DNA分子原位杂交在植物分子细胞遗传学研究中的应用

DNA分子原位杂交（in situ hybridiation）是植物分子细胞遗传学研究的重要工具。本文简要回顾了DNA分子原位杂交的起源和发展,详细综述了基因组原位杂交（genomic in situ hybridization, GISH）在植物细胞遗传学研究中的应用以及荧光原位杂交（Fluorescence in situ hybridization, FISH）在物理作图和染色体识别中的应用。文中还介绍了Fiber-FISH、BAC-FISH以及Immuno-FISH等新兴技术。最后对FISH技术进行了展望。     

对基因组原位杂交信号释译可能出现的片面性——来自一个小麦易位系(A-3)中外源遗传物质鉴定的启示

利用基因组原位杂交(GISH)和种子醇溶蛋白电泳(A-PAGE)技术对一个可能携带有10倍体长穗偃麦草(Thinopyrum ponticum (Host) Liu & Wang)遗传物质的小麦新种质A-3进行了综合鉴定. 用普通小麦"中国春"(Triticum aesticum L. cv. Chinese Spring)基因组(ABD)DNA作探针, 拟鹅观草[Pseudoroegneria stipifolia(Czern e     

基因组原位杂交技术在植物研究中的应用

基因组原位杂交是以亲本之一的总基因组DNA做探针,另一亲本的基因组DNA做封阻,在荧光原位杂交技术的基础上发展起来的一种染色体/染色质检测技术。在其发展的十几年里,已在植物的基因组研究中发挥了重要的作用。应用这一技术可对多倍体中基因组之间的亲缘关系、基因组组成及起源进行研究;对杂交种中染色体组的组成进行分析;对代换系、附加系和易位系进行有效的鉴定,并对其中的外源染色体或染色体片段的来源、大小、数目及发生位点进行检测和定位。此外,利用基因组原位杂交技术还有助于确定物种间的同源性;研究杂交种中来源不同的染色质在核中的分布;探索B染色体的起源、染色体间的配对、重组、交换等现象。随着基因组荧光原位杂交技术体系的不断发展、完善和改进,其应用范围不断拓展,在植物基因组研究领域中发挥了越来越重要的作用。     

Inheritance of traits associated with seed size in groundnut (<Emphasis Type="Italic">Arachis hypogaea</Emphasis> L.)

A new wheat-Thinopyrum substitution line AS1677, developed from a cross between wheat line ML-13 and wheat-Thinopyrum intermedium ssp. trichophorum partial amphiploid TE-3, was characterized by fluorescence in situ hybridization (FISH), sequential Giemsa-C banding, genomic in situ hybridization (GISH), seed storage protein electrophoresis, molecular marker analysis and disease resistance screening. Sequential Giemsa-C banding and GISH using Pseudoroegneria spicata genomic DNA as probe indicated that a pair of St-chromosomes with strong terminal bands were introduced into AS1677. FISH using pTa71 as a probe gave strong hybridization signals at the nuclear organization region and in the distal region of the short arms of the St chromosome. Moreover, FISH using the repetitive sequence pAs1 revealed that a pair of wheat 1D chromosomes was absent in accession AS1677. Seed storage proteins separated by acid polyacrylamide gel electrophoresis (APAGE) and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) confirmed that AS1677 lacked the gliadin and glutenin bands encoded by Gli-D1 and Glu-D1, further confirming the absence of chromosome 1D. The introduced St chromosome pair belonging to homoeologous group 1 was identified by newly produced genome specific markers. AS1677 is a new 1St (1D) substitution line. When inoculated with stripe rust and powdery mildew isolates, AS1677 expressed stripe rust resistance possibly derived from its Thinopyrum parent. AS1677 can be used as a donor source for introducing novel disease resistance genes to wheat in breeding programs aided by molecular and cytogenetic markers.     

Fluorescent in situ hybridization — a useful aid to the introduction of alien genetic variation into wheat

Summary Fluorescent in situ hybridization (FISH) of DNA to plant chromosomes has proved to be a powerful cytogenetic tool. The value of fluorescent in situ hybridization of total genomic DNA (GISH) of related species is demonstrated in the determination of wheat/alien chromosome pairing in hybrids. Its use for assessing the relative merits of the various genes that affect chromosome pairing is also shown.The ability of GISH to identify the presence in wheat of whole alien chromosomes or alien chromosome segments is illustrated. The potential of FISH for detecting repeated DNA sequences, low copy sequences and single copy genes is discussed.Abbreviations FISH fluorescent in situ hybridization - GISH genomic in situ hybridization - PRINS primer-induced in situ hybridization     

Analysis of introgression of the Tulipa fosteriana genome into Tulipa gesneriana using GISH and FISH

Southern hybridization and genomic in situ hybridization (GISH) have demonstrated that ‘Purissima’ (2n = 2x = 24) is an interspecific hybrid comprised of one genome of Tulipa (T.) gesneriana and one genome of T. fosteriana. Backcrossing T. gesneriana with ‘Purissima’ was partially successful. Simultaneous GISH and fluorescence in situ hybridization (FISH) distinguished chromosomes from both parent genomes, as well as recombinant chromosomes, in interspecific hybrids and their progeny. Chromosome recombination was observed in all cultivars except ‘Purissima’ and ‘Kouki’ (2n = 3x = 36). ‘Kouki’ (2n = 3x = 36) had two genomes of the T. gesneriana and a single genome of the T. fosteriana. The number of nonrecombinant T. fosteriana chromosomes in ‘Judith Leyster’ (2n = 4x = 48) and ‘Purissima’ progeny varied from two in ‘Hatsuzakura’ to six in ‘Kikomachi’ and ‘Momotaro’. The number and type of recombinant chromosomes also differed among cultivars. The total number of translocations ranged from one in ‘Kikomachi’ to six in ‘Hatsuzakura’. Each was a combination of a single T. fosteriana fragment and a single T. gesneriana fragment, indicating that they resulted from a single crossover event. Sequential GISH and FISH analysis with rDNA probes yielded chromosome-specific markers that were used to identify most of the chromosomes in ‘Purissima’ progeny. This is the first report of introgression of T. fosteriana chromatin into the T. gesneriana genome.     

棉花Oligo-FISH技术建立及其初步应用

【目的】荧光原位杂交技术可以实现DNA序列直观准确的染色体定位,是基因组深入研究的重要技术之一。染色体特异探针的获得是该技术应用的关键。本研究旨在建立棉花寡核苷酸荧光原位杂交技术。【方法】利用已经公布棉花基因组序列数据,采用生物信息学方法获得染色体特异的寡核苷酸库,随后用乳化聚合酶链式反应方法标记成荧光探针,在棉花有丝分裂中期染色体上进行原位杂交。【结果】建立了一套棉花寡核苷酸荧光原位杂交技术体系。【结论】该体系可用于棉花单染色体识别鉴定。     

Investigation on Evolutionary Relationships of the Subgenomes in Interspecific Triploid Cotton via Meiotic FISH

We report in this paper primary studies on interspecific species of cotton vis GISH (genomic in situ hybridization).We use interspecific triploid hybrids (F1 from hybridization of allotetraploid cultivated species with diploid A,D,or C genome species) and two cultivated tetraploids to study the chromosome paring during meiosis of pollen mother cellls (PMCs) and to estimate the consequences on synapsis between these three subgenomes after synthetic polyploid formation.     

Molecular cytogenetic characterization and SSR marker analysis of a leaf rust resistant wheat line carrying a 6G(6B) substitution from <Emphasis Type="Italic">Triticum timopheevii</Emphasis> (Zhuk.)

A disease (powdery mildew, leaf rust) resistant line was selected from the progenies of a Triticum aestivum × Triticum timopheevii amphiploid produced at Martonvásár. This line was previously identified with C-banding as a 6G(6B) substitution. In order to detect the 6G chromosome in a wheat background, fluorescence in situ hybridization (FISH) and microsatellite marker analysis were used. Ten microsatellite markers of the 43 tested generated PCR products that were polymorphic between chromosomes 6B and 6G, and four showed length-polymorphism. The FISH hybridization pattern of 6G from T. timopheevii was identified using a combination of four repetitive DNA probes (Afa-family, pSc119.2, pTa71, (GAA)₇). Genomic in situ hybridization (GISH) technique, capable of labelling the A^t and G genomes separately, was used on the same slides to differentiate the A^t and G genomes in T. timopheevii. The A^t and G genomes of T. timopheevii were grouped on the basis of the GISH patterns and a cyclic intergenomic translocation involving 6A^t-1G-4G was detected in T. timopheevii accession TRI667. The presence of 6G in the substitution line was demonstrated using FISH with the four repetitive DNA probes. Chromosome 6G was clearly identified and its FISH pattern was different from that of 6B in the parental wheat cultivar Fleischmann-481. According to field tests, the 6G(6B) substitution line has resistance to leaf rust.     

Fluorescent in situ hybridization as an aid to introducing alien genetic variation into wheat

Summary Fluorescent in situ hybridization (FISH) has been used to assess the occurrence and frequency of wheat-alien chromosome pairing in a wheat/Thinopyrum bessarabicum hybrid and in wheat/rye hybrids with different levels of chromosome pairing by examining pollen mother cells at metaphase I of meiosis. The use of FISH to identify the presence and size of alien chromatin in a wheat background is also demonstrated.The value of FISH as an aid to the introgression of alien genetic variation into wheat is discussed.Abbreviations FISH fluorescent in situ hybridization - GISH genomic in situ hybridization - PRINS primer-induced in situ hybridization     

rDNA和端粒重复序列鉴定马铃薯和茄子体细胞杂种染色体丢失和融合

植物体细胞杂交是植物种质资源创制的重要方法。体细胞杂种在原生质体再生的过程中染色体会产生非常多的遗传变异。研究体细胞杂种的染色体行为为马铃薯体细胞杂种的创制和利用提供理论基础。本研究采用rDNA和端粒重复序列作为探针进行原位杂交(fluorescence in situ hybridization),并结合基因组原位杂交(genomic in situ hybridization),对马铃薯和茄子体细胞杂种染色体组成和变异进行了分析。原位杂交结果表明,体细胞杂种中存在马铃薯和茄子融合的染色体和双着丝粒染色体,并发现部分融合染色体是由马铃薯和茄子2号染色体末端对末端融合得到的。重排的双着丝粒染色体的着丝粒一个来源于马铃薯,一个来源于茄子。此外,体细胞杂种中来源于茄子的5S rDNA在体细胞杂种再生及稳定的过程中全部丢失。研究结果表明马铃薯与茄子在进行体细胞杂交的过程中,染色体是不稳定的,容易造成融合后代出现双着丝粒和染色体重排等现象。体细胞杂种的染色体会通过染色体重排、双着丝粒、rDNA均一化等多种形式使其染色体趋于稳定。     

Protocols for Pachytene Chromosome and DNA Fiber FISH in Cotton

Fluorescence in situ hybridization (FISH) has become the most important technique in plant molecular cytogenetics research.FISH-based physical mapping provides a valuable complementary approach in genome sequeneing to measure the physical distances between adjacent BAC contigs and delineating the structure and DNA composition of genomic regions of centromere and telomere.The accuracy and precision of the FISH-base physical maps depend on the resolution power of FISH.     

Genome composition of ‘Elatior’-begonias hybrids analyzed by genomic in situ hybridisation

Interspecific hybridization of various tuberous Begonia species hybrids with Begonia socotrana results in so-called ‘Elatior’-begonias hybrids (B. × hiemalis Fotsch). In our study, genomic in situ hybridization (GISH) has been employed to assess the genome composition in eleven ‘Elatior’-begonias hybrids and their ancestor genotypes. Genomic DNA of tuberous Begonia was sonicated to 1–10-kb fragments, labelled by nick translation with digoxigenin-11-dUTP and used as a probe whereas B. socotrana DNA was autoclaved to 100 bp fragments and used as block. The genome of tuberous Begonia was clearly pronounced in ‘Elatior’-begonias when the probe concentration was ~3.75 ng/μl (150 ng/slide), with 30 times the excess of B. socotrana blocking DNA and stringency of post hybridization washings at 73% (0.1× SSC at 42°C). In ‘Elatior’-begonias hybrids GISH distinguished two groups comprising short (0.6–1.03 μm in length) and relatively longer chromosomes (1.87–3.88 μm) which represent B. socotrana and tuberous Begonia genomes, respectively. The number of chromosomes derived from tuberous Begonia ranged from 14 to 56 and for B. socotrana from 7 to 28 which suggest the presence of different ploidy levels in analyzed ‘Elatior’-begonia hybrids. Intergenomic recombination has not been detected through GISH in hybrids analyzed. Genomic in situ hybridization turned out to be useful to identify the genome constitution of ‘Elatior’-begonia hybrids and thus gain an insight into the origins of these cultivars. This knowledge on the ploidy level and genome composition is essential for further progress in breeding Begonias.     

The genus Dasypyrum—part 2. Dasypyrum villosum—a wild species used in wheat improvement

Dasypyrum villosum (L.) P. Candargy is discussed as a species commonly used in wheat improvement. Chromosomal localization of the potentially useful traits and chromosomal position of some morphological and isozyme markes are shown. The investigations using molecular RAPD, AFLP, SSR, RFLP markers and in situ (GISH, FISH) hybridization experiments on D. villosum itself and in wide hybrids with Triticum are summarized. The article also presents the information about designation of D. villosum chromosomes and the current knowledge on the phenomenon of two-coloured D. villosum caryopses.     

原位杂交技术及其在甘蔗研究中的应用

本文介绍了植物染色体原位杂交技术的产生、发展过程,以及该技术与其它生物学技术相结合而形成的一些新技术,如：细菌人工染色体荧光原位杂交、DNA纤维荧光原位杂交、基因组原位杂交、原位PCR技术等。综述了这些技术在甘蔗研究中的应用情况。     

硬粒小麦-长穗偃麦草附加系、代换系和易位系的创制

通过小麦与长穗偃麦草远缘杂交创制附加系、代换系及易位系是小麦遗传改良中利用长穗偃麦草优良基因的重要途径。本研究将长穗偃麦草特异分子标记、染色体计数、基因组原位杂交(GISH)及非变性原位杂交(ND-FISH)等多种方法相结合,对硬粒小麦Langdon(AABB)与小偃麦8801(AABBEE)的杂交后代群体进行分子细胞学鉴定,创制出硬粒小麦-长穗偃麦草3E、6E染色体双体附加系Du-DA3E和Du-DA6E,硬粒小麦-长穗偃麦草1E(1B)染色体双体代换系Du-DS1E(1B)以及硬粒小麦-长穗偃麦草1AS-1EL染色体易位系Du-T1AS·1EL。创制的4个种质中长穗偃麦草染色体均能稳定遗传,这不仅增加了硬粒小麦-长穗偃麦草附加系和代换系的类型,还为后续利用长穂偃麦草优良基因改良小麦提供了特殊种质资源。     

棉花gDNA体细胞染色体FISH技术

介绍了棉花基因组ＤＮＡ（ｇｅｎｏｍｅＤＮＡ,简为ｇＤＮＡ）体细胞染色体荧光原位杂交〔ＦＩＳＨ〕的技术流程,并着重分析和讨论了影响试验结果的关键因素,包括染色体和探针的变性条件、染色体的蛋白酶Ｋ处理技巧等。试验中作为靶ＤＮＡ的体细胞染色体采用棉属异源四倍体种海岛棉;探针和封阻均采用ｇＤＮＡ,材料是棉属二倍体种Ａ染色体组（Ａｇｅｎｏｍｅ）的棉种（亚洲棉和草棉）和Ｄ染色体组（Ｄｇｅｎｏｍｅ）的棉种（瑟伯氏棉、雷蒙德氏棉、戴维逊氏棉等）,分别交互使用。试验结果比较理想,获得良好的ＦＩＳＨ片子,而且重复性好。     

Ability of chromosome 4H to compensate for 4D in response to drought stress in a newly developed and identified wheat–barley 4H(4D) disomic substitution line

A spontaneously developed wheat–barley 4H(4D) disomic substitution line was identified cytogenetically using genomic in situ hybridization (GISH), multicolour fluorescent in situ hybridization (FISH) and microsatellite markers. The ability of the barley 4H chromosome to compensate for wheat 4D in response to mild drought stress was also investigated. In the barley cv. 'Betzes' and the 4H(4D) substitution line, mild osmotic stress induced intensive stomatal closure, resulting in reduced water loss through transpiration and unchanged relative water content in the leaves. As the CO₂ assimilation rate remained relatively high, the water use efficiency, which is an important factor associated with drought tolerance, increased extensively under mild osmotic stress in these lines. In the case of the parental wheat genotypes, however, mild drought stress induced less intense stomatal closure and a greater decrease in the CO₂ assimilation rate than in barley or in the substitution line, resulting in unaugmented or reduced water use efficiency. The results demonstrate that genes localised on the 4H chromosome of barley were able to increase the water use efficiency of the wheat substitution line, which is suitable for improving wheat drought tolerance through intergeneric crossing.     

Molecular cytogenetic characterization of wheat–<Emphasis Type="Italic">Secale africanum</Emphasis> amphiploids and derived introgression lines with stripe rust resistance

Two amphiploids, AF-1(Triticum aestivum L. cv. Anyuepaideng–Secale africanum Stapf.) and BF-1 (T. turgidum ssp. carthlicum–S. africanum), were evaluated by chromosomal banding and in situ hybridization. The individual S. africanum chromosomes were identified in the BF-1 background by sequential C-banding and genomic in situ hybridization (GISH), and were distinguishable from those of S. cereale, because they exhibited less terminal heterochromatin. Fluorescence in situ hybridization (FISH) using the tandem repeat pSc250 as a probe indicated that only 6R^a of S. africanum contained a significant hybrid signal, whereas S. cereale displayed strong hybridization at the telomeres or subtelomeres in all seven pairs of chromosomes. Extensive wheat–S. africanum non-Robertsonian translocations were observed in both AF-1 and BF-1 plants, suggesting a frequent occurrence of chromosomal recombination between wheat and S. africanum. Moreover, introgression lines selected from the progeny of wheat/AF-1 crosses were resistant when field tested with widely virulent strains of Puccinia striiformis f. sp. tritici. Three highly resistant lines were selected. GISH and C-banding revealed that resistant line L9-15 carried a pair of 1BL.1RS translocated chromosomes. This new type of S. africanum derived wheat–Secale translocation line with resistance to Yr9-virulent strains will broaden the genetic diversity of 1BL.1RS for wheat breeding.     

甘薯近缘野生种Ipomoea trifida(4x) GISH分析

以甘薯近缘野生种I. trifida (2x)为探针, 与I. trifida (4x) 2个株系“695104”和“697288”的体细胞染色体进行基因组荧光原位杂交, 结果显示, 2株系都与I. trifida (2x)有很近的亲缘关系, 但2株系的信号存在差异。“695104”几乎所有染色体整条都有均匀明亮的信号, 应为I. trifida (2x)基因组直接加倍而来;而 “697288”与“695104”不同, 虽然各条染色体也均有杂交信号, 但信号的区域与亮度有差异, 较为复杂, 可分为三种情况。第1种是整条染色体有均匀明亮的信号, 亮度与分布区域同“695104” , 有41条;第2种是几乎整条染色体有信号, 但亮度较第一种暗, 有14条;第3种为染色体部分区域有信号, 亮度较前二者更暗, 有5条。推测 “697288”是在加倍同时或之后又发生了基因组重组与部分变异。