程序性细胞死亡抑制基因Dad-1在水稻和玉米中的FISH定位(英文)

Dad-1是一种在动物和植物中都非常保守的细胞程序性死亡 (PCD) 抑制基因。作者利用 FISH (荧光原位杂交)首次把单拷贝水稻Dad-1基因物理定位在水稻第2号染色体短臂的端部(Fig.2 A,B&C)。我们还分析了它在玉米基因组中的同源序列。Southern 杂交结果显示在玉米基因组中确实存在水稻Dad-1 的同源序列(Fig.1)。FISH进一步展示了三个杂交信号分别在玉米4、5号染色体长臂和9号染色体短臂上(Fig.2 D,E&F),其信号距着丝粒的百分距离(FL值)分别为 91、98和96。其杂交位点的位置与水稻Dad-1所处的相对位置是相似的,它们都处于染色体臂的端部。这表明在一定的程度上,Dad-1基因不仅在序列同源性上而且在所处的染色体位置上具有保守性。 水稻Dad-1基因在水稻中的杂交信号检出率 (38%) 高于玉米中的。这表明与玉米相比,水稻Dad-1 基因的编码序列更容易与水稻染色体杂交;它与玉米中的相应序列可能只是部分同源。     

Cloning and characterization of maize B chromosome sequences derived from microdissection

Isolation of sequences from the maize B chromosome is always hampered by its high homology with the normal complements. In this study, this handicap was overcome by cloning the sequences from the pachytene B chromosomes dissected out of a slide by a micromanipulator followed by degenerate oligonucleotide-primed PCR. The isolated sequences were found to hybridize with genomic DNA in a B-dosage-dependent manner and with the pachytene B chromosome by fluorescence in situ hybridization (FISH), corroborating their B origin. A total of 19 B sequences were isolated, all of which are repetitive and, with one exception, are homologous to the A chromosome(s). Three sequences have strong homology to maize sequences that include two knob repeats and one zein gene (noncoding region), and 10 others are homologous to the noncoding region of Adh1, Bz1, Gag, Zein, and B centromere to a lesser degree. Six sequences have no homology to any gene. In addition to FISH, the B-specific sequence and a partially B-specific one were also mapped, by seven newly characterized TB-10L translocations, to a similar location on the central portion of the distal heterochromatic region, spreading over a region of about one-third of the B chromosome.     

A new gypsy-type retrotransposon, RIRE7: preferential insertion into the tandem repeat sequence TrsD in pericentromeric heterochromatin regions of rice chromosomes

A portion of an insertion sequence present in a member of the RIRE3 family of retrotransposons in Oryza sativa L. cv. IR36 was found to have an LTR sequence followed by a PBS sequence complementary to the 3'-end region of tRNAMet, indicative of another rice retrotransposon (named RIRE7). Cloning and sequencing of PCR-amplified fragments that made up all parts of the RIRE7 sequence showed that RIRE7 is a gypsy-type retrotransposon with partial homology in the pol region to the rice gypsy-type retrotransposons RIRE2 and RIRE3 identified in rice previously. Interestingly, various portions of the RIRE7 sequence were homologous to several DNA segments present in the centromere regions of cereal chromosomes. Further cloning and nucleotide sequencing of fragments flanking RIRE7 copies showed that RIRE7 was inserted into a site within a tandem repeat sequence that has a unit length of 155 bp. The tandem repeat sequence, named TrsD, was homologous to tandem repeat sequences RCS2 and CentC, previously identified in the centromeric regions of rice and maize chromosomes. Fluorescence in situ hybridization (FISH) analysis of the metaphase chromosomes of O. sativa cv. Nipponbare showed that both RIRE7 and TrsD sequences were present in the centromere regions of the chromosomes. The presence of RIRE7 and the TrsD sequences in the centromere regions of several chromosomes was confirmed by the identification of several YAC clones whose chromosomal locations are known. Further FISH analysis of rice pachytene chromosomes showed that the TrsD sequences were located in a pericentromeric heterochromatin region. These findings strongly suggest that RIRE7 and TrsD are components of the pericentromeric heterochromatin of rice chromosomes.     

水稻Pib基因及其连锁RFLP标记在栽培稻、药用野生稻和玉米中的比较物理定位

比较遗传学研究表明 ,禾本科不同基因组之间存在着广泛的同线性和共线性。对水稻 (OryzasativaL .)这一模式植物与其他禾本科植物的原位杂交定位可以揭示禾本科植物基因组的共同特点和进化规律 ,为建立禾本科遗传大体系积累资料。实验以图位克隆法分离的水稻Pib基因 (10 .3kb)和与之连锁的RFLP标记为探针 ,研究了Pib及与其连锁的RFLP标记在供试种中的同源性和物理位置。Southern杂交结果表明 ,Pib在玉米 (ZeamaysL .)基因组中有同源序列。进一步利用单色和双色荧光原位杂交技术确定了Pib在栽培稻 (O .sativassp .indicacv .Guangluai4)、玉米和药用野生稻 (O .officinalisWallexWatt)染色体上的物理位置。定位结果表明 ,Pib基因和与之连锁的RFLP标记在这 3个供试种基因组中具有同线性。     

Physical localization of single-copy sequences on pachytene chromosomes in maize (Zea mays L.) by chromosome in situ suppression hybridization.

A new approach for locating single-copy DNA sequences on pachytene chromosomes of maize (Zea mays L.) was developed. A cosmid clone with homologous sequences to a molecular marker (umc105a) linked to a quantitative trait locus (QTL) for resistance against sugarcane borer (SCB) was physically mapped by fluorescence in situ hybridization (FISH) to the short arm of chromosome 9. The marker umc105a was genetically placed in the centromeric region. To suppress signals generated by maize repetitive DNA, competitive in situ suppression (CISS) hybridization was necessary to obtain specific signals from umc105a. A centromere specific DNA probe (CentC) was used in a double-labeling technique as a reference marker. Fluorescence signals generated by umc105a cosmid and CentC were specific and highly reproducible. Thus the single-copy DNA sequence of umc105a was physically localized on the short arm of chromosome 9 near the telomere. This is the first report of physical localization of single-copy DNA sequence by CISS hybridization to a maize pachytene chromosome.     

水稻Pib基因及其连锁RFLP标记在栽培稻、药用野生稻和玉米中的比较物理定位

比较遗传学研究表明,禾本科不同基因组之间存在着广泛的同线性和共线性.对水稻(Oryza sativa L.)这一模式植物与其他禾本科植物的原位杂交定位可以揭示禾本科植物基因组的共同特点和进化规律,为建立禾本科遗传大体系积累资料.实验以图位克隆法分离的水稻Pib 基因(10.3 kb)和与之连锁的RFLP标记为探针, 研究了Pib及与其连锁的RFLP标记在供试种中的同源性和物理位置. Southern杂交结果表明,Pib在玉米(Zea mays L.)基因组中有同源序列.进一步利用单色和双色荧光原位杂交技术确定了Pib在栽培稻(O.sativa ssp. indica cv. Guangluai 4)、玉米和药用野生稻(O. officinalis Wall ex Watt)染色体上的物理位置.定位结果表明,Pib基因和与之连锁的RFLP标记在这3个供试种基因组中具有同线性.     

Single-gene detection and karyotyping using small-target fluorescence in situ hybridization on maize somatic chromosomes

Combined with a system for identifying each of the chromosomes in a genome, visualizing the location of individual genetic loci by fluorescence in situ hybridization (FISH) would aid in assembling physical and genetic maps. Previously, large genomic clones have been successfully used as FISH probes onto somatic chromosomes but this approach is complicated in species with abundant repetitive elements. In this study, repeat-free portions of sequences that were anchored to particular chromosomes including genes, gene clusters, large cDNAs, and portions of BACs obtained from public databases were used to label the corresponding physical location using FISH. A collection of probes that includes at least one marker on each chromosome in the maize complement was assembled, allowing a small-target karyotyping system to be developed. This set provides the foundation onto which additional loci could be added to strengthen further the ability to perform chromosomal identification in maize and its relatives. The probes were demonstrated to produce signals in several wild relatives of maize, including Zea luxurians, Z. diploperennis, and Tripsacum dactyloides.     

High-resolution single-copy gene fluorescence in situ hybridization and its use in the construction of a cytogenetic map of maize chromosome 9

High-resolution cytogenetic maps provide important biological information on genome organization and function, as they correlate genetic distance with cytological structures, and are an invaluable complement to physical sequence data. The most direct way to generate a cytogenetic map is to localize genetically mapped genes onto chromosomes by fluorescence in situ hybridization (FISH). Detection of single-copy genes on plant chromosomes has been difficult. In this study, we developed a squash FISH procedure allowing successful detection of single-copy genes on maize (Zea mays) pachytene chromosomes. Using this method, the shortest probe that can be detected is 3.1 kb, and two sequences separated by approximately 100 kb can be resolved. To show the robust nature of this protocol, we localized nine genetically mapped single-copy genes on chromosome 9 in one FISH experiment. Integration of existing information from genetic maps and the BAC contig-based physical map with the cytological structure of chromosome 9 provides a comprehensive cross-referenced cytogenetic map and shows the dramatic reduction of recombination in the pericentromeric heterochromatic region. To establish a feasible mapping system for maize, we also developed a probe cocktail for unambiguous identification of the 10 maize pachytene chromosomes. These results provide a starting point toward constructing a high-resolution integrated cytogenetic map of maize.     

玉米有丝分裂染色体上玉米BAC探针的FISH杂交体系的构建

本研究分别探讨了玉米和水稻基因组c 0t DNA对探针的封阻、杂交后洗脱的严谨度、杂交液中FAD的浓度变化对BAC-FISH杂交的影响;探讨了玉米BAC探针中重复序列含量对FISH信号的影响.初步形成了一套以玉米BAC探针在玉米有丝分裂染色体上进行FISH杂交的优化技术体系.结果表明,玉米基因组c 0t DNA对探针封阻的c 0t值应小于50;而降低杂交液中FAD浓度和适度控制杂交后洗脱的严谨度,尤其是使用水稻基因组的c 0t 100 DNA封阻探针重复序列对BAC-FISH杂交信号特异性的改善具有明显的效果;同时,验证了选择重复序列含量较少的玉米BAC作为FISH杂交的探针也是获得特异性杂交信号的重要条件.     

Bph3在栽培稻和药用野生稻中的BAC-FISH比较物理定位

用栽培稻(Oryza sativa L.)遗传图第四连锁群中与抗褐稻虱基因Bph3紧密连锁的RFLP标记RZ69及筛选出来的BAC克隆38J9作探针,对药用野生稻(O.officinalis Well ex Watt)和栽培稻荧光原位杂交,供试标记RZ69及38J9均被定位于药用野生稻和栽培稻第4染色体的短臂上,药用野生稻杂交信号的百分距分别为22.12±3.44和20.00±5.40,而栽培稻均为0.在栽培稻中,信号检出率相应地为6.29%和56.10%,在药用野生稻中则为6.14%和50.00%.BAC克隆和RFLP标记探针杂交信号的百分距十分接近,说明在栽培稻和野生稻中RFLP标记RZ69都在同一BAC克隆的大插入片段中.由此推知,药用野生稻与抗性基因Bph3的同源顺序就在第4染色体信号出现的相应位置.在未封阻的情况下,药用野生稻的BAC杂交在多条染色体上具有信号,这表明它和栽培稻的Cot-1 DNA重复顺序也在一定程度上具有同源性.药用野生稻第4染色体是根据栽培稻与药用野生稻的比较遗传图选用与Gm-6连锁的RG214通过FISH确定的.讨论了栽培稻BAC克隆对药用野生稻比较原位杂交物理作图的可行性问题.     

The pachytene chromosomes of maize as revealed by fluorescence in situ hybridization with repetitive DNA sequences

A repetitive DNA sequence, ZmCR2.6c, was isolated from maize based on centromeric sequence CCS1 of the wild grass Brachypodium sylvaticum. ZmCR2.6c is 309 bp in length and shares 65% homology to bases 421–721 of the sorghum centromeric sequence pSau3A9. Fluorescence in situ hybridization (FISH) localized ZmCR2.6c to the primary constrictions of pachytene bivalents and to the stretched regions of MI/AI chromosomes, indicating that ZmCR2.6c is an important part of the centromere. Based on measurements of chromosome lengths and the positions of FISH signals of several cells, a pachytene karyotype was constructed for maize inbred line KYS. The karyotype agrees well with those derived from traditional analyses. Four classes of tandemly repeated sequences were mapped to the karyotype by FISH. Repeats 180 bp long are present in cytologically detectable knobs on 5L, 6S, 6L, 7L, and 9S, as well as at the termini and in the interstitial regions of many chromosomes not reported previously. A most interesting finding is the presence of 180-bp repeats in the NOR-secondary constriction. TR-1 elements co-exist with 180-bp repeats in the knob on 6S and form alone a small cluster in 4L. 26S and 5S rRNA genes are located in the NOR and at 2L.88, respectively. The combination of chromosome length, centromere position, and distribution of the tandem repeats allows all chromosomes to be identified unambiguously. The results presented form an important basis for using FISH for physical mapping and for investigating genome organization in maize. Received: 29 June 1999 / Accepted: 10 November 1999     

玉米和类玉米种间杂交后代细胞学鉴定

利用组成玉米异染色质钮的180-bp重复序列和TR-1元件以及45S rDNA对玉米自交系F107、GB57、二倍体多年生类玉米及其远缘杂交后代的染色体进行荧光原位杂交,确定了3种重复序列在亲本染色体上的分布;同时对远缘杂交后代进行了细胞学鉴定,通过荧光信号在染色体上的位置,证实远缘杂交后代中异源种质的染色体来源;讨论了异染色质钮重复序列对玉米和其野生种杂交后代外源染色体整合和染色体行为等方面研究的应用。     

Chromosome Location of Two RFLP Markers umc22 and umc122 Tightly Linked to the Ht1 Gene in Maize

A biotin-labeled in situ hybridization technique was used in order to physically map two RFLP markers-umc22 and umc122-tightly linked to the Htl gene on the chromosomes of maize ( Zea mays L. ). The results showed that both markers located on the chromosomes 2,7 and umc22 also hybridized with chromosome 4, which demonstrated that the two markers were a duplicated or triplicated sequence. The average detection rate of in situ hybridization was 17.46%. The percent distances of umc22 and umc122 from centromere on the chromosome 2 were 58.36 ± 3.19 and 61.02 ± 4.32 respectively, and on the chromosome 7 were 44.70 ± 2.11 and 45.19 ± 2.27 respectively, which indicates that there are no differences between genetic and physical distances of two markers umc22 and umc122. It was deduced that the gene Htl should also have its homeologous sequence between the hybridization sites of umc22 and umc122 on 7 L besides its location between the two hybridization sites.     

水稻BAC在玉米有丝分裂染色体上FISH杂交体系的构建

 以水稻细菌人工染色体(BAC)为探针在玉米有丝分裂的细胞学制片上进行荧光原位杂交(FISH),探讨玉米基因组C_ot DNA对BAC探针重复序列的封阻、杂交后洗脱的严谨度、杂交液中FAD的浓度变化、水稻BAC探针的特异性重复序列的封阻对FISH杂交信号特异性的影响.初步形成了一套以水稻BAC探针在玉米有丝分裂染色体上进行BAC-FISH杂交的优化技术体系.研究结果表明：使用玉米基因组C_ot DNA来封阻水稻BAC探针的重复序列玉米基因组C _ot DNA的C_ot值应小于50,同时还需根据不同探针调整C_ot DNA的C_ot值及与探针的比例;而降低杂交液中FAD浓度和适度控制杂交后洗脱的严谨度,尤其是使用水稻BAC探针本身特异的重复序列的封阻对BAC-FISH杂交信号特异性的改善具有较好的效果.     

Super-stretched pachytene chromosomes for fluorescence in situ hybridization mapping and immunodetection of DNA methylation

Meiotic pachytene chromosome-based fluorescence in situ hybridization (FISH) mapping is one of the most important tools in plant molecular cytogenetic research. Here we report a simple technique that allows stretching of pachytene chromosomes of maize to up to at least 20 times their original size. A modified Carnoy's II fixative (6:1:3 ethanol:chloroform:acetic acid) was used in the procedure, and proved to be key for super-stretching of pachytene chromosomes. We demonstrate that super-stretched pachytene chromosomes provide unprecedented resolution for chromosome-based FISH mapping. DNA probes separated by as little as 50 kb can be resolved on super-stretched chromosomes. A combination of FISH with immunofluorescent detection of 5-methyl cytosine on super-stretched pachytene chromosomes provides a powerful tool to reveal DNA methylation of specific chromosomal domains, especially those associated with highly repetitive DNA sequences.     

Aneuploidy detection in human sperm nuclei using fluorescence in situ hybridization

Fluorescence in situ hybridization (FISH) was performed on human interphase sperm nuclei to determine the utility of this technique for aneuploidy detection. Repetitive DNA sequences specific for chromosomes 1, 12 and X were biotinylated and hybridized with mature sperm, which had been treated with cetyltrimethylammonium bromide and dithiothreitol to render them accessible to the probes. Detection of bound probe was accomplished with fluoresceinated avidin and antiavidin. For each of the chromosomes studied, chromosome number was determined by counting the fluorescent signals, representing hybridized regions, within the sperm nuclei. The frequencies for disomy, that is for nuclei containing two signals, for chromosomes 1, 12 and X were 0.06%, 0.04% and 0.03%, respectively. The congruence of these results with those determined by the cross-species hamster oocyte-human sperm assay, and the high efficiency of hybridization indicate that FISH is a sensitive and reliable tool for aneuploidy detection in human sperm.     

Homologous chromosomes characteristics by sequential banding procedures in rainbow trout Oncorhynchus mykiss

The development of high resolution methods of chromosome banding helped the finding of homologous chromosomes, detecting chromosomal abnormalities, and assigning the gene loci to particular chromosomes in mammals. Unfortunately, small and numerous fish chromosomes do not show GC rich and GC poor compartments, this preventing the establishment of G banding pattern. The combination of techniques enabling the identification of constitutive heterochromatin (C-banding), heterochromatin resistant to restriction endonucleas, NOR bearing chromosomes (AgNO3 banding), or AT rich regions on chromosomes (DAPI banding) in sequential staining provides a better characteristic of fish chromosomes. In this work sequentially DAPI, DdeI, AgNO3 stained chromosomes of rainbow trout resulted in the characteristic banding pattern of some homologous chromosomes. Procedure of FISH with telomere probe and DAPI as a counterstaining fluorochrome visualized simultaneous hybridization signals and DAPI banding. Possibility of detection both FISH and DAPI signals can help in procedures of gene mapping on chromosomes.     

Chromosome identification and comparative karyotypic analyses of four Pinus species

Chromosomal landmarks in four Pinus species: P. densiflora, P. thunbergii, P. sylvestris, and P. nigra were identified by fluorescence in situ hybridization (FISH) using hapten- or fluorochrome-labeled probes for the plant telomere repeat, centromeric repeat (PCSR), and rDNA. FISH landmarks were located at the interstitial and proximal regions of chromosomes and allowed us to identify nearly all of the homologous chromosomes in each species. A comparative analysis of the FISH karyotypes among the four species showed that the interstitial FISH signals obtained by hybridization with the telomere and rDNA sequences were stable and could be used to identify homologous chromosomes among species. The identification of homologous chromosomes among species facilitated a detailed comparative karyotype analysis. The results suggest that the degree of chromosomal differentiation among the four Pinus species is very low and that the proximal regions vary in their DNA sequences. The similarities and differences among FISH karyotypes are discussed in relation to phylogeny.     

Genome sequence comparison between Chinese hamster ovary (CHO) DG44 cells and mouse using end sequences of CHO BAC clones based on BAC-FISH results

Chinese hamster ovary (CHO) cells have frequently been used in biotechnology as a mammalian host cell platform for expressing genes of interest. Previously, we constructed a detailed physical chromosomal map of the CHO DG44 cell line by fluorescence in situ hybridization (FISH) imaging using 303 bacterial artificial chromosome (BAC) clones as hybridization probes (BAC-FISH). BAC-FISH results revealed that the two longest chromosomes were completely paired. However, other chromosomes featured partial deletions or rearrangements. In this study, we determined the end sequences of 303 BAC clones (BAC end sequences), which were used for BAC-FISH probes. Among 606 BAC-end sequences (BESs) (forward and reverse ends), 558 could be determined. We performed a comparison between all determined BESs and mouse genome sequences using NCBI BLAST. Among these 558 BESs, 465 showed high homology to mouse chromosomal sequences. We analyzed the locations of these BACs in chromosomes of the CHO DG44 cell line using a physical chromosomal map. From the obtained results, we investigated the regional similarities among CHO chromosomes (A–T) and mouse chromosomes (1–19 and sex) about 217 BESs (46.7% of 465 high homologous BESs). Twenty-three specific narrow regions in 13 chromosomes of the CHO DG44 cell line showed high homology to mouse chromosomes, but most of other regions did not show significant correlations with the mouse genome. These results contribute to accurate alignments of chromosomes of Chinese hamster and its genome sequence, analysis of chromosomal instability in CHO cells, and the development of target locations for gene and/or genome editing techniques.