利用Giemsa C-带和45S rDNA FISH的方法鉴定百合杂种

利用Giemsa C-带和染色体荧光原位杂交(45S rDNA FISH)的方法对‘Royal Lace’בHigh Class’的杂种后代分别进行了鉴定。结果表明：‘Royal Lace’的染色体数2n=3x=36,‘High Class’的染色体数2n=2x=24。杂种后代的染色体数出现2n=3x=36和2n=4x=48两种类型。经Giemsa C-带和45S rDNA FISH方法对两个亲本和具2n=4x=48的杂种后代分析结果表明,杂种后代的根尖染色体C-带可观察到来自双亲的可以特异追踪的染色体带纹。FISH分析表明,杂种后代中分别有两条来自‘Royal Lace’和‘High Class’的染色体。两个亲本的荧光原位杂交点数分别为10和9。杂种后代染色体为2n=36的个体有14个杂交信号,可以确定两条来自‘Royal Lace’,另外3条来自‘High Class’。杂种后代中2n=48的个体的杂交信号点为19,从而证实所获得的杂种后代的真实性。同时,对于2n=48,而杂交信号为19 的多倍体起源于未减数分裂的2n雄配子体的可能性进行了探讨。综合研究结果表明,Giemsa C-带和45S rDNA FISH方法可以准确地对百合的杂种真实性进行鉴定。     

水稻无性繁殖过程中第8号染色体单体的发现及其分子细胞学鉴定

在籼稻品种中籼3037试管苗无性繁殖过程中,发现一种无性系变异株,其植株矮小,生长势弱,高度不育．细胞学鉴定表明,该变异株体细胞中的染色体数目为2n=23,比正常植株少了一条染色体,因而在花粉母细胞减数分裂粗线期可见到一条未配对的单价染色体．用来源于水稻着丝粒特异串联重复序列RCS2以及亚端粒特异串联重复序列Os48为探针,进行荧光原位杂交,结果表明单价体染色体上的RCS2信号较弱,且位于染色体的中部,在其长臂末端有较强的Os48杂交信号,这些特征均与水稻第8号染色体的特征非常类似．进一步以水稻染色体特异的分子细胞学标记进行鉴定,确认该变异株为第8号染色体的单体．     

毛竹和厚壁毛竹的核型分析及45S和5S rDNA的FISH分析

【目的】厚壁毛竹(Phyllostachys edulis ‘Pachyloen')是毛竹(Phyllostachys edulis)的栽培品种,是具有较高经济价值的优特种质。对毛竹与厚壁毛竹进行细胞遗传学研究,了解其核型特征。【方法】采用双色荧光原位杂交技术,对毛竹、厚壁毛竹进行了核型分析,利用45S 和 5S rDNA 在其染色体上进行了物理定位。【结果】毛竹、厚壁毛竹的染色体数目都为48条,毛竹的染色体相对组成为2n=48=12L+10M₂+14M₁+12S,核型公式为 2n=48=32m+12sm+4st(2SAT); 厚壁毛竹的染色体相对组成为2n=48=12L+8M₂+16M₁+12S,核型公式为2n=48=34m+10sm+4st(2SAT)。毛竹与厚壁毛竹的染色体不对称系数分别为61.41%和59.55%,均属于“2B”型。荧光原位杂交(FISH)结果表明:毛竹、厚壁毛竹都有2个45S rDNA位点和4个5S rDNA位点。【结论】厚壁毛竹的染色体数量为48条,补充了毛竹的染色体为48条的循证依据; 获得了毛竹、厚壁毛竹45S 和 5S rDNA荧光原位杂交核型,两者的45S和5S rDNA 位点没有明显差异,具有相似的核型,显示其较近的亲缘关系。     

鮸染色体的识别及核型特征分析

鮸(Miichthys miiuy)是我国南方重要的海水养殖种类.针对鮸细胞遗传标记匮乏的问题,利用荧光染色、硝酸银染色(银染)、荧光原位杂交(FISH)等方法分析了鮸的核型特征,发现鮸的核型含24对端部着丝粒染色体(2n=48t),鮸染色体经碘化丙啶(PI)染色后,荧光强度呈现特定的二维分布模式;利用图像分析软件可转化为更直观的3D-光强度图,为鮸染色体的识别、配对提供了丰富的信息.18SrDNA和5SrDNA双色FISH结果显示,鮸的18SrDNA和5S rDNA均只有1对位点,分别位于1号染色体的近着丝粒区域和2号染色体的着丝粒区域.银染显示鮸的核仁组织区域(NOR)和4′,6-二脒基-2-苯基吲哚(DAPI)染色显示的暗带,均与18SrDNA位点的位置相同.鮸的端粒序列FISH信号分布于所有染色体的两端,未发现臂间端粒信号.综上,通过PI染色和图像分析软件解决了鮸染色体识别困难的问题,并使用FISH方法分析了鮸的核型特征,研究结果丰富了鮸的细胞遗传学标记,也为研究石首鱼类染色体进化提供了基础数据.     

青鲫（♀）×兴国红鲤（♂）杂交F1代染色体数目及组型分析

采用肾细胞染色体直接制片法及Levan提出的标准，对青鲫（♀）×兴国红鲤（♂）杂交F1代染色体数目及组型分析表明：此杂交F1代鱼的肾细胞染色体数目为150条．染色体组型按着丝点位置可分为四组，其中中部着丝粒染色体9条，亚中部着丝粒染色体15条，亚端部着丝粒染色体78条，端部着丝粒染色体48条．每个染色体小组均由3条同源染色体组成，表明此杂交鱼为三倍体，核型公式为3n=9m＋15sm＋78st＋48t．臂数NF=174．     

玉米（Zea mays L．）盐逆境胁迫蛋白基因nac1的染色体原位杂交物理定位

报道了玉米低拷贝盐逆境胁迫蛋白基因ｎａｃ１生物素标记的染色体原位杂交结果．供试探针为这个基因的ｃＤＮＡ克隆,其长度仅为５５０ｂｐ．采用酶联免疫级联放大和ＤＡＢ检测系统检测,实验结果表明,杂交信号分布在第２染色体短臂和第１０染色体长臂,与着丝粒的百分距离分别为６７．５６±３．２９１和８０．９２±２．４１１,检出率分别为５．８１％和１０．３２％．并对植物单和低拷贝基因的染色体原位杂交技术与基因染色体原位杂交作图进行了讨论     

油松及云南松染色体的荧光带型

利用两种荧光试剂ＣＭＡ和ＤＡＰＩ研究油松及云南松染色体的荧光带型。油松（２ｎ＝２４）和云南松（２ｎ＝２４）染色体的ＣＭＡ带型中均存在五种不同的ＣＭＡ带纹类型。油松的ＣＭＡ带型为３对是着丝粒和臂间处均有荧光带纹的中部着丝粒染色体（Ａ类型）,３对是臂间处有荧光带纹的中部着丝粒染色体（Ｂ类型）,２对是着丝粒处有荧光带纹的中部着丝粒染色体（Ｃ类型）,２对是无荧光带纹的近中或中部着丝粒染色体（Ｄ类型）,２对是着丝粒有荧光带纹的近中着丝粒染色体（Ｅ类型）;而云南松的ＣＭＡ带型则为Ａ类型、Ｂ类型、Ｃ类型、Ｄ类型和Ｅ类型的染色体分别有４对、２对、２对、２．５对和１．５对。这两种松树染色体的ＤＡＰＩ荧光带型与ＣＭＡ带型之间存在相反的带纹关系。最后讨论认为①利用荧光带型的不同可将二者区分开,②据荧光带型认为云南松在赤松亚组中与其它松树之间的亲缘关系可能要远一些。     

rDNA序列在多种蔬果类植物染色体上的定位

利用改进的rDNA序列标记荧光原位杂交方法,将18S-5S rDNA探针序列直接在5'端进行荧光修饰,并成功将其应用到多种植物染色体上,该方法比常规方法具有省时、经济、快速、信号强等特点,使rDNAFISH杂交更为简单,更易操作.首次利用该技术成功地对30种蔬果类植物中期染色体进行18S-5S rDNA的物理定位,初步了解了rDNA序列在这些蔬果类植物基因组中位点数目和分布特点,其中大蒜、大葱、蚕豆、萝卜(3种)茴香、红菜苔、空心菜、芥兰、莴苣、乌塌菜、雪里蕻、茼蒿、油菜、辣椒、茄子、芹菜、菜豆、花椰菜等20种植物首次确定了18S-5S rDNA在中期染色体上的位置,其余10种植物与前人的结果相吻合.该方法可以作为染色体的一个识别指标,也可为蔬果类植物属内进化和物种分化研究提供重要资料.     

一种改进的重复多色荧光原位杂交技术

多色荧光原位杂交技术是在常规荧光原位杂交技术基础上建立的一种染色体分析手段,已广泛应用于肿瘤遗传学的研究,尤其是用于染色体隐匿性重排及复杂核型分析。本研究旨在利用现有实验条件,建立一种可靠的人类全染色体分析方法——重复多色荧光原位杂交技术（repetitive multicolour fluorescence in situ hybridization,RM-FISH）。采用特异的全染色体涂染探针,分别用C3、Cy5、FITC3种荧光素直接标记,组合制备4组六色的探针池。对同一染色体片先后进行4次杂交,通过装备了4组滤镜的荧光显微镜捕获信号。RM-FISH技术结合反相DAPI显带对1例原发性食管鳞癌进行核型分析,获得了全部染色体的特异信号。本研究建立的RM-FISH简便、易行,是光谱核型分析的一种较理想的替代技术。     

着丝粒串联重复序列RCS2对亚洲栽培稻和非洲栽培稻的比较分析

为了揭示中高度重复序列在同为AA基因组的亚洲栽培稻和非洲栽培稻基因组中的差异以及重复序列在.栽培稻种的分化过程中可能起到的作用,利用水稻着丝粒串联重复序列RCS2作为探针分别对籼稻广陆矮4号、粳稻日本晴和非洲栽培稻的体细胞染色体进行荧光原位杂交(FISH)实验,并对其核型进行同源性聚类和比较分析,杂交结果显示:RCS2序列位于在3种栽培稻染色体组中,RCS2序列位于每条染色体的着丝粒位置,但有不同的分布特点,表明该3种栽培稻基因组的RCS2序列有不同的进化方向.探讨了RCS2序列结合Cot-1 DNA FISH方法对水稻染色体组进行核型分析的可行性和优势.     

Distribution of highly repeated DNA sequences in Haynaldia villosa and its application in the identification of alien chromatin

Haynaldia villosa (L.) is a wild relative species of common wheat that possesses many beneficial genes that can be used for wheat improvement. The accurate detection of H. villosa chromosomes in the genetic background of wheat is critical for transferring its beneficial genes to common wheat by chromosome engineering. The aim of the present study was to investigate the distribution patterns of two repeated DNA sequences, pSc119.2 and pAs1, as well as two rDNA multigene family sequences, 45S rDNA and 5S rDNA, in the individual chromosomes of H. villosa for the future precise identification of alien chromatin in germplasm development and breeding programs. A set of common wheat-H. villosa disomic addition 1V-7V lines was used to determine these specific signals on individual chromosomes of H. villosa. The results showed that two rDNA probes, pTa71 (45S rDNA) and pTa794 (5S rDNA), were located on 1VS and 5VS, respectively, and the signal could be discriminated exclusively in the common wheat background as effective markers of 1VS and 5VS. Furthermore, all seven chromosomes of H. villosa could be distinguished clearly by fluorescence in situ hybridization using pSc119.2 and pAs1 as probes in combination. The utilization of these cytogenetic markers of repetitive sequences, combined with other molecular markers sometimes, will make it possible for a precise identification of alien chromosomes with high efficiency.     

Primary investigation on GISH-NOR in cotton

Six loci of nucleolar organizer region (NOR) were detected in genomic in situ hybridization (GISH) of cotton (Gossypium). NOR was the characteristic of 45S rDNA but could be generated by genomic DNA (gDNA) extracted from Gossypium species as probe. With twice FISH to the same mitotic cell of G herbaceum or G hirsutum, number, position and size for NORs generated from 45S rDNA and gDNA were identified largely similar or even the same. The NORs with gDNA as probe were therefore permanently defined as GISH-NORs. GISH-NORs from G hirsutum and Graimondii mitotic images were all terminal types. Four and two GISH-NORs from G herbaceum (var. africanum) were terminal and centromere types, respectively. Six GISH-NORs in G hirsutum were chromosome mapped with two in A- and four in D-subgenomes. There were also GISH-NORs in mitotic image of G raimondii with its own gDNA as probe. From mitotic image of G herbaceum with its own gDNA as probe, GISH-NOR could not be observed but non-wholerecovery of hybridized signals was distinguished. These non-whole-recovery of hybridized signals were detected on long arm terminals of most chromosomes and especially existed in nearly half long arm of a pair of chromosomes in Gherbaceum gDNA probed itself GISH image, which may be possibly induced by low copy genes within the regions rather than inter-subgenomic segment translocations. GISH-NORs in G hirsutum mitotic images were dominantly observed when gDNAs from D and A genome species were used as probes and block, respectively, but not when the reverse probe and block gDNA from the two diploid progenitor genomes were designed. There may be two speculations to this special phenomenon: rDNA concerted evolution; content of rDNA in genome D more than genome A.     

基于核糖体小亚基DNA序列的鼠尾藻系统发生分析

通过制备野生鼠尾藻基因组DNA,用同源克隆技术获得鼠尾藻的SSU rDNA序列。该序列长度为1 784 bp,A、G、T、C 4种碱基的含量分别为24.66%、27.75%、26.35%和21.24%,序列已提交至GenBank,登录号为JN604979。选用GenBank数据库中已有的褐藻SSU rDNA序列构建系统发生树,结果显示:鼠尾藻与半叶马尾藻,铜藻以及Sargassum macrocarpum聚为一支,并且与半叶马尾藻的亲缘关系最近。     

FISH of 5S rDNA and telomeric (TTAGGG) n repeats in normal and translocated populations of the frog Quasipaa boulengeri (Anura, Ranidae)

Quasipaa boulengeri,a spiny frog,is widely distributed in the low mountain regions,around Sichuan Basin.Our previous study revealed five karyotypes,caused by a translocation,that are randomly distributed throughout different populations.5S rDNA and telomere sequence(TTAGGG) n are potential good markers for chromosome identification and karyological evolution.In this study,we examined the sequences of 14 populations using fluorescence in situ hybridization(FISH) to detect if there is any variation between karyologically normal and translocated populations.5S rDNA loci were located at the same position on chromosomes 1 in 7 translocated populations.In two of the seven normal populations,5S rDNA also occurred on chromosome 5 in addition to chromosome 1.Our findings further indicate that the 5S rDNA on No.1 most likely represents the ancestral condition,while the minor loci represent the derived state.Signal density variations of the 5S rDNA were observed beteween homologous chromosomes or sister chromatids of pair 1 in both normal and translocated populations.Telomere sequences were identically located on all ends of the 26 chromosomes in seven rearranged populations,however,no ITSs were observed on the translocated chromosomes 1 and 6.Two of the six normal populations were found to contain ITSs which indicates that populations with translocation events diverged prior to those with ITSs rearrangements.In the KKS and BF populations,the ITSs of chromosome 3 are not always found on both homologues.Inter-chromosomal signal strength of telomeric sequences commonly differs within all populations.     

5S及16S rDNA序列PCR扩增用于环境军团菌检测

军团菌病（Ｌｅｇｉｏｎｎａｉｒｅｓ’ｄｉｓｅａｓｅ）是一种严重的空气传播疾病,在世界各地都常有报道,是由军团菌（Ｌｅｇｉｏｎｅｌａ）引起,该菌迄今已鉴定出了４４个种,其中１８种具致病性,最常见的导致严重病症的种有嗜肺军团菌（Ｌ．ｐｎｅｕｍｏｐｈｉｌａ...     

一株高产抗菌活性物质链霉菌的初步分类鉴定

对一株土壤中筛选出的可以产抗菌活性物质的链霉菌进行了研究,测定了该菌的抗菌谱,发现该菌株对多种植物病原性真菌及细菌有拮抗作用,但对植物病原性真菌的抑制效果低于植物病原性细菌.通过形态特征、培养特征、生理生化特性及抑菌谱等系列比较,发现该链霉菌菌株与链霉菌属的黑胡桃链霉菌(Streptomyces nogalater)的特征基本相符;通过16S?rDNA序列比对,发现该菌株16S?rDNA与黑胡桃链霉菌的16S?rDNA同源性达99％.根据多项分类原则和系统进化树的构建分析,将该菌株暂归入黑胡桃链霉菌类.     

青菜花粉管通道法导入外源DNA的研究初报

采用花粉管通道法，将青菜株系７５Ｆ５、１４号大白菜、紫阳等三个品种的总ＤＮＡ及质粒ｐＡｃｔ１－Ｄ ＤＮＡ导入青菜６０５品系，收获青菜种子。种子发芽后分别提取约两周苗龄的小苗总ＤＮＡ，并经ＥｃｏＲⅠ酶切，以ｇｕｓ基因片段为探针做子杂交，证实了外源ｇｕｓ基因已整合到青菜６０５品系中；田间观察还获得了一株与供体大白菜花形相似的变异株。     

不结球白菜SSR引物的高效开发及其通用性研究

利用改进的ISSR-PCR分离不结球白菜基因组微卫星,进行两步PCR,分别设计出SSR两端引物IP2和IP3（即SSR引物对）,SSR引物产率为12%,明显高于传统方法.不结球白菜SSR序列以（GA）n为主,占70%.将69对SSR引物用于芸苔属其它8种作物的扩增,97%引物有显著扩增,扩增率最高的为四倍体白菜和甘蓝（85.5%）,最低的为青花菜（49.3%）.10对为通用引物,在所检测的8种作物中片段大小一致,其余的扩增片段和数目差异较大,表现出较为丰富的多态性.尤其是在C基因组的甘蓝和花椰菜上,最多可检测到5个位点,表明运用lSSR-PCR开发出的引物多态性较高.     

沈阳城市森林树种综合评价指标体系

建立了由7个抗性功能指标、5个生态功能指标、5个美学功能指标组成的城市森林树种综合评价指标体系,并运用层次分析法对沈阳42种主要城市森林乔灌木树种进行了综合评价。其中,刺槐、榆树、小叶朴、早柳等乔木树种,黄刺玫、水蜡、接骨木、鸡树条荚蓬等灌木树种获得了较高的综合评价指数。评价结果与沈阳地区的基本状况以及城市森林树种选择的基本原则有着高度的一致性,说明该研究在城市树种评价方面做了一次定性与定量、主观与客观相结合的有益尝试。     

丹参和白花丹参对黄芩化感效应的比较研究

采用室内生物测定法研究不同浓度（0、0.1、0.5、1.0、5.0 g/L）的1年生、2年生丹参和白花丹参根及根茎水浸液对黄芩种子萌发和幼苗生长的化感效应。结果表明, 丹参和白花丹参根及根茎水浸液对黄芩种子萌发率、发芽指数和活力指数表现出相似的影响趋势,即低浓度时促进种子萌发,随浓度增加化感抑制作用增强,浓度为5 g/L时抑制作用强烈。白花丹参根及根茎水浸液对黄芩种子萌发率的化感指数绝对值小于丹参。丹参和白花丹参根及根茎水浸液对黄芩幼苗苗高和根长表现出相似的影响趋势,高浓度时对幼苗鲜重的抑制作用较为强烈。白花丹参根及根茎水浸液对黄芩幼苗苗高和鲜重的化感指数绝对值小于丹参。丹参和白花丹参对黄芩种子萌发和幼苗生长均表现出低促高抑的双重效应,且白花丹参对黄芩的化感潜力小于丹参。该研究比较了丹参和白花丹参对黄芩的化感效应,可为优化复合种植模式提供科学依据。