利用粳稻基因组DNA和C_ot-1 DNA探针对普通野生稻和亚洲栽培稻的比较分析

以粳稻日本晴基因组DNA和Cot-1 DNA为探针,分别对日本晴、籼稻广陆矮4号和普通野生稻的染色体组进行了基因组原位杂交(GISH)和Cot-1 DNA荧光原位杂交(FISH)分析,并对3种染色体组进行了同源聚类和比较研究.结果表明:粳稻基因组DNA和Cot-1 DNA探针信号在3种水稻染色体组中的分布状况和覆盖率相似,Cot-1 DNA的覆盖率分别为(47.13±0.18)%、(45.89±0.22)%、(44.24±0.21)%,3种水稻基因组同源性高,亲缘关系接近.Cot-1 DNA在3种水稻染色体上的杂交信号分布各有特点,中高度重复序列的变异在普通野生稻向栽培稻进化和亚洲栽培稻籼、粳分化过程中具有重要意义,中高度重复序列含量较低的2、5、8号染色体是水稻染色体组进化过程中相对活跃的成分.     

葎草单染色体的显微分离及DOP-PCR技术体系建立

葎草是具有XX/XY1Y2性染色体系统的雌雄异株植物,是研究植物性染色体演化的模式材料之一.利用染色体显微分离技术从葎草根尖有丝分裂中期分裂相中将单条染色体进行了显微分离及DOP-PCR(Degenerate oligonucleotide primer-PCR)扩增,并构建了单染色体DOP-PCR扩增产物的荧光探针,对葎草根尖有丝分裂中期分裂相染色体进行了荧光原位杂交,其结果表明荧光信号分布在所有的染色体上,表明所建立的技术体系能够成功分离葎草单染色体并进行DNA扩增.本研究结果为进一步进行葎草X,Y染色体的细胞及分子生物学研究提供了技术支持.     

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.     

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．     

LA-PCR法制备赤麂Y2涂染探针与原位杂交

应用显微切割技术获得赤麂的Y2单条染色体,经LA-PCR（linker-adaptor PCR）扩增后用DIG标记制备探针,然后用Southern blot杂交法对所制备的探针进行验证并对毛冠鹿的中期核型进行原位杂交.结果表明：用该法制备的涂染探针是成功的,原位杂交也获得了阳性结果,可以初步验证毛冠鹿中的Y染色体.     

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。     

原位杂交在林木遗传育种上的应用现状和前景

概述了原位杂交技术的各种方法及其运用条件，分析在林木染色体水平和分析水平上的研究进展。该项技术目前主要应用于：１）鉴别种内和种间染色体间的差异，构建核型；２）探索种的起源和进化；３）结合荧光带纹研究基因组结构、有机组成和空间提列。还对该技术在林木遗传育种上的应用前景作了展望。     

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.     

香瓜属(Cucumis)植物染色体核型分析

用去壁低渗火焰干燥技术对香瓜属两个品种华兰氏和哈密瓜的染色体核型进行研究.结果表明:两种植物均具有24条染色体,中部着丝点的染色体9对,近中端着丝点的染色体3对,包括一对具随体的染色体.其核型公式均为:2n=24=18m+4sm+2sm(SAT),按照Stebbins的核型分类方法,细胞类型均为2A.通过本实验对华兰氏和哈密瓜染色体数目的确定,以及核型的分析,为今后对葫芦科尤其是香瓜属植物核型的分析研究提供了借鉴和依据.     

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。     

Repetitive DNA sequences from the X chromosome of the spiny eel (Mastacembelus aculeatus)

To investigate the characters of repetitive DNA sequence in the sex chromosomes of the spiny eel (Mastacembelus aculeatus), the X chromosomal library was screened and a family of repetitive sequence, consisting of Ma 1-Ma 6, was isolated. The fluorescence in situ hybridization (FISH) result confirmed that Ma 1 - Ma 5 dispersed over sex chromosomes and all autosomes, whereas, Ma 6 is sex chromosome-specific and distributed only on the C-band positive regions of X chromosome, and Ma 6 maybe the main components of the heterochromatic regions of X chromosome. This study provides additional information about the evolution of sex chromosomes in lower vertebrates such as fish.      

Isolation and identification of the three rice monotelosomics

In order to obtain rice monotelosomic, the progeny of 24 telotrisomics, derived from an indica rice variety, Zhongxian 3037, were screened. The variants that differed morphologically from the diploids and the original primary trisomics as well as the telotrisomics were collected for cytological identification. The variants with 24 chromosomes were selected according to the prometaphase chromosomes. From these variants, three monotelosomies with one chromosome arm deletion in each were verified by fluorescence in situ hybridization （FISH） using a rice centromeric BAC clone of 17p22 as a marker probe. The three monotelosomics were derived from telotrisomic 1S, 4L and 11L, respectively. Further identification was conducted on the prometaphase or pachytene chromosomes of the three variants, which were probed with the same centromeric BAC clone together with the corresponding chromosome arm specific makers, a0059H02 （on the short arm of chromosome 1）, a0034E24 （on the long arm of chromosome 4）, and a0071H11 （on the long arm of chromosome 11）. The results indicated that the telocentric chromosomes in the three monotelosom. ics were derived from their respective corresponding telotrisomics. According to the telocentric chromosomes of the variants, they were monotelosomic 1S （one long arm of chromosome 1 was lost）, monotelosomic 4L （one short arm of chromosome 4 was lost） and monotelosomic 11L （one short arm of chromosome 11 was lost）, respectively.     

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.     

Development and characterization of interspecific hybrids between Oryza sativa and O. latifolia by in situ hybridization

Oryza sativa and O. latifolia belong to the AA and CCDD genomes of Oryza, respectively. In this study, interspecific hybrids of these species were obtained using the embryo rescue technique. Hybrid panicle traits, such as long awns, small grain, exoteric large purple stigma, grain shattering and dispersed panicles, resemble that of the paternal parent, O. latifolia, whereas there is obvious heterosis in such respects as plant height, tillering ability and vegetative vigor. Chromosome pairing and the genomic components of the hybrid were subsequently investigated using genomic in situ hybridization (GISH) and fluorescent in situ hybridization (FISH) analysis. Based on the mitotic metaphase chromosome numbers of the root tips investigated, the hybrid is a triploid with 36 chromosomes. The genomic constitution of the hybrid is ACD. In the meiotic metaphase I of the hybrid pollen mother cell, poor chro- mosome pairing was identified and most of the chromosomes were univalent, which resulted in com- plete male sterility in the hybrid.     

Physical location of riceGm-6, Pi-5(t) genes inO. officinalis with BAC-FISH

A fluorescence in situ hybridization (FISH) procedure was adopted to physically map two rice BAC clones 24E21 and 4F22 linked to Gm-6 and Pi-5(t) in O. offi-cinalis. FISH results showed that the two BAC clones were located at 4L. The percentage distance from the centromere to the hybridization sites was 72 + 2.62 for 24E21 and 54+ 5.43 for 4F22, the detection rates were 52.70% and 61.2%. The results obtained from the BAC and plasmid clones, RG214 and RZ565 of cultivated rice and O. officinalis were the same. This suggested that the markers, RG214 and RZ565 of cultivated rice and O. officinalis were in the same BAC clones. The homologous sequences of Gm-6 and Pi-5(t) in O. officinalis were positions that signals existed on the 4L. Many signals were observed when no Cot-1 DNA blocked. This also showed that repetitive sequences were some ho-molgous between cultivated rice and O. officinalis. The identification of chromosome 4 of O. officinalis is based on Jena et al. (1994). In our study, we discussed the possibility of physical map in O. officinalis with rice BAC clones.     

Comparative physical mapping of rice BAC clones linked to resistance genes Glh,Bph-3 and xa-5 in Oryza sativa L. and O. granulata Nees et Arn. ex Watt.

Oryza granulata Nees et Arn. ex Watt. is one of the three wild relatives of rice,which are the most valuable for study and utilization in China.In this study,the homology and physical locations of three rice resistance genes,Glh,Bph-3 and xa-5 are comparatively analyzed between O.sativa and O. granulata by Southern blotting and fluorescence in situ hybridization (FISH).The results of Southern blotting indicate that there exist homologous sequences of the tested RFLP markers in O. granulata.By using three bacterial artificial chromosome (BAC) clones scanned by the tested RFLP as probes, FISH signals are detected on both mitotic and pachytene chromosomes in O.sativa and O.granulata.Dual-color FISH demonstrates that two of the three BAC clones (14E16 and 38J9) are located on the short arm of the same chromosome pair in O. granulata. Additionally, colinearity is shown for the two clones between O.sativa and O.granulata. Another BAC clone 44B4 is located on the end of the short arm of other chromosome pair in these two species.Although the phylogenetic relationship between O.sativa and O.granulata is the most distinct in Oryza and these two species have evidently different biological features and ecological habits, the relative lengths and arm ratios of the detected chromosomes and the relative positions of the tested clone signals on chromosomes in O. granulata are quite similar to those in O. sativa.     

Ipi-t,Ipi-3(t)在水稻中的物理定位及水稻第12染色体的识别

选用与水稻12染色体上Ipi-t,Ipi-3(t)和Pi-4(t)及着丝粒连锁的RFLP标记RZ670对水稻进行了荧光原位杂交。清楚显示了第12染色体着丝粒所在的位置,为水稻染色体的准确识别提供了一种新的方法。     

Physical location ofHelminthosporium carbonum susceptibility gene hm1 by FISH of a RFLP marker umc119 in Maize

A fluorescencein situ hybridization (FISH) procedure was adopted to physically map a RFLP marker, umc119 near the centromere of the long arm of linkage group1 in maize. The hm1 gene (Helminthosporium carbonum susceptibility gene) was linked closely with the marker umc119. RFLP markers are very good landmarks for mapping genes. Therefore, we also determined the position of the gene hm1 on the chromosome based on the physical location of umc119. The disease induced by infection ofHelminthosporium carbonum is one of the serious maize diseases and it distributes in many countries including China. Hybridization sites were showed on 1 L (long arm of chromosome1) and 5 L. The percentage distance from centromere to the hybridization site was 22.86 on 1 L and 58.23 on 5 L the detection rate was about 12% for mitotic cells. In interphase nuclei five hybridized sites were detected. It demonstrated that umc119 was multiplicated sequences. FISH has more advantages overin situ hybridization (ISH) detected by DAB for increasing the detection ratio and contrast between chromosomes and hybridization signals. The ability to detect the hybridization signal of a small low copy DNA sequence is a very important key towards wide application of FISH for plant genome mapping. Supported by the National Natural Science Foundation and Doctorate Vesting Point Foundation of the Education Committee of China Li Lijia: born in 1967. Ph. D.