Genetics,2001,103(4):540-546.
[86]朱元娣,李光晨,李春雨,等.苹果柱型基因的ISSR分子标记研究[J].园艺学报,2003,30(5):505-510.
[87]Yamamoto T,Kimura T,Shoda M,et al.Genetic linkage maps constructed by using an interspeciflc cross between Japanese and European pears[J].Theoretical and Applied Genetics,2002,106(1):9-18.
[88]Serquen F C,Bather J,Staub J E.Mapping and QTL analysis of horticultural traits in a narrow cross in cucumber(Cucumis sativus L.)using random-amplified polymorphic DNA markers[J].Molecular Breeding,1997,3(4):257-268.
[89]Yu Y G,Maroof M A S,Buss G R.Divergence and allelomorphic relationship of a soybean virus resistance gene based on tightly linked DNA microsatellite and RFLP markers[J].Theoretical and Applied Genetics,1996,92(1):64-69.
[90]方宣钧,宛煜嵩.我国“应县小黑豆”对SCN4号生理小种抗性的SSR及ISSR分子标记研究[J].农业生物技术学报,2002,10(1):81-85.
[91]杨传平,魏利,姜静,等.应用ISSR-PCR对西伯利亚红松19个种源的遗传多样[J].东北林业大学学报,2005,33(1):1-3.
[92]Holden M,Shipley G.Inquiry into the problems left over in PCR[J].Biotech World,2005,10:21-26.
[93](美)萨姆布鲁克(Sambrook,J.)等.分子克隆实验指南(下册)[M].黄培堂.第3版.北京:科学出版社,2002:1566-1570.
[94]林郑和.我国39个茶树品种的RAPD与ISSR分析[D].福州:福建农林大学,2006.
[95]丁晓东,吕柳新.从顽拗植物荔枝中提取基因组DNA技术的研究[J].应用与环境生物学报,2000,6(2):142-145.
[96]刘萍,代红军.DNA提取方法与植物种类的效应比较[J].宁夏农林科技,1998,(1):15-18.
[97]Porebski S,Bailey L G,Baum B R.Modification of a CTAB DNA extraction protocol for plants containing high polysaccharide and polyphenol components[J].Plant Molecular Biology Reporter,1997,15(1):8-15.
[98]Tesniere C,Vayda M E.Method for the isolation of high-quality RNA from grape berry tissues without contaminating tannins or carbohydrates[J].Plant Molecular Biology Reporter,1991,9(3):242-251.
[99]王卓伟,余茂德.PVP在桑叶总DNA提取中的应用[J].西南农业大学学报,2001,23(1):62-62.
[100]陈桂信,吕柳新,赖钟雄,等.嫩芽总RNA的提取与纯化[J].江西农业大学学报,2004,26(3):324-328.
[101]Williams J G K,Kubelik A R,Livak K J,et al.DNA polymorphisms amplified by arbitrary primers are useful as genetic markers[J].Nucleic Acids Res,1990,18(22):6531-6535.
[102]Welsh J,McClelland M.Genomic fingerprinting using arbitrarily primed PCR and a matrix of pairwise combinations of primers[J].Nucleic Acids Res,1991,19(19):5275-5279.
[103]Smith J J,Scott-Craig J S,Leadbetter J R,et al.Characterization of random amplified polymorphic DNA(RAPD)products from Xanthomonas campestris and some comments on the use of RAPD products in phylogenetic analysis[J].Mol Phylogenet Evol,1994,3(2):135-145.
[104]Ammiraju J S S,Dholakia B B,Santra D K,et al.Identification of inter simple sequence repeat (ISSR)markers associated with seed size in wheat[J].Theoretical and Applied Genetics,2001,102(5):726-732.
[105]张恒庆,安利佳.红松RAPD实验中各组成成份含量对实验结果的影响[J].植物研究,1999,19(2):183-188.
[106]彭海,张静,张梁,等.长豇豆品种ISSR-PCR最佳退火温度的筛选[J].长江蔬菜,2007,(9):3-8.
[107]Barrett B A,Kidwell K K.AFLP-based genetic diversity assessment among wheat cultivars from the Pacific Northwest[J].Crop science,1998,38(5):1261-1271.
[108]Hamrick J L,Loveless M D.The genetic structure of tropical tree populations:associations with reproductive biology[J].The Evolutionary Ecology of Plants,1989:129-146.
[109]Godwin I D,Aitken E A,Smith L W.Application of inter simple sequence repeat(ISSR)markers to plant genetics[J].Electrophoresis,1997,18(9):1524-1528.
[110]刘小梅,潘建平,曾杨,等.黄皮种质资源的RAPD分析[J].广东农业科学,2007,(2):13-15.