摘要
黄连木(Pistacia chinesis Bunge.)和阿月浑子(Pistacia vera L)是黄连木属重要的生物质能源和经济树种。本文采用形态学观察和DNA分子标记技术对不同种源的黄连木群体和引进的阿月浑子品种进行了遗传多样性和亲缘关系分析,以期为黄连木优良种质和阿月浑子优良品种的筛选提供科学依据,主要研究结果如下:
1.采用形态学观察和生理测定的方法,对不同种源黄连木种子的横纵径比、千粒重等形态学指标和发芽率、发芽势等生理指标进行了分析比较,结果表明,不同种源黄连木种子的横纵径比、千粒重、发芽率、发芽势等指标均存在显著或极显著差异。种子千粒重为37.56~56.20 g ,横纵径比为0.55~0.75 ;发芽率为15.25%~54.38%,发芽势在20.55%~54.38%之间。武安种子的千粒重为56.20 g,明显大于其它种源的种子,最小的为安徽种子。林州种源发芽率(54.38%)最高。其次是四川、房山、武安和房山,最低的是绛县。
2.利用UPGMA聚类分析,依据黄连木种子表型性状进行了分类研究。参试6个种源可分为两个组群:第一组群包括安徽滁州、四川康定、北京房山3个群体,其中四川康定与北京房山群体种子表型特征基本相同;第二组群包括山西绛县、河南林州、河北武安3个群体,其中山西绛县与河南林州群体种子表型特征较为相近。
3. DNA提取方法:用预处液(3%可溶性PVP,2%β-巯基乙醇,100mmol/L~(-1) EDTA)和CTAB提取缓冲液(2%β-巯基乙醇、水溶性PVP和4%CTAB)提取阿月浑子和黄连木基因组DNA,其OD_(260)/OD_(280)值在1.78~1.9之间,可用于PCR扩增。
4.利用正交试验设计L_(16)(4~4),对模板DNA、Taq DNA聚合酶、引物浓度、dNTP浓度进行了筛选研究,建立了适宜黄连木的RAMP反应体系。20μL PCR反应体系为:1×buffer﹢0.8μL dNTP(250μmolL~(-1))﹢0.8μL引物﹢0.8μL Taq酶。
5.不同种源黄连木的RAMP分析:13对引物组合所产生的115条DNA扩增片段中,有109条具有多态性,多态位点百分率(PPB)为94.78%;Shannon多样性指数I=0.5541,Nei's多样性指数H=0.38030,物种总基因多样性(Ht)为0.2963;种群内基因多样性(Hs)为0.3803,群体间基因分化系数(Gst)为0.2209,表明22.09%的变异存在于种群间,群体内的变异占了总变异的77.91%;基因流(Nm)为1.7633,显示种群间的基因交流顺畅。UPGMA聚类分析表明,以遗传距离0.16为阈值,可以分为两类:武安、林州、房山、滁州、绛县居群聚为一类;康定居群独自聚为一类。
5.不同阿月浑子品种亲缘关系分析:用筛选的21个随机引物对13个品种阿月浑子DNA进行PCR扩增,共扩增出137个位点,其中多态位点122个,多态位点比率占89.05%,平均每个引物扩增多态性DNA的带数为5.81条。品种间遗传距离在0.2015~0.5163之间,表明各品种间具有一定的遗传差异。UPGMA聚类分析表明,13个阿月浑子品种在遗传距离0.40处可划分为3个类群,基本反映了阿月浑子品种间的遗传多样性。
Pistacia chinesis Bunge. and pistachio (Pistacia vera L) are important energy and economic trees, this article from the seed morphological markers and DNA markers analysis of genetic diversity of Pistacia chinesis Bunge.and Pistacia vera L. genetic relationship between sub-species, and has achieved initial results.
In this paper, the length to width ratio, 1000 seeds’mass, germinating rate, germinationcharacters of seeds which originated from six different geographic of Pistacia chinensis Bunge were studied. The result showed that the range of the mass was 37.56 g~56.20 g, the range of length and width ratio of the seeds from 0.55~0.75, and the range of germinating energy from 20.25% to 54.38%, the germinating tendency from 15.25% to 54.38%. The Pistacia chinensis Bunge seeds from different geographic including vertical and length to width ratio, 1000 seeds’mass, germinating rate arrived significant or extremely significant. The WuAn 1000 seeds’mass was 56.20 g, significantly higher than other. the smallest seed was Anhui. Lin states provenance The germination rate of LinZhou was (54.38%) it is Max. Followed by Sichuan, Fangshan, Wu Ann, the lowest was JiangXian.
Pistacia chinesis Bunge. seed phenotype based on UPGMA clustering index is divided into two groups: the first group including Chuzhou, Kangding, Fangshan. The Kangding seed phenotypes similar with Fangshan; the second one including Jiangxian, Linzhou,Wuan. The seed phenotypes of Jiangxian state are more similar to Linzhou.
With a pretreatment solution (3% soluble PVP, 2%β-mercaptoethanol, 100 mmol / L-1 EDTA) and CTAB extraction buffer (2%β-mercaptoethanol, water-soluble PVP and 4% CTAB) extraction of pistachio and Pistacia chinensis Bunge genomic DNA, ultraviolet spectrophotometry agent test results OD260/OD280 values were between 1.78 to 1.9, can be a template for PCR amplification of DNA.
Using orthogonal design L16(44)to investigate four factors(TaqDNA polymerase, dNTP, primer, DNA) at four levels respectively. The RAMP reaction system suitable for Pistacia chinesis Bunge.was established, that was, 20μl amplification reactions system containing 1×PCR buffer, 250μl mol/L-1.dNTPs, 0.8μL primer, 10 ng template DNA, 0.8μL TaqDNA polymerase.
The genetic diversity of Pistacia chinesis Bunge. was analyzed using random amplified microsatellite polymorphism (RAMP) markers. The results indicated that there was relatively high genetic diversitv in china. 115 bands were amplified from 6 populations in different habitats with 13 reliable primers, 109 bands were polymorphic, The polymorphic percentage was 94.78%; The Shannon information index I was 0.5541 and Nei’s index H was 0.38030. The total gene diversity Ht was 0.2963, and the gene diversity within populations Hs was 0.3803. The Gst was 0.2209, 77.91% of genetic variation existed within populations while 22.09% of genetic variation existed among populations, and the gene flow was 1.7633. There was a strong gene flow among the populations, So the genetic drift would not cause the genetic differentiation.
In order to analyze Pistacia vera L. genetic relationship, random amplified polymorphic DNA (RAPD) was used to study the classification and identification of 13 pistachio cultivars which includs 12 exotic varities and one native selects of Xinjiang. Twenty-one 10 bp primers that selected from eighty arbitrary primers were applied for amplifying the Pistachio DNA. Total 137 bands were produced, among which 122 bands (89.05%) were polymorphic. Genetic distances among the cultivars were estimated based on the amount of band sharing and ranged from 0.2015~0.5163, it is indicated that there are genetic differences among the cultivars. UPGMA cluster analysis showed that 13 pistachio varieties can be divided into three groups in the genetic distance of 0.40 level: The first category includes eleven varieties, the second category is the‘Xinjiang selection’and the third category is‘Mateur’varieties.
引文
[1]中国森林编委会编.中国森林[M].北京:中国林业出版社,1997,6.
[2]李晓旭.不同种源的黄连木种子比较分析[D].北京林业大学硕士论文.2009.
[3]王涛.中国主要生物质燃料油木本能源植物资源概况与展望[J]科技导报,2005,23(5):12-14.
[4]刘克锋,柳振亮.黄连木容器育苗及其抗旱性研究[J].北京林业大学学报,2002, 24(2):27-30.
[5]刘启慎,谭浩亮,李建新.中国黄连木生长规律的研究[J].河南林业科技,1999, 19(2):3-6.
[6]肖彦荣,常剑文.石灰岩山地干旱阳坡造林的先锋树——黄连木[J].河北林业科技, 1995,(04).
[7]孟树标,康月兰.石灰岩山地造林的先锋树种──黄连木[J].植物杂志,1999,(1):18.
[8]肖彦荣,田玉堂,王喜成.黄连木结实期年生长规律的研究[J].河北林果研究1998, 13(1)55-57.
[9]裴宝红.黄连木的栽培技术[J].中国林副特产,2002,(2):7.
[10]候新村.生物柴油木本能源植物中国黄连木的调查与研究[D].中国林科院博士论文,2005.
[11]柳振亮,刘克锋,李树蓉等.阿月浑子及黄连木种子处理试验[J].北京农学院学报,2002,17(4):26-29.
[12]徐金燕,石玉琴.中国黄连木播种育苗技术[J].经济林研究,2001,19(4):35.
[13]毛家富.生物质能源树种黄连木的苗木资源培育技术[J].现代农业科技, 2008,(2):56-57.
[14]赵和文,柳振亮等.黄连木种子处理的研究[J].北京农学院学报2004,19(3):42-45.
[15]王花兰.黄连木大田育苗技术[J].甘肃科技,2003,20(2):148-149.
[16]赵和文,刘勇.黄连木幼苗施肥试验研究[J].北京农学院学报2005,20(1):68-70.
[17]魏玉君,贺德留.施肥对黄连木幼树生长的影响[J].河南林业科技2000,20(2):33-246.
[18]Philip McMillan Browse.Autumn glory: knowing and growing the versatile Chinese pistache[J]. American Nurseryman, 1988,167(1)115-121.
[19]刘启慎,魏玉君.中国黄连木性状变异及类型划分[J].河南林业科技,1999,19(1):1-3.
[20]史清文,左春旭.黄连木叶化学成分研究[J].中国中草药杂志,1992,17(7):422-423.
[21]杨镇,杨华生.黄连木嫩枝扦插育苗研究[J].河北林果研究,1997,12(1):31-34.
[22]Diane E.Dunn.Propagation of Pistacia chinensis by Mound Layering[J].Journal of environmental horticulture.1995,13(2)109-112.
[23]Dunn D.E., Cole J.C., Smith M.W.Position of cut, bud retention and auxins influence rooting of Pistacia chinensis[J]. Scientia horticulturae,1996, 67(1-2),105-110.
[24]Dunn DE.Timing of Pistachia chinensis Bunge.-Rooting using morphological markersassociated with calendar date and degree days[J]. Journal of the American Society for Horticultural Science,1996,121(2):269-273.
[25]Miyazaki Junji.Tissue culture of kainoki(Pistacia chinensis).Nippon Ringakkai Kyushu Shibu Kenkyu Ronbunshu,1993,(46):59-60.
[26]Michael A.Arnold:Accelerator~(TM)containers alter plant growth and the root-zone environment.Journal of environmental horticulture.1999,17(4):168-173.
[27]陈振江,范玉田,白桂芹.太行山木本野生资源的利用[J].河北林业科技,1981 (3):38-41.
[28]吴志庄.木本能源植物黄连木单株选择、类型划分与群落调查研究[D].2009.
[29]Parfitt DE.Bonsai'ornamental pistachio[J].HortScience2003,38(6):1260-1261.
[30]秦飞,郭同斌,刘忠刚.中国黄连木研究综述[J].经济林研究,2007,25(4):90-96.
[31]许荣彦.霜叶红于二月花-介绍红叶树种[J].中国花卉盆景,1995(1):12-13.
[32]贾利强,李吉跃.水分胁迫对黄连木、清香木幼苗的影响[J].北京林业大学学报,2003,25(3):55-59.
[33]杨吉华,张永涛等.栾树、黄连木、黄栌水分生理生态特性的研究[J].水土保持学报, 2002,16(4)152-158.
[34]张宏文,张慧勤等.太行山南段黄连木天然更新调查研究[J].中南林业调查规划, 1999,18(3):13-15.
[35]刘启慎.黄连木水土保持林不同径阶适宜密度的研究[J].河南林业科技.2000, 20(4):1-3.
[36]王强等.黄连木隆脉木虱空间格局的研究[J].山东农业科学.2005,(3)45-47.
[37]张玉华,赖永梅.黄连木缀叶螟的发生及防治[J].陕西林业科技,2003,(1):48-50.
[38]张玉玲,李建红.黄连木尺蛾的发生与防治[J].科技情报开发与经济.2004, 14(7):183-184.
[39]柴立英,谢金良.黄连木种子小蜂预测预报及综合治理措施[J].河南职业技术师范学院学报2003(4):33-35.
[40]柴立英,谢金良.黄连木种子小蜂预测预报及综合治理措施[J].河南职业技术师范学院学报.2003(4):33-35.
[42]袁德灿等.黄连木种子小蜂的生活习性及防治[J].林业科技通讯.1981(2):21-22.
[43]柴立英.吕文彦.杜开书.黄连木种子小蜂的生物学特性和发生规律[J].昆虫知识, 2006,43(4):567-570.
[44]田士波,靳杏蕊.黄连木种子小蜂幼虫为害特点或与防治[J].植物保护. 1994, (2):15-16.
[45]柴立英,张毅军.黄连木种子小蜂生物学特性及其综合治理技术[J].植物检疫. 2004,18(1):17-21.
[46]王玖荣.黄连木种子小蜂的发生与综合防治[J].林业实用技术2004(7):33.
[47]于桂凤.园林绿化黄连木尺蛾的发生与防治[J].山西农业科学2001,29(4):70-72.
[48]邢世岩,宋新芳,李士美等.黄连木不同优株种子发芽特性研究[J].种子(Seed),2006,25(8):32-34.
[49]宋宏伟,刘俊磊,卢绍辉.黄连木优树选择与良种繁育技术的研究[J].生物质化学工程,中国林业生物质能源发展研讨会,2006:138-141.
[50]李良厚,贾志英,付祥健.土壤水分胁迫下苗木水分参数变化的研究[J].河南农业大学学报1999,33(1):92-99.
[51]贾利强,李吉跃.水分胁迫对黄连木、清香木幼苗的影响[J].北京林业大学学报,2003, 25(3):55-59.
[52]李在军,冷平生,丛者福等.黄连木对干旱胁迫的生理响应[J].植物资源与环境学报,2006,15(3):47-50.
[53]宋宏伟,张江涛,王晶.黄连木光合特性和水势变化规律的研究[J].安徽农业科学, 2007,35(31):9924-9926.
[54]史清文,左春旭.黄连木叶化学成分研究[J].中国中草药杂志,1992,17(7):422-423.
[55]Parfitt DE et, al. Molecular phylogenetic analysis of the genus Pistacia[J]. Acta Horticulturae. 1998(470):143-151.
[56]Kafkas S.,Perl-Treves R.Interspecific relationships in Pistacia based on RAPD fingerprinting[J]. HortScience, 2002,37(1):168-171.
[57]舒常庆,杨广东,董晓明.倍子树单宁含量的分析研究[J].经济林研究,1999, 17 (2)11-12.
[58]梁宝生.夏季黄连木排放萜烯类化合物浓度日变化及排放速率的研究[J].北京大学学报(自然科学版)2003,39(4):517-521.
[59]马淑英,胡正海.西北地区漆树科四属植物分泌道的解剖学研究[J].西北植物学报,1992,12(3):180-187.
[60]Medal, JC. Host specificity of Heteroperreyia hubrichi malaise (Hymenoptera: Pergidae), a potential biological control agent of Brazilian peppertree (Schinus terebinthifolius raddi).Biological Control,1999,14(1):60-65.
[61]E.Gregory McPherson.A benefit-cost analysis of ten street tree species in Modesto, California,U.S.Journal of Arboriculture.2003,29(1):1-8.
[62]Satoshi Nishimura Structures of 4-Aryl-coumarin(Neoflavone)Dimers Isolated from Pistaciachinensis Bunge and Their Estrogen-like[J].Activity.Chemical and Pharmaceutical Bulletin.2000,48(4):505-508.
[63]舒常庆,伍华银,周继荣.黄连木五倍子生长特性的研究[J].华中农业大学学报,1998,17(4):401-403.
[64]路丙社,董源,刘忠华,白志英.阿月浑子砧木研究进展[J].果树科学,1999,16(2): 149-155.
[65]路丙社,刘忠华.阿月浑子引种研究[M].中国林业出版社2006.
[66]张文越,王钧毅.以黄连木为砧木嫁接阿月浑子试验[J].山东林业科技,2002 (2):15-16.
[67]Chen Y-W(陈岳武),Chen W-M(陈维民).1982.Gene diversity in populations andindividuals of masson pine of Fujian province[J]. J Nanjing Techn Coll For Prod(南京林产工业学院学报),2:29~42(in Chinese).
[68]董玉琛.生物多样性及作物遗传多样性检测[J].植物品种资源,1995,(3):1~5.
[69]曹家树,曹寿椿,易清明.白菜及其相邻类群基因组DNA的RAPD分析[J].园艺学报.1995, 22(1):47-50.
[70]李斌,顾万春,卢宝明.白皮松天然群体种实性状表型多样性研究[J].生物多样性, 2002,10(2):181-188.
[71]罗建勋,李晓清,孙鹏等.云杉天然群体的表型变异[J].东北林业大学学报, 2003,31(1):9-11.
[72]李文英.蒙古栎天然群体遗传多样性研究[D].北京林业大学博士论文,2003.
[73]毛钧,张明宇,虞泓.泸定百合普洱群体遗传与变异研究[J].云南大学学报(自然科学版),2003,25(增刊):91-96.
[74]周世良,张方,王中仁等.杭州石荠苎和石香薷的遗传多样性研究[J].遗传学报,1998, 25(2):173-180.
[75]顾万春,李斌,游应天等.生态梯度轴(EGA)区划林木育种区的研究[J].生态学报,1997,17(2):159-169.
[76]程诗明,顾万春.苦楝表型性状梯度变异的研究[J],林业科学,2006,42(5):29-35.
[77]李文英,顾万春.蒙古栎天然群体表型多样性研究[J],林业科学,2005,41(1):49-56.
[78]任旭琴.遗传多样性及其研究方法[J].淮阴工学院学报.2002,11(5):6-8.
[79]刘华,贾继增.指纹图谱在作物品种鉴定中的应用[J].作物品种资源,1997(2):45~48.
[80]姜自锋,林乃锉,徐梅.RAPD技术及其应用中的一些问题[J].福建农林大学学报(自然科学版).2002, 31 (3):356-360.
[81]李文英.蒙古栎天然群体遗传多样性研究[D].北京林业大学博士论文,2003.
[82]Davila J.A, Sanchez de la, Hoz M P, et al. The use of random amplified microsatellite polymorphic DNA and coefficients of parentage to determine genetic relationships in barley[J]. Genome, 1998,41(4):477-486.
[83]赵欢,吴卫,郑有良等.应用RAMP分子标记研究红花资源遗传多样性[J].植物遗传资源学报, 2007, 8(1):64-71.
[84]佘跃辉,荣廷昭,粟生群等.应用RAMP分子标记分析小豆栽培型种质资源遗传多样性[J].作物学报: 2006, 32(2):217-222.
[85]尚海英,郑有良,魏育明等.应用RAMP标记研究黑麦属遗传多样性[J].农业生物技术学报, 2003,11(6):566-571.
[86]潘志刚,游应天.中国主要外来树种引种栽培.北京:中国科学技术出版社. 1994, 523-525.
[87]路丙社,白志英,孙浩元,李会平,刘忠华,董源.土壤含水量对阿月浑子叶片净光合速率及叶绿素荧光参数的影响[J].园艺学报. 2004. 31(6):727-731.
[88]路丙社,白志英,梁海永,吴京民,董源.阿月浑子叶片光合作用的光抑制[J].园艺学报,2002.29(4): 313-316.
[89]周如久,倪志云,路丙社,白志英,李献明.阿月浑子花序生长和花期物候学研究[J].西北植物学报. 2006.26(8):1579-1583.
[90]毕庆雨.数理统计[M].中国林业出版社.1990.
[91]E. Silfverberg-Dilworth,C. L. Matasci,W. E. Van de Weg,et al.Microsatellite markers spanning the apple (Malus domestica Borkh.) genome[J].Tree Genetics & Genomes,2006,2: 202-224.
[92]Gianfranceschi L, Seglias N, Tarchini R,et al.Simple sequence repeats for the genetic analysis of apple[J].Theor Appl Genet,1998,96:1069–1076.
[93]Wang X Q,Zou Y P,Zhang D M,et al. Problems in the use of RAPD to the study of genetic diversity and systematics [J].Acta Botanica Sinica, 1996, 38(12):954-962.
[94]Rannala B, Yang Z. Probability distribution of molercular evolutionary trees: a new method of phylogenetic inference [J]. J Mol Evol, 1996, 43(3):304-311.
[95]Rohlf F J.NTSYS-pc Numerical taxonomy and multivariate analysis system.Version 2.01.Exter Software, Setauket, New York, 1998.
[96]裴会明,陈明琦.黄连木的开发利用[J].中国野生植物资源,2005,24(1): 43-44.
[97]郭杰.不同种源苦楝种苗特性和耐盐能力差异的研究[D].南京:南京林业大学15-16.
[98]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.
[99]安泽伟,黄华孙.一种提取橡胶树叶中总DNA的方法[J].植物生理学通讯,2005,41(4):513-515.
[100]张军,深州杨嵌合特性的初步研究(D)河北农业大学.2006.
[101]杨谷良,谭晓风,乌云塔娜.应用CTAB法对砂梨品种DNA提取效果的研究[J].生物技术,2005,15(5):41-43.
[102]曾杰,邹喻苹,白嘉雨等.顽拗植物类群的总DNA制备[J].植物学报,2002, 44(6):694-697.
[103]李莉,彭建营.不同方法对枣叶片总DNA提取效果的影响[J]果树科学.2007, 24(3):389-392.
[104]陈永华,相阳.4种不同广玉兰基因组DNA提取方法的比较[J].中南林业科技大学学报,2008,28(3)45-48
[105]陈析丰,查笑君,范文杰等.山茶花叶片DNA提取及RAPD反应体系的研究[J].植物研究,2007,27(2):218-223.
[106]曹庆芹,徐月,冯永庆等.板栗基因组DNA不同提取方法的比[J].农业生物技术科学,2007,23(6):160-163.
[107] Karoly R. Black locust (Robinia pseudoacaciaL. ) improvement and management in Hungary[J]. Forestry Studies in China, 1999, 11(2):42-46.
[108]王滑,郝俊民,王宝庆等.中国核桃8个天然居群遗传多样性分析[J].林业科学,2007,43(7):120-124.
[109] Cozzolino S, Cafasso D, Pellegrino G. Fine-scale phylogeographical analysis of Mediterranean Anacamptis palustris (Orchidaceae) populations based on chloroplast m in isatellite and microsatellite variation[J]. Molecular Ecology, 2003, 12 (2): 783-792.
[110]沈镝,方智远,戚春章等.西双版纳黄瓜群体遗传多样性的SSR分析[J].园艺学报, 2009, 36 (10):1457-1464.
[111]Hartl D L, Clark A G. Principles of population genetics (2ndedn. ) [M]. Sunderland, MA: Sinauer Associates, 1989.
[112]郭先锋,王莲英.部分芍药种质资源的RAPD分析[J].园艺学报. 2007. 34(5): 1321-1326.
[113]胡薇,黄儒珠,潘晓华,李锦凤,孙端.建兰38个品种的RAPD分析.园艺学报[J]. 2008.35 (2): 289-294.
[114]Salih Kafka, Hakan Ozkan, Bekir Erol Ak, Izzet Acar, Halit Seyfettin Atli, Sonay Koyuncu. 2006. Detecting DNA Polymorphism and Genetic diversity in a wide pistachio germplasm: Comparison of AFLP,ISSR and RAPD markers[J]. J Amer. Soc. Hort. Sci. 131(4): 522-529.
[115]Hormaza, Pinney, Polito. Genetic diversity of pistachio germplasm based on randomly amplified polymorphic DNA(RAPD)markers[J]. Economic Botany, 1998.52(1): 78-87.