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.

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

Oryza sativa (一个染色体) 和 O 的 genomic 结构。meyeriana (G 染色体) 比较地在 situ 杂交(GISH ) 用二色的 genomic 被学习。GISH 清楚地能在 O 的染色体之间区别。sativa 和 O。在没有堵住 DNA 的种间的 F1 混血儿的 meyeriana，和合作杂交几乎没被检测。O 的平均有丝分裂的染色体长度。meyeriana 被发现是乘 O 的的 1.69。sativa。染色的 4,6-diamidino-2-phenylindole 的比较出现了 O 的染色体。meyeriana 更广泛地被标记，建议 G 染色体与更重复的序列比被放大一个染色体。在分裂期间原子核， 9-12 多彩石印版中心通常被检测，将近，所有多彩石印版中心组成了 G 染色体特定的 DNA。中心由相应于 G 染色体的染色质压缩形成了的更多和更大的多彩石印版与它的父母相比在混血儿被检测。在 F1 混血儿的 pachytene 期间， A 和 G 的大多数染色体互相，除了 1-2，染色体在他们的手臂的结束配对的触处。在 meiotic 中期我， chromosomal 协会的三种类型，即 O。sativa-O。sativa (A-A ) ， O。sativa-O。meyeriana (A-G ) 和 O。meyeriana-O。meyeriana (G-G ) ，在 F1 混血儿被观察。配对配置的 A-G 染色体包括了 bivalents 和 trivalents。结果向学习染色体组织和 O 的进化提供了一个基础。meyeriana。

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.

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.

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.

DETECTION OF CYTOMEGALOVIRUS GENOME BY IN SITU HYBRIDIZATION IN PARAFFIN EMBEDDED ENDOMYOCARDIAL BIOPSY SPECIMENS OF VIRAL MYOCARDITIS

Cytomegalovirus (CMV) genes were detected by in situ hybridization in 25 Chinese patients with viral myocarditis (VMC). The positive hybridization signals werre found in cardiomyocytes (6 cases, 24%), capillary endothelial cells (4 cases, 16%) and interstitial cells (7 cases, 28%). The difference between VMC and control group (16 cases died of brain trauma and 10 cases of congenital heart diseases was statistically significant. There was no definite pathomorphological relationship between the detection of CMV genes and myocardial lesions. The results suggest that CMV infection may be one of the causes of myocarditis and chronic stimulation of the immune system induced by CMV may be a possible pathogenesis of this disease.

Identification and cell wall analysis of interspecific hybrids between Oryza sativa and Oryza ridleyi

Oryza ridleyi is an allotetraploid wild species with the HHJJ genome, and Oryza sativa is a diploid cultivated rice that has the AA genome. Although the wide hybrid between the two species is difficult to obtain, we overcome this difficulty by young embryo rescue. An obvious heterosis was primarily found for the plant height, tillering ability, vegetative vigor, etc. However, the hybrid panicle and culm traits were found to resemble that of the wild rice parent, O. ridleyi, for the long awns, exoteric purple stigma, grain shattering, dispersed panicles, and culm mechanical strength. Genomic in situ hybridization （GISH） analysis was subsequently performed on the mitotic metaphase chromosome of the root tips, and we determined that the hybrid is an allotriploid with 36 chromosomes and its genomic constitution is AHJ. Chemical analyses conducted on the culm of O. sativa, O. ridleyi, and their interspecific hybrids showed that major changes occurred in the xylose, glucose, and arabinose concentrations, which are correlated with the specific hemicellulose polymer and cellulose components that are important in the primary cell walls of green plants. Meanwhile, the culm anatomical analyses indicated that additional large vascular bundles and an extra sclerenchyma cell layer were found in O. ridleyi. Additionally, further thickening of the secondary cell walls of the cortical fiber sclerenchyma cells and the phloem companion cells was discovered in O. ridleyi and in the interspecific hybrids. These results imply that there may be a potential link between culm mechanical strength and culm anatomical structure.

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

黑壳楠染色体核型分析及基因组Survey预测

【目的】明确黑壳楠(Lindera megaphylla)染色体形态结构特征和基因组基本信息。【方法】以野生黑壳楠为材料,采用荧光原位杂交法,对染色体进行核型分析,并通过基因组survey预测了黑壳楠基因组的基本信息。【结果】黑壳楠染色体数目为2n=24,核型公式为2n=2X=24=18m(2SAT)+6sm,9号染色体存在1对随体,属于2B型染色体。染色体组绝对长度变异范围为2.92~5.83,相对长度的变化范围为5.33%~11.11%,核型不对称系数为59.49%。研究还发现,存在1对5S rDNA杂交位点和1对18S rDNA杂交位点,分别位于5号和9号染色体。18S rDNA杂交位点2个杂交信号位置相同、强度近似。基因组survey结果显示,黑壳楠基因大小为1.38 Gb,杂合率为0.8%,重复序列比例为70%。【结论】黑壳楠为2倍体,染色体数目为24条,核型相对对称。黑壳楠在樟科的进化中属于较为原始的物种,且染色体未发生重组、变异或者种内杂交等现象。黑壳楠为大基因组,杂合率较高,重复序列多的物种。

异源三倍体普通烟草(SST)减数分裂期的分子细胞学研究

烟草属野生种是栽培烟草品种改良的重要种质来源,通过对烟草种间杂种的创建及其减数分裂期分子细胞学分析,为后续优异野生种质的有效转移和利用提供基础。以林烟草Q67(Nicotiana sylvestris)和普通烟草K326(Nicotiana tabacum L.cv.K326)为父母本创建异源三倍体种间杂种,采取常规细胞学观察、基因组原位杂交及实时定量PCR等方法对其染色体组成、减数分裂时期染色体行为及相关基因的表达进行调查。结果表明,68株种间杂种体染色体条数为36条,染色体组成为SST,其整体表型兼具双亲特征。种间杂种的花粉母细胞减数分裂期异常细胞占比为29.26%,主要异常行为为减数分裂I和II中期的T染色体组来源的染色体滞后,落后染色体条数为1-3条,其中,减数分裂中期I和中期II落后类型最多的分别为落后1条T染色体和落后2条T染色体的细胞,分别占该时期携带落后染色体细胞总量的29.09%和30.88%;减数分裂I后期和减数分裂II后期及末期的异常类型主要为不均等分离、落后染色体和微核,其中,减数分裂I后期统计到多种异常分离比,但分离后染色体组成仍接近2∶1(S∶T)。相较于对照K326,在减数分裂I前期,来源于S染色体组的CDKA:5319表达显著上调和CYCA:6720表达极显著上调;在减数分裂I中期至减数分裂II末期,CENH3和来源于T基因组的CYCA:0111表达显著上调,来源于S基因组的CDKA:5319和CYCA:6720均呈极显著上调。表明转录水平上减数分裂过程的紊乱,相关基因的表达变化可能是减数分裂异常的原因。

基于流式细胞术与基因组Survey分析白及基因组大小及特征

目的研究白及的核型、基因组大小等特征信息。方法双色荧光原位杂交分析白及的染色体核型;以番茄为内参,流式细胞术检测白及基因组大小;基因组Survey分析获得白及基因组大小、杂合率、GC含量等生物学信息。结果DAPI荧光染色及端粒荧光原位杂交发现白及染色体为二倍体,染色体类型为sm和st型,核型类型为2C。rDNA荧光原位杂交发现白及染色体有1对显示较强的5S rDNA位点和1对18S rDNA位点,5S rDNA位点位于2个同源染色体间隙,18S rDNA位点位于染色体短臂的次缢痕部位。白及基因组大小为2.37 Gb,同时全基因Survey分析得到有效数据60.11 Gb,经过K-mer分析修正及Genomescope评估,白及基因组大小约为2.53 Gb,杂合率约为1.099%,重复序列约占67.45%,GC含量为36.11%,总测序深度为23.73。结论本研究首次获得白及基于5S rDNA和18S rDNA荧光原位杂交核型及增补了白及属植物的基因组大小数据,为白及后续的物种分类、种群进化研究及全基因组测序、组装及去冗余处理等工作提供基础参考数据。

基因组原位杂交辨别芸薹属异源四倍体AA、BB、CC基因组研究

利用基因组原位杂交 (GISH)技术 ,以标记的白菜型油菜 (AA ,2n =2 0 )的基因组总DNA为探针 ,分别同芥菜型油菜 (AABB ,2n =36 )和甘蓝型油菜 (AACC ,2n =38)的中期染色体和间期核杂交 ,结果芥菜型油菜有 2 0条染色体表现大而明亮的杂交信号 ,其它染色体上信号很弱或无 ,可以区分A、B基因组 ;甘蓝型油菜染色体上表现有38个信号 ,A、C基因组不能区分开来。以黑芥 (BB ,2n =16 )的基因组总DNA为探针与埃塞俄比亚芥 (BBCC ,2n =34)的中期染色体和间期核杂交 ,16条染色体上显示明显的杂交信号 ,其它染色体上信号很弱。说明在长期的进化过程中 ,芸薹属中BB与AA和CC基因组间分化程度较大 ,而AA与CC基因组分化较小。基因组原位杂交表明最强的信号多集中分布于着丝粒区 。

利用离果山羊草3C染色体诱导簇毛麦4V染色体结构变异

通过普通小麦农林 2 6 离果山羊草 3C异附加系与普通小麦 簇毛麦 4V(4D)代换系杂交 ,杂交F1代与普通小麦回交 ,综合运用染色体构型分析、C 分带和荧光原位杂交等技术从BC1F2 、BC1F3 代中鉴定出涉及簇毛麦 4V染色体的易位系、端体、等臂染色体系等变异植株 ,表明离果山羊草 3C染色体可有效诱发簇毛麦 4V染色体结构变异 ,是创造小麦 簇毛麦

甘蓝与芸薹属5个近缘物种的基因组原位杂交分析

利用基因组原位杂交(GISH)技术,研究了甘蓝基因组与芸薹属其它5个近缘物种基因组间的相互关系。以标记的甘蓝(CC,2n=18)的基因组总 DNA为探针,分别同二倍体甘蓝、白菜型油菜(AA,2n=20)、黑芥(BB,2n=16)和异源四倍体芥菜型油莱(AABB,2n=36)、埃塞俄比亚芥(BBCC,2n=34)、甘蓝型油莱(AACC,2n=38)的中期染色体杂交,甘蓝、白菜型油菜和甘蓝型油菜的所有染色体上都有杂交信号,不能区分A、C基因组。黑芥染色体上只有零星的弱小信号,埃塞俄比亚芥的中期染色体显示出18个明显的信号,可以区分出C、B基因组;芥菜型油菜的中期染色体显示出20个明显的信号,其余染色体上信号很弱或无,可以区分出A、B基因组。说明在长期的进化过程中,甘蓝与白菜型油菜、甘蓝型油菜的亲缘关系较近,基因组的分化程度较小,而甘蓝与黑芥的亲缘关系较远,基因组的分化程度较前者高;甘蓝与芥菜型油菜和埃塞俄比亚芥间的亲缘关系介于上述二者之间。

应用基因组原位杂交及RFLP标记鉴定小麦中的大麦染色体

用生物素（Biotin-16-dUTP)标记的大麦Betzes基因组DNA作探针，以普通小麦中国春总DNA作封阻进行基因组原位杂交（Genome in situ hybridization,简称GISH），从13株小麦-大麦杂交后代中鉴定出2个含有3条大麦Betzes 2H染色体的材料（2n＝43）；2个2H单体异代换系（2n＝42）；7个2H二体异代换系（2n＝42）。用已定位在小麦第2部分同源群短臂上的探针psr131进行RFLP分析，结果表明大麦Betzes、代换系A5有1条区别于小麦中国春的特异带，A5的2条2A染色体被大麦Betzes的2条2H染色体所代换。GISH及RFLP的准确鉴定及小麦－大麦2H二体异代换系的首次获得，为向小麦导入2H染色体上的有用基因提供了宝贵材料。

大白菜和结球甘蓝基因组原位杂交及核型分析

为研究A、C基因组间的亲缘关系和识别不同染色体,分别以大白菜(AA)和结球甘蓝(BB)的基因组总DNA为探针,与大白菜和结球甘蓝的中期染色体杂交。结果表明,两种基因组总DNA在大白菜20条染色体和结球甘蓝18条染色体上都有杂交信号,但信号图型有明显差异。大白菜基因组总DNA在大白菜和结球甘蓝染色体上的杂交信号几乎都只集中于近着丝粒区和核仁组织区,但在大白菜染色体上的分布区域略大;结球甘蓝基因组总DNA在其染色体上的杂交信号分散布满其全长,但在着丝粒区和核仁组织区显示增强的信号带,而在大白菜中期染色体上则主要集中于着丝粒和近着丝粒区,且强度弱于大白菜基因组总DNA为探针的杂交信号。基于大白菜基因组DNA的GISH信号对大白菜和结球甘蓝的核型进行了分析。该研究结果为鉴别其种间杂种及其染色体的组成和同源关系提供了分子细胞学依据。