菠菜rDNA及端粒多色荧光原位杂交分析

以拟南芥25S rDNA、5S rDNA以及端粒序列为探针对菠菜的中期染色体进行了多色荧光原位杂交分析, 其中25S rDNA用生物素标记, 端粒序列用地高辛标记, 5S rDNA用生物素和地高辛共同标记。杂交结果显示, 菠菜中期染色体25S rDNA杂交位点为6个, 分别位于3号、5号和6号染色体的随体部位; 5S rDNA杂交位点为4个, 分别位于3号和5号染色体长臂, 端粒序列杂交位点位于6号染色体端部以及其余染色体的端部和着丝粒部位。     

基于荧光原位杂交的藜属植物核型分析

为精确地识别藜属植物染色体组的核型特征,该文研究了4种来自青海高原的野生藜属植物(灰绿藜、藜、菊叶香藜及杂配藜)和1种从美国引进的栽培藜麦品种PI614932-HX(3)基于染色体荧光原位杂交(rDNA FISH)的核型。利用5S rDNA和45S rDNA对5种藜属植物有丝分裂中期的染色体进行FISH研究。藜属植物的核型分析结果表明:(1)藜属植物中存在二倍体(2n=2x=18)和四倍体(2n=4x=36)两种倍性,藜麦和灰绿藜为四倍体,其余3种为二倍体。(2)藜麦、灰绿藜、藜、菊叶香藜及杂配藜的核型公式分别为2n=4x=36=34m(2AST)+2sm,2n=4x=36=32m(4AST)+4sm,2n=2x=18=16m(4AST)+2sm,2n=2x=18=18m及2n=2x=18=16m+2sm。(3)染色体由大部分的中部着丝粒染色体(m)和少部分近中部着丝粒染色体(sm)组成。(4)核型类型除了菊叶香藜为1B以外,其余均属于2B类型。(5)在藜麦、灰绿藜及藜中具有分布位置不同、数量不等的双随体。5S rDNA、45S rDNA FISH结果表明:(1)藜麦和灰绿藜的染色体上存在2对5S rDNA位点和1对45S rDNA位点,藜、杂配藜的染色体上存在1对5S rDNA位点和1对45S rDNA位点,菊叶香藜的染色体上只存在1对5S rDNA位点。(2)5S rDNA和45S rDNA位点均位于染色体的短臂上。该研究首次获得了藜属植物基于5S rDNA和45S rDNA荧光原位杂交核型,为藜属植物亲缘关系研究和细胞生物学研究提供了分子细胞遗传学依据。     

豆科三属八种植物的核型及rDNA定位研究

对豆科槐属的国槐(SophorajaponicaL.)、五叶槐(S.japonicaL.f.oligophyllaFranch.)、龙爪槐(S.japonicaL.f·pendulaLoud.)、黄金槐(S.xanthanthaC.Y.Ma.)(以上均为四倍体,2n=4x=28)、红花槐(S.rubrifloraTsoong.,2n=3x=21),刺槐属的刺槐(Robiniapseudoa-caciaL.,2n=2x=22)、毛洋槐(R.hispidaL.,2n=2x=30)和紫穗槐属的紫穗槐(AmorphafruticosaL.,2n=2x=40)核型进行了分析,并应用荧光原位杂交技术进行了45SrDNA的染色体定位。槐属4个四倍体种各具4个45SrDNA位点,位于两对染色体的着丝粒周围;红花槐,3个45SrDNA位点位于第5组染色体随体区域。刺槐,4个rDNA位点位于两对随体染色体端部;毛洋槐,8个rDNA位点,4个位于两对染色体的随体及次缢痕,另4个位于两对染色体着丝粒周围。紫穗槐,6个45SrDNA位点,分别位于3对染色体的着丝粒和随体。本文对rDNA作为染色体标记在核型分析中的应用及在基因组中的分布特点进行了讨论。     

不同地域乌拉尔甘草基因组的FISH分析与染色体识别

在核型分析与染色体识别基础上,分别以番茄45S和5S rDNA为探针,对3种不同地域的乌拉尔甘草进行FISH分析.结果表明:内蒙古鄂托克前旗的乌拉尔甘草核型公式为2n=2x=16=6m+10sm (2SAT),新疆阿勒泰地区的乌拉尔甘草核型公式为2n=2x=16=4m+12sm(2SAT),内蒙古喀喇沁旗乌拉尔甘草核型公式为2n=2x=16=4m+12sm(2SAT);其第8染色体均带有随体.3种乌拉尔甘草基因组内均有1对5S rDNA和1对45S rDNA杂交位点.核型分析显示,5S rDNA杂交位点均位于第2染色体的短臂部位,45S rDNA杂交位点均位于第8染色体的次缢痕和随体部位.45S与5S rDNA在3种乌拉尔甘草中期分裂相上的位点数和分布情况高度一致,表明来自3种不同地域的乌拉尔甘草在染色体结构水平上没有较大的分化.     

薏苡45S和5S rDNA的染色体定位研究(简报)

通过荧光原位杂交的方法确定了45S和5S正NA序列在薏苡前中期染色体上的位置.尽管具有20条染色体的薏苡是四倍体植物,但它的基因组中只有一个45S和5S rDNA位点.根据薏苡前中期染色体的核型,确定45S rDNA序列位于薏苡第2号染色体短臂上的次级缢痕区和随体上,5S rDNA序列位于第7号染色体长臂靠近着丝粒处,5S rDNA位点到着丝粒的百分距离是29.13±1.76.     

金鱼草自交不亲和位点位于着丝粒的周边区

在蔷薇科、茄科和玄参科配子体自交不亲和中,编码花柱的S RNase控制花柱的自交不亲和性.在前两科植物中,自交不亲和(S)位点定位于着丝粒的附近,但在玄参科植物金鱼草(Antirrhinum)中自交不亲和位点至今未知.为了确定它在染色体上的位置和基因组结构,以基因型S2S5金鱼草根尖为材料,进行染色体的制备观察,利用地高辛标记的S2 RNase和含有其全长的BAC克隆(S2 BAC)为探针进行荧光染色体原位杂交(FISH),发现S2RNase杂交信号位于染色体的着丝粒附近,而S2 BAC的杂交信号位于每条染色体的着丝粒的周边区,呈对称的4个,表明金鱼草S位点位于着丝粒的周边区.对S2BAC预测基因的分析表明,发现一个金鱼草新的反转座子(RIS1).结果显示,金鱼草S位点位于染色体着丝粒的周边区,富含转座子和反转座子,和其他两类配子体自交不亲和的位置类似,预示它们的共同起源和具有抑制重组的功能.     

金鱼草自交不亲和位点位于着丝粒的周边区

在蔷薇科,茄科和玄参科配子体自交不亲和中,编码花柱的SRNase控制花柱的自交不亲和性,在前两科植物中,自交不亲和（S）位点定位于着丝粒的附近,但在玄参科植物金鱼草（Antirrhinum)中自交不亲和位点至今未知,为了确定它在染色体上的位置和基因组结构,以基因型S2S5金鱼草根尖为材料,进行染色体的制备观察,利用地高辛标记的S2RNase和含有其全长的BAC克隆（S2BAC）为探针进行荧光染色体原位杂交（FISH）,发现S2RNase杂交信号位于染色体的着丝粒附近,而S2BAC的杂交信号位于每条染色体的着丝粒的周边区,呈对称的4个,表明金鱼草S位点位于着丝粒的周边区,对S2BAC预测基因的分析表明,发现一个金鱼草新的反转座子（RIS1）。结果显示,金鱼草S位点位于染色体着丝粒的周边区,富含转座子和反转座子,和其他两类配子体自交不亲和的位置类似,预示它们的共同起源和具有抑制重组的功能。     

薄片牡蛎的核型及18S-28S核糖体rRNA基因的染色体定位

以薄片牡蛎(Dendostrea folium)成体鳃组织为材料制备有丝分裂中期染色体标本,对其染色体核型进行了分析,并运用荧光原位杂交技术(FISH)将18S-28S核糖体RNA基因定位于中期染色体上。FISH探针是通过PCR扩增介于18S-28S rRNA基因之间的ITS和5.8S rRNA基因序列,并在PCR扩增过程中掺入了Biotin-11-dUTP进行生物素标记。结果显示,薄片牡蛎的单倍染色体数目为n=10,全部为中部着丝粒染色体。与大多数已知巨蛎属牡蛎的染色体核型相似。ITS探针在薄片牡蛎中期分裂体相上产生两簇FISH信号,分别杂交于2号染色体短臂的近端粒区域。本研究首次报道了薄片牡蛎的中期染色体核型以及18S-28S核糖体RNA基因在染色体上的定位。     

甘薯栽培种及其近缘野生种的DAPI核型及rDNA FISH分析

利用DAPI显带和rDNA-FISH技术对栽培种甘薯(‘徐薯18’)(Ipomoea batatas cv.Xushu No.18)及2种不同产地近缘野生种(Ipomoea hederacea Jacq.)进行了细胞遗传学研究。DAPI核型分析表明,‘徐薯18’核型公式为2n=6x=90=72m+18sm(18SAT),随体位于第1、3、6染色体上;美国近缘野生种核型公式为2n=2x=30=30m(4SAT),香港近缘野生种核型公式为2n=2x=30=20m+10sm(4SAT),随体均位于第6、12染色体上。rD-NA-FISH结果显示,栽培种甘薯基因组中含有3对5SrDNA位点,分别位于着丝粒区、亚着丝粒区和染色体端部;美国近缘野生种基因组中含有2对5SrDNA位点,香港近缘野生种基因组中含有1对5SrDNA位点,均位于随体部位;两种不同地域来源的近缘野生种基因组中均含有2对45SrDNA位点,分别位于第6和第12染色体上。     

黄褐棉45S rDNA的FISH定位及核型分析

黄褐棉是棉属5个四倍体棉种之一,利用荧光原位杂交技术将45S rDNA定位在黄褐棉2、4、9号染色体,2号染色体上的45S rDNA特别大,信号位于随体并覆盖了染色体的短臂,比二倍体和四倍体棉种的45SrDNA都要大得多;另外的2对信号很小,形状与陆地棉中的弱信号类似。黄褐棉的核型公式为：2n=4x=52=50m（2SAT）＋2sm,属于2B类型,第2对染色体为亚中着丝粒染色体,其余都为中部着丝粒染色体。黄褐棉的核型、随体数、45S rDNA与其他四倍体棉种区别很大,黄褐棉是一个非常特殊的四倍体棉种。     

Fluorescence in situ hybridization of rDNA, telomeric (TTAGGG)n and (GATA)n repeats in the red abalone Haliotis rufescens (Archaeogastropoda: Haliotidae)

The physical location of 18S-5.8S-28S rDNA, telomeric sequences with (TTAGGG)n DNA probe and (GATA)n microsatellites were performed by fluorescence in situ hybridization in chromosomes of red abalone Haliotis rufescens. The karyotype of red abalone showed a diploid number of 36 (8M+9SM+1ST). FISH performed with rDNA probe, showed the location of major ribosomal clusters in the terminal region of the large arms of two submetacentric pairs (chromosome 4 and 5). Localization of heteromorphisms of FISH-rDNA was found between chromosome homologues and sister chromatids in all metaphases analyzed. This indicates that rDNA clusters are variable within the red abalone genome. The variability in the NOR-bearing reported using silver staining in other gastropods and our result are discussed. In addition, the presence of microsatellite (TTAGGG)n and (GATA)n was demonstrated after FISH treatment by DNA probes. The telomeric sequence occurred at the ends of all mitotic chromosomes, while the (GATA)n repetitive was found on chromosomal interstitial zones as well as at the telomeres in abalone chromosomes.     

Karyotype of maize (Zea mays L.) mitotic metaphase chromosomes as revealed by fluorescencein situ hybridization (FISH) with cytogenetic DNA markers

Here we demonstrate fluorescencein situ hybridization (FISH) of chromosome-specific cytogenetic DNA markers for chromosome identification in maize using repetitive and single copy probes. The fluorescently labeled probes, CentC and pZm4–21, were shown to be reliable cytogenetic markers in the maize inbred line KYS for identification of mitotic metaphase chromosomes. The fluorescent strength of CentC signal, relative position, knob presence, size and location were used for the karyotyping. Based on direct visual analysis of chromosome length and position of FISH signals, a metaphase karyotype was constructed for maize inbred line KYS. All chromosomes could be identified unambiguously. The knob positions in the karyotype agreed well with those derived from traditional cytological analyses except chromosomes 3, 4 and 8. One chromosome with a telomeric knob on the short arm was assigned to 3. A chromosome with a knob in the middle of the long arm was assigned number 4 by simultaneous hybridization with a knob-specific probe pZm4–21 and a chromosome 4-specific probe Cent 4. On chromosome 8, we found an additional small telomeric knob on the short arm. In addition, chromosome-specific probes were employed to identify chromosome 6 (45S rDNA) and chromosome 9 (single-copy probeumc105a cosmid).     

Cytogenetic characterization of the bay scallop, Argopecten irradians irradians, by multiple staining techniques and fluorescence in situ hybridization

The chromosomes of Argopecten irradians irradians were studied by various cytogenetic approaches. Conventional chromosome characterization built on C-banding, DAPI-staining, and silver staining was complemented by the physical mapping of ribosomal DNA and telomeric sequence (TTAGGG)n by FISH. Results showed that the constitutive heterochromatin revealed by C-banding was mainly distributed at telomeric and centromeric regions. However, interstitial C-bands were also observed. The pattern of DAPI banding was almost consistent with that of C-banding. Silver staining revealed that NORs were located on the short arms of chromosome 3 and 10, and this was further confirmed by FISH using 18S-28S rDNA. 5S rDNA was mapped as two distinguishable loci on the long arm of chromosome 11. 18S-28S and 5S rDNA were located on different chromosomes by sequential FISH. FISH also showed that the vertebrate telomeric sequence (TTAGGG)n was located on both ends of each chromosome and no interstitial signals were detected. Sequential 18S-28S rDNA and (TTAGGG)n FISH demonstrated that repeated units of the two multicopy families were closely associated on the same chromosome pair.     

Ribosomal, telomeric and heterochromatin sequences localization in the karyotype of Anemone hortensis

The karyotype of the Mediterranean species Anemone hortensis L. (Ranunculaceae) was characterized with emphasis on heterochromatin distribution and localization of ribosomal (18S−5.8S−26S and 5S rDNA) and telomeric repeats (TTTAGGG). Diploid chromosome complement, 2 n  = 2 x  = 16, common to all investigated populations, consisted of three acrocentric, one meta-submetacentric and four metacentric chromosomes ranging in size from 6.34 to 10.47 µm. Fluorescence in situ hybridization (FISH) with 18S and 5S rDNA probes revealed two 18S−5.8S−26S rDNA loci on a satellite and secondary constriction of acrocentric chromosome pair 2 and terminally on acrocentric chromosome pair 3, and two 5S rDNA loci in the pericentromeric region of meta-submetacentric chromosome pair 4 and in the proximity of the 18S−5.8S−26S rDNA locus on chromosome pair 2. The only GC-rich heterochromatin, as revealed by fluorochrome Chromomycin A3 staining, was that associated with nucleolar organizer regions, whereas AT-rich heterochromatin, stained with 4,6-diamino-2-phenylindole (DAPI), was distributed intercalarly and terminally on the long arm of all three acrocentric chromosomes, and terminally on chromosomes 4 and 5. FISH with Arabidopsis -type telomeric repeats (TTTAGGG) as a probe revealed two classes of signals, small dot-like and large bands, at chromosome termini exclusively, where they corresponded to terminal DAPI-stained heterochromatin. Heteromorphism of chromosome pair 4, which refers to terminal DAPI bands and FISH signals, was observed in populations of Anemone hortensis . Chromosome pairing during meiosis was regular with formation of localized chiasmata proximal to the centromere.  © 2006 The Linnean Society of London, Botanical Journal of the Linnean Society , 2006, 150 , 177–186.     

FISH mapping of 18S-28S and 5S ribosomal DNA, (GATA)n and (TTAGGG)n telomeric repeats in the periwinkle Melarhaphe neritoides (Prosobranchia, Gastropoda, Caenogastropoda)

Spermatocyte chromosomes of Melarhaphe neritoides (Mollusca, Prosobranchia, Caenogastropoda) were studied using fluorescent in situ hybridization (FISH) with four repetitive DNA probes (18S rDNA, 5S rDNA, (TTAGGG)n and (GATA)n). Single-colour FISH consistently mapped one chromosome pair per spread using either 18S or 5S rDNA as probes. The telomeric sequence (TTAGGG)n hybridized with termini of all chromosomes whereas the (GATA)n probe did not label any areas. Simultaneous 18S-5S rDNA and 18S-(TTAGGG)n FISH demonstrated that repeated units of the three multicopy families are closely associated on the same chromosome pair.     

Characterization of Japanese flounder karyotype by chromosome bandings and fluorescence in situ hybridization with DNA markers

The chromosomes of Japanese flounder, Paralichthys olivaceus, were examined by conventional differential staining methods including G-, Q-, C-, silver (Ag)-, fluorochrome, and replication R-bandings and by fluorescence in situ hybridization (FISH) with 5S and 18S rDNAs and telomeric DNA as probes. Replication R-banding substantially made it possible to identify 24 homologous pairs by their RBG-banding pattern and relative length. Both rDNA loci were mapped to chromosome 1, where 5S and 18S rDNA loci were located at the centromeric region and secondary constriction, respectively. C-banding revealed that both rDNA loci were heterochromatic, and 18S rDNA loci were positive for chromomycin A₃ but negative for 4′,6-diamidino-2-phenylindole (DAPI) staining. Telomeric FISH signals were observed at all chromosome ends and at the interstitial region of some chromosomes. The observed results were discussed in relation to the karyotype evolution in the order Pleuronectiformes.     

Mapping of ribosomal DNA and (TTAGGG)n telomeric sequence by FISH in the bivalve Patinopecten yessoensis (Jay, 1857)

The chromosome set of Patinopecten yessoensis (Jay, 1857) wascharacterized using Giemsa staining, DAPI staining and fluorescencein situ hybridization (FISH) with three repetitive DNA probes[18S–28S rDNA, 5S rDNA and telomeric (TTAGGG)_n]. DAPIstaining showed that AT-rich regions were located on the centromereof almost all chromosomes and interstitial banding was not observed.FISH showed that 18S–28S rDNA spread over the short armsof two subtelocentric chromosome pairs and 5S rDNA was locatedon the long arm of one subtelocentric chromosome pair. SequentialFISH demonstrated that 18S–28S and 5S rDNA were locatedon different chromosomes. FISH also showed that the vertebratetelomeric sequence (TTAGGG)_n was located on both ends of eachchromosome and no interstitial signals were detected. Sequential18S–28S rDNA and (TTAGGG)_n FISH indicated that repeatedunits of the two multicopy families were closely associatedon the same chromosome pair. (Received 4 January 2007; accepted 1 September 2007)     

Karyotype of mitotic metaphase and meiotic diakinesis in non-heading Chinese cabbage

Non-heading Chinese cabbage [Brassica rapa L. ssp. chinensis (L.) Hanelt] is one of the most popular leafy vegetables. Despite the economic importance of non-heading Chinese cabbage, little attention has been given to its cytogenetic profile. This study reveals the karyotype of non-heading Chinese cabbage. Fluorescence in situ hybridization (FISH) with 45S and 5S rDNA probes was performed on mitotic metaphase complementary regions. We located 45S rDNA on the centromeric or adjacent region of chromosomes A1 and A2, with the largest on the satellite of chromosome A5. Meanwhile, 5S rDNA co-localized with 45S rDNA on chromosomes A2 and A5, and on the telomeric region of chromosome A10. We performed DAPI fluorescence banding on the same metaphase chromosomes to identify homologous chromosomes. The DAPI fluorescence pattern was observed mainly on the centromeric heterochromatin regions of each chromosome. However, the lengths of chromosomes A2 and A6 were completely stained, except for their telomeric regions. Meiotic diakinesis chromosomes as new substrates in FISH-developed karyotype were revealed for the first time. The karyotype of non-heading Chinese cabbage reveals that it contains eight submetacentric chromosomes, one subtelocentric chromosome (bearing satellite), and one telocentric chromosome. Diakinetic chromosome pairing can overcome the difficulty of unlabeled chromosome identification. This study provided valuable information for cytogenetic research and molecular breeding of non-heading Chinese cabbage by using the combination of FISH and DAPI fluorescence patterns on mitotic and meiotic chromosomes.     

rDNA (18S-28S and 5S) colocalization and linkage between ribosomal genes and (TTAGGG)(n) telomeric sequence in the earthworm,Octodrilus complanatus (Annelida: Oligochaeta: Lumbricidae), revealed by single- and double-color FISH

Spermatogonial and metaphase I chromosomes of the lumbricid earthworm Octodrilus complanatus (Annelida: Oligochaeta) were examined using fluorescent in situ hybridization (FISH) with three repetitive DNA probes-5S rDNA, 18S-26S rDNA, and (TTAGGG)(n). Single-color FISH consistently mapped one chromosome pair per spread using either 5S rDNA or 18S-26S rDNA as probes. Simultaneous (18S-26S)-5S and (18S-26S)-(TTAGGG)(n) FISH demonstrated that repeated units of the two ribosomal families were overlapped and closely associated with telomeric sequences.     

Karyotype analysis of a Korean cucumber cultivar (Cucumis sativus L. cv. Winter Long) using C-banding and bicolor fluorescence in situ hybridization

An intensive karyotype analysis of a Korean cucumber cultivar (Cucumis sativus L. cv. Winter Long) was carried out with three different methods. These included Feulgen staining, Giemsa C-banding, and fluorescence in situ hybridization (FISH). The mitotic chromosomes of the cucumber (2n = 2x = 14) were characterized, based on the length and arm ratio values. A C-banding analysis showed dark stains on the centromeric, telomeric, and intercalary regions of the chromosomes, except that chromosome 2 had a heavy staining in the long arm. Bicolor FISH, using 45S and 5S rDNA probes, provided additional information to identify cucumber chromosomes. The signals for 45S rDNA were detected on the pericentromeric regions of chromosomes 1, 2, and 4. The signals for 5S rDNA were on the short arm of chromosome 5. Similar band patterns (as the C-banding) were observed when the chromosomes were counter-stained with 4',6-diamidino-2-phenyoindole (DAPI). The data implied that the karyotype of the Korean cucumber cultivar is peculiar and different from previous reports.