植物核型分析方法的研究进展_英文_胡进耀
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核型分析方法研究及进展窦笑菊【摘要】染色体核型分析是生物学研究中常用的实验方法。
核型分析技术区别于传统的形态鉴别,在细胞水平上研究染色体特征,并以此为依据对植物进行分类,结果更加真实准确,对于植物分类、进化过程、以及属种间关系具有重要的应用价值。
该文综述了核型分析研究的发展过程,指出了目前实验中存在的问题,并对其发展前景进行展望。
%Karyotype analysis method is a basic technology in plants taxonomy. Different from traditional morphology, Karyo- type analysis research plants chromosome at cellular level. The result is more reliable. It also has a great application value in the research of plants taxonomy, evolution and the relationship of different genus. In this paper a summary was focus on the progress of karyotype analysis, pointing out the shortage and prospecting its further development.【期刊名称】《安徽农学通报》【年(卷),期】2012(018)013【总页数】3页(P32-34)【关键词】染色体;核型分析;显带技术【作者】窦笑菊【作者单位】西藏职业技术学院农林系,西藏拉萨850000【正文语种】中文【中图分类】Q947核型分析是遗传学的基本技术之一。
近60~70a来,核型分析在人体和动物中的应用获得了长足的进步。
人类基因组计划的实施,使得对人类染色体的研究,由单纯的形态描述深入到对结构、功能的探索。
植物染色体的核型分析一实验原理任何一种生物的细胞都有一定数目、一定大小和形态的染色体,便构成了生物体特有的核型。
核型是指染色体组在有丝分裂中期的表型。
包括染色体的数目、大小和形态的总和。
不同的生物,其核型是不同的。
核型分析是在对有丝分裂中期染色体进行测量、计算的基础上,进行配对,按一定原则编号(从大到小)、分组、排列,并进行形态分析的过程。
核型分析可以为细胞遗传分类、物种间亲缘的关系、以及染色体数目和结构变异的研究提供重要依据。
因此,在细胞遗传研究领域中具有重要意义。
二实验目的了解核型分析的过程,学习核型分析的方法。
三实验材料放大的蚕豆根尖染色体照片四实验器具及药品1 器具毫米尺、计算器、剪刀、镊子、胶水(制染色体标本片的器具同有丝分裂,外加摄影显微镜、放大机、相纸、暗室设备等)2 药品制染色体标本片的药品同有丝分裂,外加前处理用的秋水仙素五实验步骤1 取根尖→秋水仙素预处理(增加中期分裂相)→固定→解离→水洗后染色压片→观察:选染色体形态好、分散好、且完整的细胞进行显微摄影→冲洗胶卷→放大成照片(已作)2对照片上分散的染色体随机编号,打一草表,测量、记录每条染色体的长臂、短臂、臂比、全长和相对长度。
相对长度=每条染色体的长度/单倍染色体组长度(2N总长度/2)X1003配对根据测定的每条染色体的相对长度和臂比,将大小和形态相近的两条染色体配对成一对同源染色体。
4分类和排序染色体的分类根据Levan(1964)的分类标准,根据臂比大小不同分成:m、sm、st、t四类。
根据相对长度的大小,将配对后的染色体从大到小编号排序。
1 2 3 4 5 6 7大麦根尖有丝分裂核型。
DOI:10.16246/j.issn.1673-5072.2002.03.006 第23卷 第3期四川师范学院学报(自然科学版)2002年9月 Vol.23 No.3Journal of Sichuan Teachers College(Natural Science)Sep.2002文章编号:1001-8220(2002)03-0239-06Progress of Karyotype Analysis Method inPlants ResearchHU Jin_Yao,SU Zhi_Xian,YUE Bao_Liang,ZHANG Qiao_Ying(Sichuan Provincial Key Laboratory of Environmental Science and Biodiversity Conservation,Research Centerof Biodiversit y,Sichuan Teachers College,Nanc hong,637002,China)A bstract:Karyotype anal ysis method of plants is a basic technique in taxonomy and other related subjects.Its research progressstages are divided into two parts:chromosome morphological mark analysis and banding analysis.The methods in these two stagesare elaborated respectively.Its prospects are also discussed.We have analysed the factors restricting the progress of karyotypeanalysis methods in plant.Karyotype analysis method in plants will not breakthrought until its application range is expanded andnew research projects is brought up with and combinaed with other experi mental techniques and theory methods in other subjects.Key words:karyotype;banding technique;chromosome.CLC number:Q343.21 Document code:AKaryotype analysis is a basic work in cell genetics.In recent six to seven decades,especially after Hsu et al invent-ed tissue culture hyotonic,the karyotype research in human and ma mmals has made wonder ful progress.The international standards of human chromosome r esearch make human cell genetics into an applied new stage.But karyotype research methods in plants have made little improvement and la gged human chromosome research by far.No matter whether to make plates or make mold picture,plants kar yotype research is mor e difficult than that of animals.So it is necessar y to overview the research methods and impr ove mor e widely applications and more innovations of kar yotype analysis methods in plant research..1 History of karyotype analysis methods in plantsThe research history of karyotype analysis methods of plants can be divided into two stages:chromosome morphologi-cal mar k analysis stage and banding analysis stage.1.1 Chromosome morphological mark analysis stageWithout the help of microscope,computer etc,and lack of some related subjects such as statistics,in the initial stage of the development of karyotype analysis technique,scientists analyzed karyotype only by utilizing chromosome mor-phology mark(length of chr omosomes,arm ratio,satellites,etc.)In this stage,we adopted plants chromosome common pressing plates method in research karyotype.This technique is invented by Beilin in1921[1].It has been used and improved for many years.No w it has bec ome one of the most pop-ular basic technique in cell genetics and cell taxonomy.And it cannot be substituted by other ne w techniques.For several decades,although common pressing plates method has made some improvements,great breakthrough has not appeared yet.Chromosome morphological mark analysis method is too rough and simple to survey single chromosome.①Received date:2002-03-12As for measures ,taking photomicrograph ,magnifying ,cutting ,dividing into groups ,pasting ,taking photo again ,then making into karyotype photos are the popular methods used in this stage .The precision of this method is very lo w .It needs too much work ,so it is ver y easy to make mistakes and picture effects are also bad [2].In this stage ,some karyotype standards make research more convenient .In 1955,Darlingtond et al [3]gave some kar yotype standards .Then in 1971,Stebbins G L et al [4]integrated some standards .In 1964,Levan et al [5]introduced some methods to calculate ,classify and na me chromosomes .These all improved the development of karyotype analysis .1.2 Banding analysis stageThe invention of highly distinguishable electronic micr oscope ,powerful computer and software pr ovide an probability for the development of karyotype analysis methods .In this sta ge ,the invention of banding technique set kar yotype analy -sis in the way to more micro and precise research .In the research of secale cereale ,dividual chr omosome cannot be dis -tinguished by common morphological methods .B ut by banding technique ,it can be discerned .The result of secale ce -reale C -band research by different researchers is the same by large ,but there are also a few differences .So did in the same plants species but different in dividuals .That is to sa y secale c ereale has band pattern polymorphism .According to the difference of homologous chromosomed ,we can easily judge it is a cross -pollination plants [1].So banding technique makes kar yotype analysis be used in the research of botony systematics ,population ecology ,genetics ,and so on .Plants banding technique includes fluorescent banding and Giemsa banding .In 1968,T .O .Caspersson invented Q -band technique .And he used it to survey broad bean mitotic chromosomes .In 1971,Arrighi and Hsu invented C -band technique .In 1972,Vosa introduced it into plants research field .The result showed C -band was more obvious in plants heterochromatin than in animals '.After then ,there are other banding techniques invented .They all belong to Gie msa banding with C -band .But their importance is junior to C -band technique .According to the incomplete statis -tics by 1980,more than 60genuses have got C -band [6].Although Q -banding technique which belongs to fluorescent banding is the earliest one applied in plants karyotype analysis ,now Giemsa banding technique is more popular in the re -search of plants chromosomes .These two banding techniques have different merits respectively (Table 1).Table 1 Comparison of fluorescent banding and Giemsa bandingcharacterFluores cent banding Giems a banding Theorembanding by utilizing fluorescent materials banding by certain chemis try methods Operation conditionNeed fluores cent ins trument and medicine Not need fluorescent ins trument and medicine Operation diffic ultyProcedures are simple Procedures are more compl icated Special band patternCan sho w special band pattern Need special dis position ,or it can not sho w special band pattern EffectMore s table L ess stable Preservation Cannot make into permanent plates Can make into permanent plates .It can s ho w different band if c hange dis pos ition procedures There is a very significant difficult problem that plants chromosome specimen cannot be made as well as that of ani -mals for the limiting of by cell wall .Common pr essing plates method has many shortages such as the difficulty of dispers -ing chromosomes ,overlap and transfor mation of chromosomes ,etc .In the research of plants chromosome G -band ,be -cause common pr essing plates method is hard to completely get rid of the c over of cell wall and cytoplasm thor oughly ,the repeatability of Giemsa band is low .It also has influences on the research of submicrostructure .So it is very emer gent to innovat plants chr omosomes plates making methods to promot plants chromosomes research ,especially for the research of plants chromosome submicrostructure and Giemsa banding .The wall degradation hyotonic method developed a way to solve this problem .The prime process is using enzyme to degradate cell wall ,hyotonic and dry with fire .The wall degra -dation hyotonic method is superior to common plates methods :①It can get many dispersed cells efficiently .②Chromo -somes are integrate and clear .③Procedure is simple and no need of special instruments .Because of these merits ,now 240 四川师范学院学报(自然科学版)2002年the wall degradation hyotonic method is used widely [7-9].As a basic technique ,common pressing plates method is still used widely .In this stage ,common plates has devel -oped too .Yue Aiqin et al [10]innovated plates making methods of three types chromosomes in leguminous by researching kar yotype analysis method .Table 2 Comparison among several karyotype analytic softwaremerits and shortages PC s y stem and Photoshop software General microcomputer soft ware forkaryotype anal ysis Semi -automatic plants chromosomes images karyotype analysis systemcommon merits Not need some special instruments :dark room ,etc ;Workload is less than common method ;Accuracy is higher thancommon methodmerits The capital is less than commonmethods and special analysis softwareCheaper than abroad soft ware ;inter -changeable ,automatic common op -eration ,output large information ;Can intervene by person Plants chromosomes are its direct re -search objects ,so it is much powerful in research plants karyotype shortagesLess power than abroad automaticchromosomes analysis s ystem Use in DOS operation system ,inter -face isn 't friendly Can not be used in the research of an -imal and hu man chromosomes In this stage ,some new methods were invented in measur e and analysis .Jiang Shanshan et al [2]used personal com -puter system and Photoshop software to measure and analyze chr omosomes .Liu Dongxu [11]translated General micr ocom -puter software for kar yotype analysis .Liuquan et al [12]introduced an applied Semi -automatic plants chromosomes images kar yotype analysis system .There are also some abr oad special kar yotype analysis software [13].Each of the software has its merits and shortages (Table 2).In the past ,most pursuers reached conclusions directly ,after simply calculating the data without using of statistic exa mination [14-16]. So their results are unreliable .Now some researchers have noticed this prob -lem .Yang Xiuying et al [7]estimated the believable scope of chr omosomes arm rate .2 Prospect2.1 The expansion of application rangeKaryotype analysis technique has been born for nearly one centur y .Plants kar yotype analysis methods has made some progress .But there are many difficulties to make a wonderful breakthrough .We drew 40domestic articles radomly on plants karyotype analysis between 1995_2001,in which the object of about thirty articles is to accumulate research data .Their research results are one or several karyotype for mula or kar yotype mode pictures [14,15,17].The more complex articles are only to compare karyotype of different plants in the same group [16,18,19].However kar yotype analysis has entered applied stage in animal and human chromosome research .For ex -ample ,using kar yotype analysis to identify hereditar y disease and analyzing the mechanism of pathological change [20,21].Narr ow research objects may be the lethal limiting to innovating karyotype analysis methods .In the future ,ho w to bring for ward new objects is an urgent task .Abroad circumstances are more optimistic .In the first half of the twentieth century ,c ytologists such as Darlington 1931;Darlington and La Cour 1942,Kihara 1924,and Sears 1941studied plant chromosomes ,making key disc overies such as the importance of polyploidy in plant evolution and showing that whole chromosomes carried similar groups of genes in different species .Soon thereafter ,an understanding of evolutionar y changes at the cytological level was estab -lished that included an appreciation for chromosome translocation ,fusion ,fission ,and the correlation between chromoso -mal particularities and phylogeny .Recently ,Luca Comaia et al studied evolution and hybrid superiority mechanism in 第23卷第3期胡进耀,等:植物核型分析方法的研究进展241 plant by means of karyotype analysis technique .The basis of this technique in the field is that many wild and cultivated allopolyploids that originated in prehistoric times are fertile ,well adapted ,and genetically stable ,allopolyploids of more recent origin commonly display genomic and phenotypic instability (Pope and Love 1952;Allard 1960;Gupta and Red -dy 1991).2.2 The innovation of experiment techniqueUp to now ,all plants banding techniques except Q -band are invented in animal research firstly .E ven C -band ,which is the most popular technique used in plants ,is also the continuator of BSG technique which is originated from ani -mal research .Plants research is junior to animals 'on banding techniques and the diversity of banding pattern .The re -search achievements in the banding mechanism are mostly obtained from animal research .Standard band patterns were es -tablished for animal chr omosomes .But plants research lags behind animal 's .Now native researchers usually adopt Steb -bins [4],Li Maoxue ,Chen Ruiyang 's [22,23]standard and Levan system [5]to name chromosomes .At present ,ther e ar e no plants banding patterns ,which can parallel with animal 's G -band .Undoubtedly ,this is a great obstacle to combine chromosome banding with genetic analysis .It is also much unfavorable to bring up ne w objects of plants kar ytype analy -sis .In r ecent year ,human chr omosomes fluorescence banding technique made a rapid progress .Follo wing the invention of FI SH technique and chromosome scribbling dyeing plates tec hnique which is developing from it ,ThomaRied and E vetin research groups in NCHGR in American NIH invented a spectrum imaging method which can mar k 24human chromosome individually with special fluorescence [24,25].In fact ,this method can mark 2n -1(n on behalf of the types of fluores -cence dye )chromosomes ,undoubtedly this is a wonder ful breakthr ough on the basic of FISH technique .This also brings a hope for the innovation of plants fluorescence banding technique ,which is invented earlier than Giemsa banding while less used than the latter .But it needs further research whether the method can be used in plants chromosome analysis .Data statistic and analysis methods need further improvement too .For example ,translate mor e powerful and c onve -nient software for analysis and cut down the capital etc .Some researcher invented some new methods by combining related techniques in other subject with common methods in karyotype analysis .In early 1980s Macdonald ,Shimizu et al consid -er ed that PFGE (pulsed field gel electrophoresis )can be used in kar yotype analysis [26-27].Later ,they applied it to study plants karyotype [28-30].Native r esearchers have done similar work (Shi Liangen et al [31]).Bian Yinbing et al [32]ana -lyzed electrophoretic kar yotype of jell basidiomycetes auricularia auricula by using of contour -cla mped homogeneous electric field (CHEF )electrophoresis .The r esult is fairly good .Luca Comaia et al studied hybrid genetic polymorphism by combination karyotype analysis with molecular biology .There 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要:植物核型分析方法是植物系统学等学科的一门基本技术.本文将植物核型分析方法的发展过程划分为两个阶段:染色体形态标志分析时期和分带分析时期,并分别阐述了这两个时期在核型分析方法上的创新,以及这些方法的优点和不足之处.同时对其今后的发展方向作一展望.分析了限制植物核型分析方法发展的几个因素,认为核型分析方法要取得突破性进展,第一必须扩大其应用范围,提出新的研究课题,第二必须结合动物和人体核型分析的最新研究方法以及其它学科的实验技术和理论方法.关键词:核型分析;分带技术;染色体中图分类号:Q343.21 文献标识码:A①收稿日期:2002-03-12作者简介:胡进耀(1978-),男,浙江兰溪人,四川师范学院环境科学专业硕士研究生,主要从事生殖生态学方面研究工作.244 四川师范学院学报(自然科学版)2002年[7]沈玉芳.熔沸点比较七则[J ].中学化学教学参考,1992,1-2(22).[8]聂保民.熔沸点比较中的误解[J ].中学化学教学参考,1993,5(27).[9]邓 存.Sn 和Pb 哪个金属性强[J ].中学化学教学参考,1988,3(28).[10]武艳妮,赵中华.如何判断氧化—还原反应的生成物[J ].中学化学教学参考,1992,4(10).Application of Normal Data of Chemistry in TeachingYU Zheng -qi 1,MA Hong -yan 2,ZOU Rong -xian3(1.Nanchong Station of Educational Instrument ,Nanchong 637000,China ;2.Nanchong Senior Middle School ,Nancong 637000,China ;3.Depart ment of Chemistry ,Sichuan Teachers College ,Nanchong 637002,China )A bstract :Nor mal data of chemistry are very widely applied in teac hing ,research work ,production and other fields and lots of discoverices of theories ,principles and la ws are the results of analyses and summaries of these data materials .Somaking full use of these data in chemistry teaching can promote students 'ability of analysing and solving problems in practice .Key words :nor mal data of chemistr y ;teaching ;application 318 四川师范学院学报(自然科学版) 2002年。
文章编号㊀1672G6634(2019)06G0097G14D O I ㊀10.19728/j.i s s n 1672G6634.2019.06.014苹果属植物的核型分析华利源㊀王㊀轩㊀钱关泽(聊城大学生命科学学院,山东聊城252059)摘㊀要㊀因为种间杂交㊁多倍化和无融合生殖等现象,苹果属植物以难于分类和鉴定而闻名,其中尤以山荆子种复合体为甚.本文针对山荆子种复合体中的主要自然分布植物和关系可能较密切的花楸苹果组植物进行了染色体分析,十五个苹果属分类群包括一个四倍体,四个三倍体,十个二倍体,天目山湖北海棠㊁泰山海棠㊁日瓦海棠(新拟)㊁紫花海棠㊁冬红果染色体数目为首次报道;核型多为中部着丝点染色体(m )㊁亚中部着丝点染色体(s m ),属较原始的类型,沧江海棠核型为1A ,最为原始,三叶海棠核型为3B ,最为进化.试验结果提示分布于天目山顶部的很可能并非湖北海棠,而可能是一个独立的种,它可能与平邑甜茶和泰山海棠的杂交亲本有关;山荆子也有可能与其亲本有关;四川西部发现的日瓦海棠与锡金海棠近似,但其核型及染色体数目差异很大,因此也支持该种成立.核型分析结果也为后续的基因组原位杂交研究积累了资料.关键词㊀苹果属;去壁低渗;染色体;核型分析中图分类号㊀Q 944文献标识码㊀A 0㊀引言人类染色体制片技术是从上个世纪70年代开始引用到植物材料中的[1G5].染色体作为生物遗传信息的载体,生物细胞中最重要的成分,具有极高的稳定性,一般来讲,它的数目和结构是特定的,在进行植物分类研究中弄清楚染色体数目,对其核型进行分析是十分重要的.核型分析的研究对象为处于分裂中期的染色体,通过对苹果属植物染色体的数目㊁长度㊁形态㊁着丝点位置㊁长短臂比例㊁随体的有无等特征的研究,为细胞遗传分类㊁物种间的亲缘关系以及染色体数目和结构变异的研究提供重要依据.苹果属植物染色体小,数目多,进行核型分析难度大.目前,染色体分析技术应用在苹果属植物中的报道依然很少,以梁国鲁和陈瑞阳为主要代表.梁国鲁[6]对苹果属植物31个种类的核型分析结果为其核型均为中部着丝点染色体(m )和近中部着丝点染色体(s m );梁国鲁和陈瑞阳[7]对其染色体数目观察结果均发现苹果属植物存在混倍体现象;肖艳[8]等研究发现不同生活环境会引起苹果属植物染色体倍性变异.参考前人的研究结果,可以看出苹果属植物核型的复杂性,本试验对所采集的试验材料进行染色体数目观察及核型分析,补充苹果属植物核型资料,为苹果属植物分类提供参考依据.1㊀材料和方法1.1㊀材料试验所用材料为十五个苹果属分类群种子萌发的根尖,具体采集时间㊁采集地点等(表1).将采集的种子用75%酒精消毒后,着床于培养皿中的湿纱布内进行变温萌发试验,纱布及培养皿经高温高压灭菌处理.待种子萌发,选取长至1G2c m 的根尖制作染色体制片,发芽培养箱为杭州托普仪器有限公司G T O P G450B H 光照培养箱.1.2㊀方法本试验对比压片法[9G19]和涂片法[20,21]发现,涂片法制备的染色体在形态和分散性上相较于压片法好.结合去壁低渗法[22G28]涂片法制备根尖染色体制片,主要试验过程为:(1)取材,在每天上午9:00G11:00根尖细()预处理,为了同步化染色体,获取更多的染色体中第32卷㊀第6期2019年12月㊀㊀㊀聊城大学学报(自然科学版)J o u r n a l o fL i a o c h e n g U n i v e r s i t y (N a t .S c i .)V o l .32N o .6D e c .201989㊀聊城大学学报(自然科学版)期分裂相,将取好的根尖放在4ħ冰水中处理24h或放在0.2%8G羟基喹啉中处理4h.(3)固定,将预处理好的根尖放在卡诺固定液(乙醇:冰醋酸=3:1)中固定24h.(4)前低渗,将固定好的根尖取出,去离子水冲洗3次后,再用去离子水浸泡10m i n.(5)酶解,取浓度为2.5%的果胶酶纤维素酶混合液40μL,将前低渗处理之后的根尖用去离子水冲洗3次后去除根冠,将白色分生组织切成小块放在酶液中37ħ处理60G90m i n.(6)后低渗,将酶解之后的根尖细胞在室温下用去离子水浸泡20G30m i n,让根尖细胞吸水更加膨胀,使细胞壁破裂,细胞质破散,染色体更容易分散,细胞膜失去约束容易破裂,所以后低渗时间应控制在一定范围,最合适的后低渗时间也因试验材料不同略有差异.(7)制片,取处理好的根尖在载玻片上,镊子捣碎,加60%乙酸在55ħ烤片机上涂片,用卡诺固定液冲洗一遍,室温下干燥;晾干后的玻片用稀释的吉姆萨染液染色5G10m i n,去离子水冲掉多余的液体,室温下晾干.(8)镜检,将做好的染色体制片在N I K O NE C L I P S E80i显微镜下镜检并在图像分析系统下拍照.(9)染色体核型分析,每种试验材料拍摄50张中期分裂相图像进行计数,85%以上的细胞染色体数目为一固定数值,则为该试验材料的染色体数目.十五个苹果属分类群染色体中期分裂相,如图1(a).表1㊀试验材料采集信息种名采集日期采集地点种名采集日期采集地点平邑甜茶2017.10.05平邑蒙山沧江海棠2018.10.25云南省维西县泰山海棠2017.10.20山东泰山山荆子2017.10.29陕西省黄龙县湖北海棠2018.12.05浙江天目山台湾林檎2017.10.15广西省柳州市丽江山荆子2018.10.16云南香省格里拉县西府海棠2017.10.26聊城大学三叶海棠2018.10.21云南省维西县紫花海棠2017.10.24聊城大学变叶海棠2018.10.26四川省雅江县冬红果2017.10.30聊城大学锡金海棠2018.10.15云南省德钦县八棱海棠2017.10.15北京妙峰山日瓦海棠2018.10.24四川省稻城县㊀㊀核型分析按照李懋学和陈瑞阳的标准;按照L e v a n[29]等的标准计算染色体的相对长度㊁臂比值和类型;根据S t e b b i n s[30]的分类标准进行核型分类;用A r a n o[31]的方法计算核型不对称系数.选出染色体长度适中㊁形态较好㊁分散良好的5个中期染色体图片,用p h o t o s h o p C S5软件处理,染色体核型图,如图1(b).选取染色体分散良好,缢痕明显的中期分裂相,测量每条染色体长度(长短臂长度,随体不计内),进行核型分析,各项核型参数(表2).每种试验材料的核型组成主要为中部着丝粒染色体(m)和近中部着丝粒染色体(s m).根据染色体相对长度指数(I.R.L)将每个种的染色体组组成分为四个组:长染色体(L)㊁中长染色体(M2)㊁中短染色体(M1)和短染色体(S).利用E x c e l软件制作的十五个苹果属分类群核型模式图(图2).2㊀结果分析2.1㊀染色体数目山荆子(M.b a c c a t a(L.)B o r k h)㊁丽江山荆子(M.r o c k i i R e h d e r)㊁日瓦海棠(R i w a c r a b a p p l e)㊁天目山湖北海棠(T i a n m u s h a nc r a b a p p l e)㊁沧江海棠(M.o m b r o p h i l a H a n d.GM a z z.)㊁台湾林檎(M.d o u m e r i (B o i s.)C h e v.)和栽培种八棱海棠(M.ˑr o b u s t a R e h d.)㊁西府海棠(M.m i c r o m a l u s M a k i n o)㊁紫花海棠(M.p u r p u r e a)㊁冬红果(M. D o n g h o n g g u o )的染色体数目为34条,为二倍体,即2n=2x=34;平邑甜茶(M.h u p e h e n s i s(P a m p.)R e h d.v a r.m e n g s h a n e n s i s)㊁泰山海棠(M.h u p e h e n s i s R e h d.v a r.t a i e n s i s)㊁三叶海棠(M.t o r i n g o D eV r i e s e)㊁变叶海棠(M.t o r i n g o i d e s(R e h d.)H u g h e s)染色体数目为51条,为三倍体,即2n=3x=51;锡金海棠(M.s i k k i m e n s i s(W e n z.)K o e h n e)染色体数目为68条,为四倍体,即2n=4x=68.2.2㊀十五个苹果属分类群的核型比较本试验结果显示,十五个苹果属分类群中,锡金海棠为四倍体,平邑甜茶㊁泰山海棠㊁三叶海棠和变叶海棠为三倍体,其余皆为二倍体;染色体类型多为中部着丝点染色体(m)和近中部着丝点染色体(s m),三叶体,其他试验未发现随体;染色体相对长度组成分别为山荆子:2n =34=4L +14M 2+12M 1+4S ,平邑甜茶:2n =51=12L +9M 2+27M 1+3S ,泰山海棠:2n =51=12L+6M 2+24M 1+9S ,八棱海棠:2n =34=6L+6M 2+20M 1+2S ,台湾林檎:2n =34=6L +8M 2+12M 1+8S ,三叶海棠:2n =51=6L+12M 2+27M 1+6S ,变叶海棠:2n =51=9L +12M 2+21M 1+9S ,锡金海棠:2n =68=8L +20M 2+32M 1+8S ,丽江山荆子:2n =34=6L +8M 2+16M 1+4S ,日瓦海棠:2n =34=4L+10M 2+16M 1+4S ,湖北海棠:2n =34=4L+8M 2+20M 1+2S ,沧江海棠:2n =34=4L+10M 2+18M 1+2S ,西府海棠:2n =34=2L+16M 2+14M 1+2S ,紫花海棠:2n =34=4L +10M 2+18M 1+2S ,冬红果:2n =34=4L+12M 2+12M 1+6S ,十五个苹果属植物试验材料核型比较(表3).(a)99第6期华利源,等:苹果属植物的核型分析3㊀讨论苹果属植物种子存在休眠现象,所以,探究打破种子休眠的条件是获取试验材料最重要的一步.本研究发现4ħ培养21天左右基本能打破种子休眠,之后28ħ恒温培养,种子即可萌发[32],不同的种所需打破休眠时间存在差异.获取材料后染色体制片是本试验最关键的一步,作为木本植物,苹果属植物木质化严重,具有坚实的细胞壁,酶解去壁低渗法能有效去除细胞壁,获取较好的染色体中期分裂相.同时优化了染色体制片过程中的试验条件,相较于8G羟基喹啉,冰水预处理获得的染色体分散效果更好,前低渗时间10m i n ,苹果属植物根尖酶解时间90m i n ,后低渗时间20m i n 效果最佳,不同试验材料的最佳试验条件略有差异.本试验所研究的十五个苹果属植物试验材料包括十一个野生种材料(平邑甜茶㊁泰山海棠㊁天目山湖北海棠㊁山荆子㊁丽江山荆子㊁湖北海棠㊁三叶海棠㊁变叶海棠㊁锡金海棠㊁日瓦海棠㊁沧江海棠)和四个栽培种材料(八棱海棠㊁西府海棠㊁紫花海棠㊁冬红果),天目山湖北海棠㊁泰山海棠㊁日瓦海棠(新拟)㊁紫花海棠㊁冬红果染色体数目为首次报道.在苹果属植物研究中,湖北海棠是典型的异源三倍体,存在无融合生殖现象,泰山海棠和平邑甜茶为其两个变种.本研究中采自天目山的湖北海棠为二倍体,与湖北海棠在染色体数目上存在较大差异,故其很可能并非湖北海棠,而是一个独立的种,但考虑其各项核型参数与平邑甜茶和泰山海棠相近,且在形态上与两者相似,故推测其与平邑甜茶和泰山海棠的杂交亲本有关;除天目山湖北海棠外,本试验中的山荆子在各项核型参数上也与平邑甜茶和泰山海棠相近,且在形态上也与两者存在很多相似的地方,故推测其可能也与两者的杂交亲本有关;日瓦海棠为二倍体,与锡金海棠形态相似,但果实和叶等特征有明显不同,且两者核型及染色体数目差别很大,因此,本文作者支持该种成立;变叶海棠㊁三叶海棠㊁丽江山荆子㊁锡金海棠和西府海棠的染色体数目除本研究所得出的结果外,还存在其他倍性,梁国鲁对其进行了统计[33],如变叶海棠和三叶海棠还存在二倍体和四倍体,丽江山荆子还有三倍体㊁四倍体存在,锡金海棠还存在二倍体和三倍体,西府海棠也有三倍体存在.一般认为,核型是由对称向不对称进化发展的[34],即植物越原始,核型越对称,植物越进化,核型对称程度越低,本研究中苹果属植物从染色体进化程度上看,大多为中部染色体和近中部染色体,核型多为2B ,属较为原始的类型,沧江海棠核型为1A ,最为原始,三叶海棠核型为3B ,最为进化.中国苹果属植物种类繁多,分类困难,仅靠传统分类法难以得出准确的分类结果,分子标记技术虽然在苹果属植物中获得广泛应用,但提供的证据依然有限[35G38].核型分析技术是细胞分类学研究中不可缺少的手段,本研究通过对部分苹果属植物进行核型分析,为他们的亲缘关系和进化程度提供了参考依据,在接下来的研究中,还将借助分子生物学技术获得更直观可靠的证据,完善苹果属植物分类系统.表2㊀十五个苹果属分类群染色体各项参数种染色体编号相对长度(S +L )/%相对长度系数(I .R.L )臂比(L /S )着丝粒型山荆子(M .b a c c a t a (L .)B o r k h)12.56+5.62=8.181.392.20s m 22.66+4.76=7.421.261.79s m 32.76+4.36=7.121.211.58m 42.94+4.14=7.081.201.41m 52.53+3.85=6.381.081.52m62.24+4.04=6.281.071.80s m 72.33+3.84=6.171.051.65m 82.66+3.50=6.161.051.32m 91.92+4.04=5.961.012.10s m10∗2.35+3.33=5.680.971.42m 112.23+3.34=5.570.951.50m 122.02+3.23=5.250.891.60m 132.02+3.17=5.190.881.57m 001㊀聊城大学学报(自然科学版)(续)表2㊀十五个苹果属分类群染色体各项参数种染色体编号相对长度(S +L )/%相对长度系数(I .R.L )臂比(L /S )着丝粒型平邑甜茶(M .h u pe Gh e n s i s (P a m p .)R e Gh d .v a r .m e n gs h a n e n Gs i s)12.36+5.84=8.201.392.47s m 22.57+4.88=7.451.271.90s m 32.63+4.80=7.431.261.83s m 42.98+4.44=7.421.261.49m 52.07+4.42=6.491.102.14s m 62.15+4.31=6.461.102.00s m 71.98+4.14=6.121.042.09s m 82.01+3.72=5.730.971.85s m 91.78+3.81=5.590.952.14s m 101.67+3.80=5.470.932.28s m 112.11+3.11=5.220.891.47m 121.94+3.19=5.130.871.64m 132.11+2.98=5.090.871.41m 142.15+2.86=5.010.851.33m 151.95+2.65=4.600.781.36m 162.03+2.56=4.590.781.26m 171.83+2.19=4.020.681.20m 泰山海棠(M .h u pe Gh e n s i s R e h d .v a r .t a i e n s i s)12.64+6.94=9.581.632.63s m 22.61+5.60=8.211.402.15s m 32.13+4.87=7.001.192.29s m 42.32+4.38=6.701.401.89s m 52.49+3.71=6.201.051.49m 62.25+3.83=6.081.031.70m 72.01+3.77=5.780.981.88s m 82.19+3.53=5.720.971.61m 92.61+3.10=5.710.971.19m 102.66+3.04=5.700.971.14m 112.13+3.28=5.410.921.54m 122.17+3.15=5.320.901.45m 132.19+2.98=5.170.881.36m 142.13+2.80=4.930.841.31m 101第6期华利源,等:苹果属植物的核型分析(续)表2㊀十五个苹果属分类群染色体各项参数种染色体编号相对长度(S +L )/%相对长度系数(I .R.L )臂比(L /S )着丝粒型八棱海棠(M .ˑr o Gb u s t a R e h d .)14.06+5.49=9.551.621.35m 22.32+5.49=7.811.332.37s m 32.54+4.84=7.381.261.91s m 41.76+4.42=6.181.052.51s m 5∗2.09+4.06=6.151.041.94s m 61.73+4.28=6.011.022.47s m 71.98+3.74=5.720.971.89s m 82.00+3.71=5.710.971.86s m 92.16+3.54=5.700.971.64m 102.09+3.52=5.610.951.68m 111.87+3.40=5.270.901.82s m 122.14+3.07=5.210.891.43m 131.56+3.63=5.190.882.33s m 141.82+3.29=5.110.871.81s m 151.76+3.19=4.950.841.81s m 161.76+2.75=4.510.771.56m 171.76+2.41=4.170.671.37m 台湾林檎(M .d o u m e r i (B o i s .)C h e v .)13.45+6.20=9.651.641.80s m 22.65+4.98=7.631.301.88s m 32.91+4.70=7.611.291.62m 42.76+4.59=7.351.251.66m 52.30+4.37=6.671.131.90s m 62.32+4.26=6.581.121.84s m 72.88+3.46=6.341.081.20m 82.30+3.55=5.850.991.54m 92.53+3.21=5.740.981.27m 102.53+2.77=5.300.901.09m 112.53+2.76=5.290.901.09m 122.30+2.90=5.200.881.26m 131.91+2.91=4.820.821.52m 141.61+2.75=4.360.741.71s m 201㊀聊城大学学报(自然科学版)(续)表2㊀十五个苹果属分类群染色体各项参数种染色体编号相对长度(S +L )/%相对长度系数(I .R.L )臂比(L /S )着丝粒型三叶海棠(M .t o r i n go D eV r i e s e)13.56+5.35=8.911.511.50m 22.11+5.45=7.561.292.58s m 31.68+5.69=7.381.253.39s t 42.14+4.89=7.031.202.29s m 52.42+4.29=6.711.141.77s m 61.80+4.33=6.131.042.40s m 71.71+4.08=5.790.982.38s m 81.54+4.20=5.740.982.72s m 91.95+3.75=5.700.971.93s m 101.58+3.92=5.510.942.48s m 112.22+3.23=5.460.931.46m 121.56+3.70=5.270.902.37s m 131.83+3.37=5.200.881.85s m 141.76+3.06=4.820.821.74s m 151.53+3.26=4.790.812.13s m 161.60+2.53=4.130.701.58m 171.54+2.34=3.880.661.51m 变叶海棠(M .t o r i n go i d e s (R e h d .)H u gh e s )12.38+7.20=9.581.633.03s t 22.17+6.00=8.161.392.76s m 32.88+4.61=7.491.271.60m 42.20+5.09=7.291.242.31s m 52.15+4.66=6.811.162.17s m 62.55+3.68=6.231.061.44m 71.73+4.40=6.131.042.54s m 82.23+3.45=5.670.961.55m 92.07+3.58=5.650.961.72s m 101.64+3.90=5.550.942.37s m 112.29+2.77=5.070.861.21m 121.87+3.10=4.970.841.66m 131.82+2.80=4.620.791.54m 141.74+2.72=4.470.761.57m 301第6期华利源,等:苹果属植物的核型分析(续)表2㊀十五个苹果属分类群染色体各项参数种染色体编号相对长度(S +L )/%相对长度系数(I .R.L )臂比(L /S )着丝粒型锡金海棠(M.s i k k i m e n Gs i s (W e n z .)K o e h n e)12.82+6.61=9.441.602.34s m 22.60+5.00=7.601.291.93s m 32.49+4.81=7.301.241.94s m 42.34+4.47=6.811.161.91s m 52.88+3.90=6.781.151.35m 62.58+3.74=6.321.071.45m 71.99+4.10=6.091.042.06s m 81.93+3.98=5.911.002.06s m 92.51+3.22=5.730.971.29m 101.83+3.80=5.630.962.08s m 112.23+2.98=5.210.891.34m 122.01+2.98=4.990.851.49m 132.03+2.78=4.810.821.37m 142.11+2.68=4.790.811.27m 151.79+2.69=4.480.761.51m 161.93+2.43=4.360.741.26m 171.63+2.13=3.760.641.30m 丽江山荆子(M.r o c k i iR e h d e r)13.04+5.40=8.441.431.77s m 22.87+5.40=8.271.411.88s m 32.53+4.90=7.431.261.93s m 43.04+4.05=7.091.211.33m 52.53+4.39=6.921.181.73s m 62.62+3.97=6.591.121.52m 72.87+3.71=6.581.121.29m 82.36+3.29=5.660.961.39m 92.36+2.95=5.320.901.25m 102.20+3.05=5.250.901.39m 112.15+3.08=5.230.891.43m 122.24+2.62=4.850.821.17m 132.11+2.62=4.730.801.24m 141.98+2.58=4.560.781.30m 401㊀聊城大学学报(自然科学版)(续)表2㊀十五个苹果属分类群染色体各项参数种染色体编号相对长度(S +L )/%相对长度系数(I .R.L )臂比(L /S )着丝粒型日瓦海棠(R i w ac r a b a p pl e )12.81+6.27=9.081.542.23s m 22.09+5.60=7.701.312.68s m 32.13+4.95=7.071.202.32s m 42.41+4.57=6.991.191.90s m 52.38+4.26=6.631.131.79s m 62.84+3.62=6.461.101.27m 71.84+4.11=5.961.012.23s m 82.16+3.72=5.891.001.72s m 91.83+3.74=5.570.952.05s m 102.09+3.37=5.460.931.61m 111.67+3.44=5.110.872.06s m 121.95+3.09=5.040.861.58m 132.09+2.87=4.960.841.37m 141.99+2.87=4.860.831.45m 151.74+2.94=4.680.801.69m 161.63+2.73=4.360.741.67m 171.95+2.23=4.190.711.14m 湖北海棠(天目山)(T i a n m u s h a nc r a b a pGpl e )12.35+7.48=9.831.673.18s t 22.24+5.93=8.171.392.65s m 32.13+5.13=7.261.232.41s m 42.69+3.78=6.461.101.41m 52.17+4.22=6.391.091.95s m 62.31+3.86=6.171.051.67m 72.60+3.28=5.881.001.26m 82.35+3.32=5.670.961.42m 92.13+3.14=5.270.901.47m 101.77+3.43=5.200.881.94s m 111.81+3.28=5.090.871.82s m 121.88+3.14=5.020.851.67m 131.96+2.92=4.870.831.49m 142.17+2.67=4.840.821.23m 501第6期华利源,等:苹果属植物的核型分析(续)表2㊀十五个苹果属分类群染色体各项参数种染色体编号相对长度(S +L )/%相对长度系数(I .R.L )臂比(L /S )着丝粒型沧江海棠(M.o m b r o ph i l a H a n d .GM a z z .)13.00+4.70=7.711.311.57m 22.62+4.90=7.511.281.87s m 32.81+4.51=7.321.241.60m 42.67+4.27=6.941.181.60m 53.05+3.50=6.551.111.15m 62.81+3.59=6.411.091.28m 72.62+3.74=6.361.081.43m 82.28+3.59=5.881.001.57m 91.94+3.69=5.640.961.90s m 101.99+3.50=5.490.931.75s m 111.99+3.35=5.350.911.68m 122.23+2.97=5.200.881.33m 132.15+2.96=5.110.871.38m 142.04+3.06=5.110.871.50m 151.90+2.83=4.730.801.49m 161.75+2.82=4.580.781.61m 171.90+2.25=4.140.701.18m 西府海棠(M.m i Gc r o m a l u s M a k i n o)12.97+4.74=7.711.311.60m 22.47+4.91=7.381.251.99s m 32.22+4.74=6.961.182.13s m 42.18+4.70=6.881.172.15s m 52.81+3.70=6.501.111.32m 62.26+4.12=6.381.081.82s m 72.63+3.58=6.211.061.36m 82.10+4.07=6.171.051.94s m 92.18+3.82=6.001.021.75s m 101.88+3.87=5.750.982.05s m 112.34+3.03=5.380.911.30m 121.89+3.24=5.130.871.72s m 131.64+3.49=5.130.872.13s m 142.18+2.91=5.080.861.33m 601㊀聊城大学学报(自然科学版)(续)表2㊀十五个苹果属分类群染色体各项参数种染色体编号相对长度(S +L )/%相对长度系数(I .R.L )臂比(L /S )着丝粒型紫花海棠(M .pu r Gpu r e a )12.23+5.75=7.991.362.58s m 22.75+4.83=7.581.291.76s m 31.90+4.83=6.741.152.54s m 42.38+4.32=6.701.141.81s m 51.98+4.61=6.591.122.33s m 62.64+3.80=6.441.091.44m 72.46+3.47=5.931.011.41m 81.90+3.88=5.780.982.04s m 91.76+3.96=5.720.972.26s m 102.02+3.62=5.630.961.79s m 112.01+3.54=5.560.951.76s m 122.07+3.38=5.460.931.63m 131.83+3.32=5.150.881.82s m 142.12+3.00=5.120.881.42m 152.13+2.69=4.820.821.26m 161.67+2.93=4.600.781.75s m 171.56+2.63=4.200.711.68m 冬红果(M . D o n gGh o n g gu o )12.76+6.83=9.601.632.47s m 22.20+5.52=7.721.312.51s m 31.89+4.84=6.731.142.56s m 41.84+4.67=6.521.112.53s m 52.37+4.07=6.451.101.72s m 62.16+4.11=6.271.071.90s m 73.05+3.12=6.161.051.02m 82.13+3.86=5.981.021.81s m 92.50+3.21=5.710.971.28m 101.91+3.72=5.630.961.94s m 112.15+3.30=5.450.931.53m 122.43+2.81=5.240.891.16m 132.18+2.82=4.990.851.29m 142.27+2.61=4.890.831.15m 152.06+2.31=4.370.741.12m 161.66+2.69=4.360.741.62m 171.43+2.50=3.930.671.75s m㊀㊀㊀注1.m :中部着丝粒;s m :近中部着丝粒;∗:具随体染色体;2.随体长度不计入染色体全长.701第6期华利源,等:苹果属植物的核型分析㊀㊀注:(A )山荆子,(B )平邑甜茶,(C )泰山海棠,(D )八棱海棠,(E )台湾林檎,(F )三叶海棠,(G )变叶海棠,(H )锡金海棠,(I)丽江山荆子,(J)日瓦海棠,(K )湖北海棠,(L )沧江海棠,(M )西府海棠,(N )紫花海棠,(O )冬红果.图2㊀十五个苹果属分类群染色体核型图㊀801㊀聊城大学学报(自然科学版)表3㊀十五个苹果属分类群核型参数种核型公式相对长度范围/%臂比范围平均臂比臂比>2的比例/%核型不对称系数(A s k)/%类型山荆子2n =2x =34=22m+12s m 3.88G8.181.31G2.201.6311.7661.902B 平邑甜茶2n =3x =51=24m+27s m (3s a t )4.02G8.201.20G2.471.7629.4163.702B 泰山海棠2n =3x =51=36m+15s m 3.77G9.581.12G2.631.6117.6561.872B 八棱海棠2n =2x =34=12m+22s m (2s a t )4.17G9.551.35G2.511.8723.5363.762B 台湾林檎2n =2x =34=24m+10s m3.52G9.651.09G1.901.49059.921B 三叶海棠2n =3x =51=12m+36s m+3s t 3.88G8.911.46G3.392.1252.9467.463B 变叶海棠2n =3x =51=30m+18s m+3s t 3.86G9.581.21G3.031.8835.2965.382B 锡金海棠2n =4x =68=40m+28s m 3.76G9.441.26G2.341.6423.5362.322B 丽江山荆子2n =2x =34=26m+8s m4.22G8.441.17G1.931.44059.311B 日瓦海棠2n =2x =34=16m+18s m 4.19G9.081.14G2.681.8135.2964.402B 湖北海棠2n =2x =34=22m+10s m+2s t 4.30G9.831.22G3.181.7417.6563.432B沧江海棠2n =2x =34=28m+6s m 4.14G7.711.15G1.901.52060.241A 西府海棠2n =2x =34=14m+20s m 3.92G7.711.30G2.151.7629.4163.472A 紫花海棠2n =2x =34=12m+22s m 4.20G7.991.26G2.581.8429.4164.582A 冬红果2n =2x =34=16m+18s m3.93G9.601.02G2.561.7323.5363.002B 参㊀考㊀文㊀献[1]㊀陈瑞阳,宋文芹.植物G i e m s a 分带技术的研究[J ].植物学报,1979,21(1):11G18.[2]B i k r a mSG ,K i m b e rG.T h eG i e m s aC Gb a n d i n g k a r y o t y p e o f r ye [J ].P r o cN a tA c a dS c iU S A ,1974,71:1247G1249.[3]K u r a t aN ,O m u r aT.K a r y o t y p e a n a l y s i s i n r i c e 1An e w m e t h o 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s M i l lHU AL iGy u a n㊀WA N G X u a n㊀Q I A N G u a nGz e(S c h o o l o fL i f eS c i e n c e,L i a o c h e n g U n i v e r s i t y,L i a o c h e n g252059,C h i n a)A b s t r a c t㊀T h e r ee x i s t i n t e r s p e c i f i ch y b r i d i z a t i o n,p o l y p l o i d y a n da p o m i x i s p h e n o m e n o n i nt h e M a l u s M i l l.s o t h e y a r e k n o w n f o r t h e i r d i f f i c u l t y i n c l a s s i f i c a t i o n a n d i d e n t i f i c a t i o n,t h e c o m p l e x s p e c i e s o f M a l u s b a c c a t a(L.)B o r k h i s e v e nm o r e d i f f i c u l t.I n t h i s p a p e r,c h r o m o s o m e a n a l y s i sw a s c a r r i e do u t o n t h em a i n p l a n t s o f t h e c o m p l e x s p e c i e s o f M a l u s b a c c a t a(L.)B o r k h a n d t h e S e c t.S o r b o m a l u s z a b e l,w h i c h i s p o s s iGb l y c l o s e l y r e l a t e d p l a n t s.F i f t e e nt a x ao f t h e M a l u s M i l l.c o n s i s t o f o n e t e t r a p l o i d,f o u r t r i p l o i d sa n dt e n d i p l o i d s.T h e c h r o m o s o m em a t e r i a l so fT i a n m u s h a nc r a b a p p l e,M a l u s h u p e h e n s i s R e h d.v a r.t a i e n s i s G.Z.Q i a n,R i w a c r a b a p p l e,M a l u s p u r p u r e a a n d M a l u s D o n g h o n g g u o w e r e p u b l i s h e df o r t h e f i r s t t i m e.T h e k a r y o t y p e s a r e m o s t l y b e l o n g t o m e t a c e n t r i cc h r o m o s o m e(m)a n ds u b m e t a c e n t r i cc h r o m o s o m e(s m), w h i c hb e l o n g t o t h em o r e p r i m i t i v e t y p e.T h e k a r y o t y p e o f M a l u s o m b r o p h i l a H a n d.GM a z z.i s1A,w h i c h i s t h em o s t p r i m i t i v e,a n dt h ek a r y o t y p eo f M a l u s t o r i n g o D eV r i e s e i s3B,w h i c hi s t h e m o s te v o l u t i o n a r y o n e.T h e e x p e r i m e n t a l r e s u l t s s u g g e s t e d t h a t i t i s p r o b a b l y n o t t h e M a l u s h u p e h e n s i s(P a m p.)R e h d.t h a td i s t r i b u te s a t t h e t o p o fT i a n m u M o u n t a i n,b u t i t c o u l db e a n i n d e p e n d e n t s p e c i e s,i t i s r e l a t e d t o t h e p a rGe n t s of M a l u s h u p e h e n s i s(P a m p.)R e h d.v a r.m e ng sh a n e n si s G.Z.Q i a n e tW.H.S h a o a n d M a l u s h u p e h e nGs i s R e h d.v a r.t a i e n s i s G.Z.Q i a n;M a l u s b a c c a t a(L.)B o r k h p r o b a b l y i s r e l a t e d t o t h e p a r e n t s t o o;t h eR i w a c r a b a p p l e,w h i c h f o u n d i n t h ew e s t e r n o f S i c h u a n i s r e s e m b l ew i t h M a l u s s i k k i m e n s i s(W e n z.)K o e h n e,b u t t h e r e a r e g r e a t d i f f e r e n c e s i nk a r y o t y p e a n d c h r o m o s o m en u m b e r,s o i t a l s os u p p o r t s t h e i n d e p e n d e n t s p eGc i e s.T h e r e s u l t s o f k a r y o t y p e a n a l y s i s a l s o a c c u m u l a t e dd a t a s f o r f u r t h e r s t u d i e s o f g e n o m i c i n s i t uh y b r i dGi z a t i o n.K e y w o r d s㊀M a l u s M i l l;w a l l d e g r a d a t i o na n dh y p o t o m i sm e t h o d s;c h r o m o s o m e;k a r y o t y p i n g。
DOI:10.16246/j.issn.1673-5072.2002.03.006 第23卷 第3期四川师范学院学报(自然科学版)2002年9月 Vol.23 No.3Journal of Sichuan Teachers College(Natural Science)Sep.2002文章编号:1001-8220(2002)03-0239-06Progress of Karyotype Analysis Method inPlants ResearchHU Jin_Yao,SU Zhi_Xian,YUE Bao_Liang,ZHANG Qiao_Ying(Sichuan Provincial Key Laboratory of Environmental Science and Biodiversity Conservation,Research Centerof Biodiversit y,Sichuan Teachers College,Nanc hong,637002,China)A bstract:Karyotype anal ysis method of plants is a basic technique in taxonomy and other related subjects.Its research progressstages are divided into two parts:chromosome morphological mark analysis and banding analysis.The methods in these two stagesare elaborated respectively.Its prospects are also discussed.We have analysed the factors restricting the progress of karyotypeanalysis methods in plant.Karyotype analysis method in plants will not breakthrought until its application range is expanded andnew research projects is brought up with and combinaed with other experi mental techniques and theory methods in other subjects.Key words:karyotype;banding technique;chromosome.CLC number:Q343.21 Document code:AKaryotype analysis is a basic work in cell genetics.In recent six to seven decades,especially after Hsu et al invent-ed tissue culture hyotonic,the karyotype research in human and ma mmals has made wonder ful progress.The international standards of human chromosome r esearch make human cell genetics into an applied new stage.But karyotype research methods in plants have made little improvement and la gged human chromosome research by far.No matter whether to make plates or make mold picture,plants kar yotype research is mor e difficult than that of animals.So it is necessar y to overview the research methods and impr ove mor e widely applications and more innovations of kar yotype analysis methods in plant research..1 History of karyotype analysis methods in plantsThe research history of karyotype analysis methods of plants can be divided into two stages:chromosome morphologi-cal mar k analysis stage and banding analysis stage.1.1 Chromosome morphological mark analysis stageWithout the help of microscope,computer etc,and lack of some related subjects such as statistics,in the initial stage of the development of karyotype analysis technique,scientists analyzed karyotype only by utilizing chromosome mor-phology mark(length of chr omosomes,arm ratio,satellites,etc.)In this stage,we adopted plants chromosome common pressing plates method in research karyotype.This technique is invented by Beilin in1921[1].It has been used and improved for many years.No w it has bec ome one of the most pop-ular basic technique in cell genetics and cell taxonomy.And it cannot be substituted by other ne w techniques.For several decades,although common pressing plates method has made some improvements,great breakthrough has not appeared yet.Chromosome morphological mark analysis method is too rough and simple to survey single chromosome.①Received date:2002-03-12As for measures ,taking photomicrograph ,magnifying ,cutting ,dividing into groups ,pasting ,taking photo again ,then making into karyotype photos are the popular methods used in this stage .The precision of this method is very lo w .It needs too much work ,so it is ver y easy to make mistakes and picture effects are also bad [2].In this stage ,some karyotype standards make research more convenient .In 1955,Darlingtond et al [3]gave some kar yotype standards .Then in 1971,Stebbins G L et al [4]integrated some standards .In 1964,Levan et al [5]introduced some methods to calculate ,classify and na me chromosomes .These all improved the development of karyotype analysis .1.2 Banding analysis stageThe invention of highly distinguishable electronic micr oscope ,powerful computer and software pr ovide an probability for the development of karyotype analysis methods .In this sta ge ,the invention of banding technique set kar yotype analy -sis in the way to more micro and precise research .In the research of secale cereale ,dividual chr omosome cannot be dis -tinguished by common morphological methods .B ut by banding technique ,it can be discerned .The result of secale ce -reale C -band research by different researchers is the same by large ,but there are also a few differences .So did in the same plants species but different in dividuals .That is to sa y secale c ereale has band pattern polymorphism .According to the difference of homologous chromosomed ,we can easily judge it is a cross -pollination plants [1].So banding technique makes kar yotype analysis be used in the research of botony systematics ,population ecology ,genetics ,and so on .Plants banding technique includes fluorescent banding and Giemsa banding .In 1968,T .O .Caspersson invented Q -band technique .And he used it to survey broad bean mitotic chromosomes .In 1971,Arrighi and Hsu invented C -band technique .In 1972,Vosa introduced it into plants research field .The result showed C -band was more obvious in plants heterochromatin than in animals '.After then ,there are other banding techniques invented .They all belong to Gie msa banding with C -band .But their importance is junior to C -band technique .According to the incomplete statis -tics by 1980,more than 60genuses have got C -band [6].Although Q -banding technique which belongs to fluorescent banding is the earliest one applied in plants karyotype analysis ,now Giemsa banding technique is more popular in the re -search of plants chromosomes .These two banding techniques have different merits respectively (Table 1).Table 1 Comparison of fluorescent banding and Giemsa bandingcharacterFluores cent banding Giems a banding Theorembanding by utilizing fluorescent materials banding by certain chemis try methods Operation conditionNeed fluores cent ins trument and medicine Not need fluorescent ins trument and medicine Operation diffic ultyProcedures are simple Procedures are more compl icated Special band patternCan sho w special band pattern Need special dis position ,or it can not sho w special band pattern EffectMore s table L ess stable Preservation Cannot make into permanent plates Can make into permanent plates .It can s ho w different band if c hange dis pos ition procedures There is a very significant difficult problem that plants chromosome specimen cannot be made as well as that of ani -mals for the limiting of by cell wall .Common pr essing plates method has many shortages such as the difficulty of dispers -ing chromosomes ,overlap and transfor mation of chromosomes ,etc .In the research of plants chromosome G -band ,be -cause common pr essing plates method is hard to completely get rid of the c over of cell wall and cytoplasm thor oughly ,the repeatability of Giemsa band is low .It also has influences on the research of submicrostructure .So it is very emer gent to innovat plants chr omosomes plates making methods to promot plants chromosomes research ,especially for the research of plants chromosome submicrostructure and Giemsa banding .The wall degradation hyotonic method developed a way to solve this problem .The prime process is using enzyme to degradate cell wall ,hyotonic and dry with fire .The wall degra -dation hyotonic method is superior to common plates methods :①It can get many dispersed cells efficiently .②Chromo -somes are integrate and clear .③Procedure is simple and no need of special instruments .Because of these merits ,now 240 四川师范学院学报(自然科学版)2002年the wall degradation hyotonic method is used widely [7-9].As a basic technique ,common pressing plates method is still used widely .In this stage ,common plates has devel -oped too .Yue Aiqin et al [10]innovated plates making methods of three types chromosomes in leguminous by researching kar yotype analysis method .Table 2 Comparison among several karyotype analytic softwaremerits and shortages PC s y stem and Photoshop software General microcomputer soft ware forkaryotype anal ysis Semi -automatic plants chromosomes images karyotype analysis systemcommon merits Not need some special instruments :dark room ,etc ;Workload is less than common method ;Accuracy is higher thancommon methodmerits The capital is less than commonmethods and special analysis softwareCheaper than abroad soft ware ;inter -changeable ,automatic common op -eration ,output large information ;Can intervene by person Plants chromosomes are its direct re -search objects ,so it is much powerful in research plants karyotype shortagesLess power than abroad automaticchromosomes analysis s ystem Use in DOS operation system ,inter -face isn 't friendly Can not be used in the research of an -imal and hu man chromosomes In this stage ,some new methods were invented in measur e and analysis .Jiang Shanshan et al [2]used personal com -puter system and Photoshop software to measure and analyze chr omosomes .Liu Dongxu [11]translated General micr ocom -puter software for kar yotype analysis .Liuquan et al [12]introduced an applied Semi -automatic plants chromosomes images kar yotype analysis system .There are also some abr oad special kar yotype analysis software [13].Each of the software has its merits and shortages (Table 2).In the past ,most pursuers reached conclusions directly ,after simply calculating the data without using of statistic exa mination [14-16]. So their results are unreliable .Now some researchers have noticed this prob -lem .Yang Xiuying et al [7]estimated the believable scope of chr omosomes arm rate .2 Prospect2.1 The expansion of application rangeKaryotype analysis technique has been born for nearly one centur y .Plants kar yotype analysis methods has made some progress .But there are many difficulties to make a wonderful breakthrough .We drew 40domestic articles radomly on plants karyotype analysis between 1995_2001,in which the object of about thirty articles is to accumulate research data .Their research results are one or several karyotype for mula or kar yotype mode pictures [14,15,17].The more complex articles are only to compare karyotype of different plants in the same group [16,18,19].However kar yotype analysis has entered applied stage in animal and human chromosome research .For ex -ample ,using kar yotype analysis to identify hereditar y disease and analyzing the mechanism of pathological change [20,21].Narr ow research objects may be the lethal limiting to innovating karyotype analysis methods .In the future ,ho w to bring for ward new objects is an urgent task .Abroad circumstances are more optimistic .In the first half of the twentieth century ,c ytologists such as Darlington 1931;Darlington and La Cour 1942,Kihara 1924,and Sears 1941studied plant chromosomes ,making key disc overies such as the importance of polyploidy in plant evolution and showing that whole chromosomes carried similar groups of genes in different species .Soon thereafter ,an understanding of evolutionar y changes at the cytological level was estab -lished that included an appreciation for chromosome translocation ,fusion ,fission ,and the correlation between chromoso -mal particularities and phylogeny .Recently ,Luca Comaia et al studied evolution and hybrid superiority mechanism in 第23卷第3期胡进耀,等:植物核型分析方法的研究进展241 plant by means of karyotype analysis technique .The basis of this technique in the field is that many wild and cultivated allopolyploids that originated in prehistoric times are fertile ,well adapted ,and genetically stable ,allopolyploids of more recent origin commonly display genomic and phenotypic instability (Pope and Love 1952;Allard 1960;Gupta and Red -dy 1991).2.2 The innovation of experiment techniqueUp to now ,all plants banding techniques except Q -band are invented in animal research firstly .E ven C -band ,which is the most popular technique used in plants ,is also the continuator of BSG technique which is originated from ani -mal research .Plants research is junior to animals 'on banding techniques and the diversity of banding pattern .The re -search achievements in the banding mechanism are mostly obtained from animal research .Standard band patterns were es -tablished for animal chr omosomes .But plants research lags behind animal 's .Now native researchers usually adopt Steb -bins [4],Li Maoxue ,Chen Ruiyang 's [22,23]standard and Levan system [5]to name chromosomes .At present ,ther e ar e no plants banding patterns ,which can parallel with animal 's G -band .Undoubtedly ,this is a great obstacle to combine chromosome banding with genetic analysis .It is also much unfavorable to bring up ne w objects of plants kar ytype analy -sis .In r ecent year ,human chr omosomes fluorescence banding technique made a rapid progress .Follo wing the invention of FI SH technique and chromosome scribbling dyeing plates tec hnique which is developing from it ,ThomaRied and E vetin research groups in NCHGR in American NIH invented a spectrum imaging method which can mar k 24human chromosome individually with special fluorescence [24,25].In fact ,this method can mark 2n -1(n on behalf of the types of fluores -cence dye )chromosomes ,undoubtedly this is a wonder ful breakthr ough on the basic of FISH technique .This also brings a hope for the innovation of plants fluorescence banding technique ,which is invented earlier than Giemsa banding while less used than the latter .But it needs further research whether the method can be used in plants chromosome analysis .Data statistic and analysis methods need further improvement too .For example ,translate mor e powerful and c onve -nient software for analysis and cut down the capital etc .Some researcher invented some new methods by combining related techniques in other subject with common methods in karyotype analysis .In early 1980s Macdonald ,Shimizu et al consid -er ed that PFGE (pulsed field gel electrophoresis )can be used in kar yotype analysis [26-27].Later ,they applied it to study plants karyotype [28-30].Native r esearchers have done similar work (Shi Liangen et al [31]).Bian Yinbing et al [32]ana -lyzed electrophoretic kar yotype of jell basidiomycetes auricularia auricula by using of contour -cla mped homogeneous electric field (CHEF )electrophoresis .The r esult is fairly good .Luca Comaia et al studied hybrid genetic polymorphism by combination karyotype analysis with molecular biology .There 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,MORINAGA T .Electrophoretic Karyot ype of Lentinula Ed odes [J ],Trans .Mycol Soc .Japan ,1993,34:481-485.[37] SAGAWA I ,NNGATA Y .Anal ysis of Chromosomal DNA of Mushroom in Genus Pleurotus b y Pulsed Field Gel Electrophoresis [J ].J 第23卷第3期胡进耀,等:植物核型分析方法的研究进展243Gen Appl .Microbiol ,1992,38:47-52.[38] ROYER J C ,HINTZ W E ,HORGE N P A .Electrophoretic Karyotype Analysis of Button Mushroom [J ].Agaricus Bisporus .Genome ,1992,35:649-698.[39] SHI L G ,XU J L .Studies on the Regeneration -Reversion of Protoplasts and Karyotype of Beauveria Bassiana [J ].Mycos ystema ,2000,19(2):223-229.[40] BIAN Y B ,LOU X C .WANG B .Electrophoretic Karyotype Analysis of Auricularia Auricula [J ].Mycosystema ,2000,19(1):78-80.[41] GU H Y ,QU L J .Plant Molecular Biology -a Laboratory M anual [M ].Advanced Education Press ,1998.植物核型分析方法的研究进展胡进耀,苏智先,岳宝良,张桥英(四川省环境科学与生物多样性保护重点实验室,四川师范学院生物多样性研究中心,四川南充637002)摘 要:植物核型分析方法是植物系统学等学科的一门基本技术.本文将植物核型分析方法的发展过程划分为两个阶段:染色体形态标志分析时期和分带分析时期,并分别阐述了这两个时期在核型分析方法上的创新,以及这些方法的优点和不足之处.同时对其今后的发展方向作一展望.分析了限制植物核型分析方法发展的几个因素,认为核型分析方法要取得突破性进展,第一必须扩大其应用范围,提出新的研究课题,第二必须结合动物和人体核型分析的最新研究方法以及其它学科的实验技术和理论方法.关键词:核型分析;分带技术;染色体中图分类号:Q343.21 文献标识码:A①收稿日期:2002-03-12作者简介:胡进耀(1978-),男,浙江兰溪人,四川师范学院环境科学专业硕士研究生,主要从事生殖生态学方面研究工作.244 四川师范学院学报(自然科学版)2002年[7]沈玉芳.熔沸点比较七则[J ].中学化学教学参考,1992,1-2(22).[8]聂保民.熔沸点比较中的误解[J ].中学化学教学参考,1993,5(27).[9]邓 存.Sn 和Pb 哪个金属性强[J ].中学化学教学参考,1988,3(28).[10]武艳妮,赵中华.如何判断氧化—还原反应的生成物[J ].中学化学教学参考,1992,4(10).Application of Normal Data of Chemistry in TeachingYU Zheng -qi 1,MA Hong -yan 2,ZOU Rong -xian3(1.Nanchong Station of Educational Instrument ,Nanchong 637000,China ;2.Nanchong Senior Middle School ,Nancong 637000,China ;3.Depart ment of Chemistry ,Sichuan Teachers College ,Nanchong 637002,China )A bstract :Nor mal data of chemistry are very widely applied in teac hing ,research work ,production and other fields and lots of discoverices of theories ,principles and la ws are the results of analyses and summaries of these data materials .Somaking full use of these data in chemistry teaching can promote students 'ability of analysing and solving problems in practice .Key words :nor mal data of chemistr y ;teaching ;application 318 四川师范学院学报(自然科学版) 2002年。