Detection of Differentiation Among BB, CC and EE Genomes in the Genus Oryza by Two-probe Genomic in situ Hybridization (GISH)

The genus Oryza consists of two cultivated species (O. sativa L. and O. glaberrima Steud.) and approximately 20 wild relative species widely distributed in the pan-tropics. These species have been classified into four complexes following the Vaughan's taxonomic system([1]). The O. officinalis complex is the largest complex in the genus, which includes ten species, having BE, CC, on, and EE genomes in the diploids as well as BBCC and CCDD genomes in the tetraploids. The relationships among the BE, CC, and EE genomes still remain unclear, although previous studies have indicated certain affinities of these genomes([2-4]). Genomic in situ hybridization (GISH) is a powerful technique to detect the relationships among the related genomes at chromosome and DNA levels. The objective of the present study was to investigate the relationships among the BE, CC and EE genomes in the genus Oryza by the two-probe GISH.

The Distribution of Repetitive DNAs Along Chromosomes in Plants Revealed by Self-genomic in situ Hybridization

The distribution of repetitive DNAs along chromosomes is one of the crucial elements for understanding the organization and the evolution of plant genomes. Using a modified genomic in situ hybridization （GISH） procedure, fluorescence in situ hybridization （FISH） with genomic DNA to their own chromosomes （called self-genomic in situ hybridization, self-GISH） was carried out in six selected plant species with different genome size and amount of repetitive DNA. Nonuniform distribution of the fluorescent labeled probe DNA was observed on the chromosomes of all the species that were tested. The signal patterns varied among species and were related to the genome size. The chromosomes of the small Arabidopsis genome were labeled almost only in the pericentromeric regions and the nucleolus organizer regions （NORs）. The signals in the relatively small genomes, rice, sorghum, and Brassica oleracea var. capitata L., were dispersed along the chromosome lengths, with a predominant distribution in the pericentromeric or proximal regions and some heterochromatic arms. All chromosomes of the large genomes, maize and barley, were densely labeled with strongly labeled regions and weakly labeled or unlabeled regions being arranged alternatively throughout the lengths. In addition, enhanced signal bands were shown in all pericentromeres and the NORs in B. oleracea var. capitata, and in all pericentromeric regions and certain intercalary sites in barley. The enhanced signal band pattern in barley was found consistent with the N-banding pattern of this species. The GISH with self-genomic DNA was compared with FISH with Cot-1 DNA in rice, and their signal patterns are found to be basically consistent. Our results showed that the self-GISH signals actually reflected the hybridization of genomic repetitive DNAs to the chromosomes, thus the self-GISH technique would be useful for revealing the distribution of the regions where repetitive DNAs concentrate along chromosomes and some chromatin differentiation associated with repetitive DNAs in plants.

Analysis of the meiosis in the F_1 hybrids of Longiflorum × Asiatic(LA) of lilies(Lilium) using genomic in situ hybridization

Longiflorum and Asiatic lilies of the genus Lilium of the family Liliaceae are two important groups of modem lily cultivars. One of the main trends of lily breeding is to realize introgression between these groups. With cut style pollination and embryo rescue, distant hybrids between the two groups have been obtained. However, the FI hybrids are highly sterile or some of them could produce a small number of 2n gametes, and their BC1 progenies are usually triploids. Dutch lily breeders have selected many cultivars from these BC1 progenies based on their variation. It is presumably suggested that such variation could be caused by intergenomic recombination and abnormal meiosis during gamete formation in F1 hybrids of Longiflorum × Asiatic （LA） hybrids in Lilium. Therefore, the meiotic process of ten F1 LA hybrids was cytologically investigated using genomic in situ hybridization and traditional cytological methods in the present research. The results showed that： at metaphase I, the homoeologous chromosome pairing among different F1 hybrids ranged from 2.0 to 11.4 bivalents formed by homoeologous chromosomes per pollen mother cell （PMC）, and very few multivalents, and even very few bivalents were formed by two chromosomes within one genome rather than homoeologous chromosomes in some PMCs; at anaphase I, all biva- lents were disjoined and most univalents were divided. Both the disjoined bivalents （half-bivalents） and the divided univalents （sister chromatids） moved to the opposite poles, and then formed two groups of chromosomes; because the two resulting half-bivalents retained their axes in the cell undisturbed, many crossover types, including single crossovers, three strand double crossovers, four strand double crossovers, four strand triple crossovers, and four strand multiple crossovers between the non-sister chromatids in the tetrads of bivalents, were clearly inferred by analyzing the breakpoints on the disjoined bivalents. The present investigation not only explained the reason for sterility of the Fl LA hybrids and the variation of their BCx progenies, but also provided a new method to analyze crossover types in other F1 interspecific hybrids as well.

Cytogenetic comparisons between A and G genomes in Oryza using genomic in situ hybridization

The genomic structures of Oryza sativa （A genome） and O. meyeriana （G genome） were comparatively studied using bicolor genomic in situ hybridization （GISH）. GISH was clearly able to discriminate between the chromosomes of O. sativa and O. meyeriana in the interspecific F1 hybrids without blocking DNA, and co-hybridization was hardly detected. The average mitotic chromosome length of O. meyeriana was found to be 1.69 times that of O. sativa. A comparison of 4,6-diamidino-2-phenylindole staining showed that the chromosomes of O. meyeriana were more extensively labelled, suggesting that the G genome is amplified with more repetitive sequences than the A genome. In interphase nuclei, 9-12 chromocenters were normally detected and nearly all the chromocenters constituted the G genome-specific DNA. More and larger chromocenters formed by chromatin compaction corresponding to the G genome were detected in the hybrid compared with its parents. During pachytene of the F1 hybrid, most chromosomes of A and G did not synapse each other except for 1-2 chromosomes paired at the end of their arms. At meiotic metaphase I, three types of chromosomal associations, i.e.O, sativa-O, sativa （A-A）, O. sativa-O, meyeriana （A-G） and O. meyeriana-O, meyeriana （G-G）, were observed in the F1 hybrid. The A-G chromosome pairing configurations included bivalents and trivalents. The results provided a foundation toward studying genome organization and evolution of O. meyeriana.

Identification of Intergeneric Hybrid Plants Between Oryza sativa and O.minuta via GISH and RAPD

To transfer desirable resistance traits from O. minuta to O. sativa, intergeneric hybrid plants between O. sativa (AA, 2n=2X=24) and O. minuta (BBCC, 2n=4X=48) were produced by embryo rescue after sexual cross. Morphological observation and chromosome counts indicated their hybrid status (ABC, 2n=3X=36). Genomic in situ hybridization (GISH) was further applied to confirm the parentage of the chromosomes of F 1 hybrids. Chromosomes of O. minuta and O. sativa were distinguishable in the hybrids in different fluorescence colors. GISH indicated that A and BC chromosomes were not randomly assembled in a cell. RAPD profiles unequivocally revealed their hybrids with double parent patterns. The results of blast tests showed that the hybrids had obtained disease resistance from O. minuta, and had a level of susceptibility between the parents.

Uniqueness of the Gossypium mustelinum Genome Revealed by GISH and 45S rDNA FISH

Gossypium mustelinum [(AD)4] is one of five tetraploid species in Gossypium.Three pairs of nucleolar organizer regions(NOR) in(AD)4 were detected by FISH with 45S rDNA as a probe,they also were observed with genomic DNA(gDNA) from Gossypium D genome species as probes.Of

Genetic Relationships Among Five Basic Genomes St, E, A, B and D in Triticeae Revealed by Genomic Southern and in situ Hybridization

The St and E are two important basic genomes in the perennial tribe Triticeae （Poaceae）. They exist in many perennial species and are very closely related to the A, B and D genomes of bread wheat （Triticum aestivum L.）. Genomic Southern hybridization and genomic in situ hybridization （GISH） were used to analyze the genomic relationships between the two genomes （St and E） and the three basic genomes （A, B and D） of T. aestivum. The semi-quantitative analysis of the Southern hybridization suggested that both St and E genomes are most closely related to the D genome, then the A genome, and relatively distant to the B genome. GISH analysis using St and E genomic DNA as probes further confirmed the conclusion. St and E are the two basic genomes of Thinopyrum ponticum （StStE^eE^bE^x） and Th. intermedium （StE^eE^b）, two perennial species successfully used in wheat improvement. Therefore, this paper provides a possible answer as to why most of the spontaneous wheat-Thinopyrum translocations and substitutions usually happen in the D genome, some in the A genome and rarely in the B genome. This would develop further use of alien species for wheat improvement, especially those containing St or E in their genome components.

Comparative genome research between maize and rice using genomic in situ hybridization

Using the genomic DNAs of maize and rice as probes respectively, the homology of maize and rice genomes was assessed by genomic in situ hybridization. When rice genomic DNAs were hybridized to maize, all chromosomes displayed many multiple discrete regions, while each rice chromosome delineated a single consecutive chromosomal region after they were hybridized with maize genomic DNAs. The results indicate that the genomes of maize and rice share high homology, and confirm the proposal that maize and rice are diverged from a common ancestor.

Establishment of a Multi-color Genomic in situ Hybridization Technique to Simultaneously Discriminate the Three Interspecific Hybrid Genomes in Gossypium

To identify alien chromosomes in recipient progenies and to analyze genome components in polyploidy, a genomic in situ hybridization （GISH） technique that is suitable for cotton was developed using increased stringency conditions. The increased stringency conditions were a combination of the four factors in the following optimized state： 100：1 ratio of blocking DNA to probe, 60% formamide wash solution, 43 ℃ temperature wash and a 13 min wash. Under these specific conditions using gDNA from Gossypium sturtianum （C1 C1 ） as a probe, strong hybridization signals were only observed on chromosomes from the C1 genome in somatic cells of the hybrid F1 （G. hirsutum x G. sturtianum） （AtDtC1）. Therefore, GISH was able to discriminate parental chromosomes in the hybrid. Further, we developed a multi-color GISH to simultaneously discriminate the three genomes of the above hybrid. The results repeatedly displayed the three genomes, At, Dt, and C1, and each set of chromosomes with a unique color, making them easy to identify. The power of the multi-color GISH was proven by analysis of the hexaploid hybrid F1 （G. hirsutum x G. australe） （AtAtDtDtG2G2）. We believe that the powerful multi-color GISH technique could be applied extensively to analyze the genome component in polyploidy and to identify alien chromosomes in the recipient progenies.

Characterization of Interspecific Hybrids Between Oryza sativa L, and Three Wild Rice Species of China by Genomic In Situ Hybridization

In the genus Oryza, interspecific hybrids are useful bridges for transferring the desired genes from wild species to cultivated rice （Oryza sativa L.）. In the present study, hybrids between O. sativa （AA genome） and three Chinese wild rices, namely O. rufipogon （AA genome）, O. officinalis （CC genome）, and O. meyeriana （GG genome）, were produced. Agricultural traits of the F1 hybrids surveyed were intermediate between their parents and appreciably resembled wild rice parents. Except for the O. sativa × O. rufipogon hybrid, the other F1 hybrids were completely sterile. Genomic in situ hybridization （GISH） was used for hybrid verification. Wild rice genomic DNAs were used as probes and cultivated rice DNA was used as a block. With the exception of O. rufipogon chromosomes, this method distinguished the other two wild rice and cultivated rice chromosomes at the stage of mitotic metaphase with different blocking ratios. The results suggest that a more distant phylogenetic relationship exists between O. meyeriana and O. sativa and that O. rufipogon and O. sativa share a high degree of sequence homology. The average mitotic chromosome length of O. officinalis and O. meyeriana was 1.25- and 1.51-fold that of O. sativa, respectively. 4＇,6＇-Diamidino- 2-phenylindole staining showed that the chromosomes of O. officinalis and O. meyeriana harbored more heterochromatin, suggesting that the C and G genomes were amplified with repetitive sequences compared with the A genome. Although chromocenters formed by chromatin compaction were detected with wild rice-specific signals corresponding to the C and G genomes in discrete domains of the F1 hybrid interphase nuclei, the size and number of O. meyeriana chromocenters were bigger and greater than those of O. officinalis. The present results provide an important understanding of the genomic relationships and a tool for the transfer of useful genes from three native wild rice species in China to cultivars.

Discrimination of Repetitive Sequences Polymorphism in Secale cereale by Genomic In Situ Hybridization-Banding

Genomic in situ hybridization banding （GISH-banding）, a technique slightly modified from conventional GISH, was used to probe the Chinese native rye （Secale cereale L.） DNA, and enabled us to visualize the individual rye chromosomes and create a universal reference karyotype of the S. cereale chromosome 1R to 7R. The GISH-banding approach used in the present study was able to discriminate S. cereale chromosomes or segments in the wheat （Triticum aestivum L.） background, including the Triticale, wheat-rye addition and translocation lines. Moreover, the GISH-banding pattern of S. cereale subsp. Afghanicum chromosomes was consistent with that of Chinese native rye cv. Jingzhou rye; whereas the GISH-banding pattern of Secale vavilovii was different from that of S. cereale, indicating that GISH-banding can be used to study evolutionary polymorphism in species or subspecies of Secale. In addition, the production and application of GISH-banding to the study of adenine-thymine-riched heterochromaUn is discussed.

沙田柚多倍体的获得与基因组原位杂交(GISH)分析

【目的】沙田柚是中国特有的柚类名优品种,但种子多,一般100粒左右。为创新三倍体无核品种积累育种材料,本研究通过实生筛选获得不同倍性沙田柚新种质,同时运用基因组原位杂交(GISH)技术分析天然与人工四倍体新种质的染色体组组成。【方法】随机采集沙田柚自然授粉果实,萌发种子检测其染色体数目获得倍性变异植株;以2x母株gDNA为探针,同获得的4x植株中期染色体杂交进行GISH分析。【结果】从6000粒沙田柚种子中共获得三倍体5株,四倍体9株;对沙田柚天然与人工四倍体新种质的基因组原位杂交(GISH)分析表明,天然四倍体中有7株为异源四倍体,2株为同源四倍体,秋水仙碱诱导获得的人工四倍体4株均为同源四倍体;初步观察显示;与二倍体相比,四倍体植株生长缓慢,树冠较小,枝短而密生,叶片浓绿,宽度变宽,叶形指数减小,叶片厚度增加明显。【结论】多倍体单胚柚新种质的获得为进一步选育无核品种奠定了基础,同时GISH分析证实了沙田柚雌性未减数配子的存在。

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。

应用GISH与STS标记鉴定小麦-中间偃麦草抗黄矮病端体系

由大麦黄矮病毒引起的小麦黄矮病毒病是一个严重病害,至今在小麦属内还没有发现抗源。中间偃麦草2Ai-2染色体携带1个高抗黄矮病基因,对该基因的染色体臂定位将为制定抗病基因向小麦转移策略,筛选、开发特定的、与抗性连锁的分子标记的研究提供重要信息。本文对由小麦-中间偃麦草二体附加系Z6衍生的3个抗黄矮病端体系进行鉴定,通过分析端体的遗传构成、筛选与端体共分离的STS标记以确定端体在遗传上的染色体臂归属,从而明确BYDV抗病基因的染色体位置。以拟鹅冠草基因组[Pseudoroegneria strigosa(M.Bieb.).L.o.ve,St]DNA为探针,中国春基因组(TriticumaestivumL.,ABD)DNA作封阻分别对抗病亲本Z6及抗病端体系N530的根尖体细胞染色体进行原位杂交,结果表明,N530体细胞中有2个端体显示出与Z6中外源染色体2Ai-2短臂相似,而与长臂不同的杂交信号。以小麦第2同源群的5个RFLP探针的DNA序列为基础,设计了6对PCR引物,对小麦-中间偃麦草二体异附加系、二体代换系和端体系进行扩增,结果表明,基于短臂探针psr126,psr131序列设计的2对引物,可在含有2Ai-2染色体及端体的抗黄矮病材料中特异扩增,而基于长臂探针psr112序列设计的1对引物,可在含有2Ai-2染色体的抗黄矮病材料中特异扩增,但不能在端体系进行特异扩增,证明外源端体为2Ai-2染色体的短臂。本研究不仅将黄矮病抗性基因定位于2Ai-2染色体的短臂上,而且由RFLP探针psr126、psr131和psr112序列转化的标记STS126(sequence tagged site)STS131和STS112还可分别作为追踪2Ai-2染色体短臂和长臂的分子标记,用于抗病易位系辅助选择。

普通小麦×大麦杂交后代中间材料的GISH及PAGE鉴定

利用基因组荧光原位杂交 (GISH)及种子贮藏蛋白聚丙烯酰胺凝胶电泳 (PAGE)对普通小麦×大麦杂交后代中间材料进行了鉴定分析。GISH结果表明 ,WBA984和WBA9812为二体小大麦异附加系 ,WBS0 2 15和WBS0 2 6 4为小大麦二体异代换系 ,WBT0 2 12 5和WBT0 2 183为端部易位系 ;种子贮藏蛋白PAGE分析表明 ,WBA9812和WBS0 2 6 4含有大麦特有的高分子量麦谷蛋白亚基和在γ区含有大麦特有的醇溶蛋白带型 ,WBA9812为大麦 5H附加系 ,WBS0 2 6 4为 1B/ 5H代换系 ,WBT0 2 12 5为 1BL/ 5HL端部易位系。

基于GISH的甘蔗与斑茅F_1染色体遗传与核型分析

斑茅在甘蔗育种的利用是现代甘蔗育种种质创新的热点，育种者期望把斑茅中优异的特性通过杂交渗透到甘蔗中。甘蔗与斑茅的F1是斑茅利用研究的难点也是基础。本研究利用基因组原位杂交技术（GISH）分析甘蔗与斑茅的F1染色体构成和核型，探讨甘蔗与斑茅F1染色体的遗传行为。GISH结果表明，甘蔗与斑茅杂交F1的染色体众数68-69条，其中40条来自甘蔗热带种Badila，28-29来自海南斑茅，未发现有染色体的交换或易位现象。参试材料大部分染色体都属于中部着丝点（m）的染色体，少数为近中部着丝点（sm），YCE95-41核型属2B型，其余的核型都属为1B型。甘蔗与斑茅的染色体按n ＋ n的方式传递给F1，本研究结果为斑茅种质在甘蔗育种中的利用及其杂交后代染色体细胞遗研究提供参考依据。

栉孔扇贝和虾夷扇贝杂交子代的GISH鉴定及其免疫学特性

以栉孔扇贝[Chlamys farreri(Jones et Preston)](♀)和虾夷扇贝(Patinopecten yessoensis)(♂)杂交子代担轮幼虫为材料,分别用栉孔扇贝和虾夷扇贝基因组作探针,采用基因组荧光原位杂交(GISH)的方法,对杂交后代杂交子的确切身份进行初步鉴定。结果表明,子代分别继承了双亲各一套染色体(n=19),为真正的杂交种。为了解杂交扇贝的免疫学特性,在自然海域栉孔扇贝大规模死亡的情况下,分别对杂交扇贝及其亲本3个扇贝群体血细胞的胞内活性氧含量(ROIs)、血清凝集素效价(HA)、溶菌酶活力(LSZ)、抑菌活力、酚氧化酶活性(PO)、过氧化氢酶(CAT)、超氧化物歧化酶(SOD)以及酸性磷酸酶(ACP)和碱性磷酸酶(ALP)等9种非特异性免疫学指标进行测定。结果表明,栉孔扇贝除ROIs、SOD、ACP等3个指标显著低于虾夷扇贝外(P<0.01),其他6种指标均高于虾夷扇贝,且除血清凝集素效价外均达显著水平(P<0.05)。在杂交子代中,上述免疫指标除SOD活性低于低值亲本外(P>0.6),其余8种免疫指标均介于双亲之间。杂交子代在9种免疫指标中有8种与母本无显著性差异,而子代与父本之间9种免疫指标中有7种达显著差异(P<0.05)。这些结果说明,杂交扇贝在非特异性免疫上存在明显的偏母性特征,这点与子代在外形特征上的偏母性相吻合。因此杂交扇贝相对于其母本在生产实践中表现出的一定程度的抗逆优势可能与非特异性免疫无明显关系。[中国水产科学,2006,13(4):597-602]

萝卜-芥蓝异源四倍体的合成及GISH分析

通过萝卜（Raphanus sativus L．，2n=18，RR）与白花芥蓝（Brassica alboglabra Bailey，2n=18，CC）杂交，F1经秋水仙碱加倍合成萝卜-芥蓝异源四倍体（Raphanobrassica，2n=36，RRCC）。经F4-F10代连续育性选择，F10单株种子产量达32．3g，每角粒数达14．9。基因组原位杂交显示F10减数分裂行为类似于二倍体物种，表明该异源四倍体的细胞学行为已经稳定。育性观察表明，可育花粉足够各代生产种子，但低世代杂种出现高频瘪粒种子，胚珠败孕可能是其主要原因。该萝卜-芥蓝异源四倍体可以用作向油菜（B．napus L．，2n=38，AACC）转移萝卜基因的遗传桥梁。

甘蓝-白芥单体异附加系自交后代的GISH分析

将甘蓝-白芥单体异附加系自交,获得了其自交后代.利用基因组原位杂交(genomicin situhy-bridization,GISH),结合双色荧光原位杂交(dual-colour fluorescencein situhybridization,dcFISH)技术,从这些自交后代中鉴定出了纯合的甘蓝-白芥二体异附加系植株.GISH分析结果表明,甘蓝-白芥二体异附加系有丝分裂中期具有18条甘蓝染色体及2条白芥染色体,减数分裂中期I表现为9个C染色体二价体及1个S染色体二价体,减数分裂后期I会出现落后的1对S染色体,有时落后的1对S染色体形成染色体桥.

柑橘基因组原位杂交(GISH)技术体系的建立

通过改进实验方法,建立起柑橘基因组原位杂交(Genomicinsituhybridization,GISH)分析技术,并成功地应用于属间有性杂种鉴定,为进一步分析柑橘体细胞杂种核基因组组成奠定了基础。柑橘的染色体制片以去壁低渗-火焰干燥法较好,容易获得大量清晰、分散的有丝分裂中期相,且杂质较少。切刻平移法生物素(Biotin)标记探针,标记基因组总DNA与封阻基因组总DNA的浓度比例为1:50时,能有效分开属间杂种枳橙中来自双亲的染色体。