应用分子原位杂交技术解析小麦-天兰冰草部分双二倍体──远中2号的染色体构成

为分析普通小麦（Ｔｒｉｔｉｃｕｍａｅｓｔｉｖｕｍ）－天兰冰草（Ａｇｒｏｐｙｒｏｎｉｎｔｅｒｍｅｄｉｕｍ）部分双二倍体──远中２号（２ｎ＝５４）的染色体构成,用生物素（ｂｉｏｔｉｎ－１６－ｄＵＴＰ）标记天兰冰草染色体组ＤＮＡ作为探针,以普通小麦品种中国春染色体组ＤＮＡ为封闭ＤＮＡ（ｂｌｏｃｋｉｎｇＤＮＡ）,与远中２号的有丝分裂中期染色体ＤＮＡ进行了分子原位杂交。证明远中２号除具有普通小麦的２１对染色体外,附加了１对小麦－天兰冰草易位染色体（即天兰冰草染色体片段易位到小麦染色体的两臂端部）、５对天兰冰草染色体。说明小麦－天兰冰草部分双二倍体在形成过程中染色体行为是比较复杂的,不仅可能产生小麦－天兰冰草染色体间易位,而且小麦染色体也可能与天兰冰草染色体的３种染色休组染色体共同参与组建新的染色体组附加到小麦中去。     

应用分子原位杂交技术解析小麦—天兰冰草部分双二倍体——远中2号的…

为分析普通小麦－天兰冰草部分双二倍体－远中２号的染色体构成,用生物素标记天兰冰草染色体组ＤＮＡ俄为探针,,以普通小麦品种中国春染色体组ＤＮＡ为封闭ＤＮＡ,与远中２号的有丝分裂中期染色体ＤＮＡ进行了分子原位杂是明远中２号除具有普通小麦的２１对染色体外,附加了１对小麦－地冰草易位染色体（妈卫兰冰草染色体片段易位到小麦染色体的两臂端部）,５对天兰冰草染色体。说明小麦－天兰冰草部分双二倍体在形成过程中当构     

利用电离辐射处理整臂易位系成熟雌配子诱导外源染色体小片段易位

簇毛麦（Haynaldia villosa）是小麦（Triticum aestivum）改良的重要遗传资源．培育小片段易位,特别是中间插入易位,有助于更好地利用外源有益基因．已报道的小麦-簇毛麦易位系大多数属于整臂易位或大片段易位．本研究以60Co-γ谢线（剂量率：1．6Gy／min,三种剂量：16．0,19．2和22．4Gy）处理整臂易位系的成熟雌配子,随后选取适龄穗子去雄套袋,2～3天后再用普通小麦品种“中国春”的新鲜成熟花粉授粉．通过M1植株根尖细胞有丝分裂中期染色体基因组原位杂交（genomic in situ hybridization,GISH）,从534株M1材料中检测到97株涉及6V染色体短臂（6VS）的小片段结构变异,包括小片段中间插入易位染色体80条、末端易位染色体57条和6VS缺失55条．在22．4Gy处理中这三种结构变异的诱变频率分别为21．02％,14．01％和14．65％,远远高于已报道的结果．获得了涉及146条6VS小片段结构变异的74株M1材料的回交种子．M1植株中的小片段结构变异可通过回交传递给后代．利用电离辐射处理整臂易位系成熟雌配子是一种高效诱导染色体小片段结构变异、特别是中间插入易位的新方法．     

用分子原位杂交（GISH）鉴定小麦－簇毛麦双倍体、附加系、代换系和易位系

用生物素标记的簇毛麦（Ｈａｙｎａｌｄｉａｖｉｌｌｏｓａ）染色体组ＤＮＡ（ｔｏｔａｌｇｅｎｏｍｉｃＤＮＡ）作探针,以普通小麦染色体组ＤＮＡ作遮盖（用量１：２００左右）,进行有丝分裂中期和减数分裂中期Ｉ染色体的分子原位杂交（ＧＩＳＨ）,经抗生物素蛋白－辣根过氧化物酶复合物（ｂｉｏ－ｓｔｒｅｐｔａｖｉｄｉｎ－ｈｏｒｓｅｒａｄｉｓｈｐｅｒｏｘｉｄａｓｅ）和联苯胺四盐酸（ＤＡＢ）检测显色后,小麦－簇毛麦双倍体、附加系、代换系和易位系中的簇毛麦染色体及染色体片段显棕色,与显浅蓝色的小麦染色体可明显区分。用ＧＩＳＨ不仅可以检测导入小麦中的簇毛麦染色质,而且可以清楚地显示出易位染色体断裂点的确切位置。将ＧＩＳＨ用于减数分裂期染色体配对分析,还可以清晰形象地显示出同源和非同源染色体之间的配对和分离情况。     

普通小麦-大赖草易位系NAU601和NAU618的选育及双端二体测交分析

利用根尖体细胞有丝分裂中期染色体Giemsa C-分带和荧光原位杂交从普通小麦-大赖草Lr.2、Ir.7异附加系辐射后代中选育出2个纯合易位系：（1）易位系NAU618(MS142-3),2n=44,易位染色体由大赖草Lr.7染色体的大部分（约5/6,包括着丝粒）及小麦染色体1A短臂的一部分（近端1/3）组成,外源染色体片段的长度约占易位染色体总长度的4/5;（2）易位系MAU601（MS101-4）,2n=42,易位染色体由小麦染色体4B的整个短臂和4B长臂近着丝粒部分（1/3）及大赖草Lr.2短臂的绝大部分组成,外源染色体片段占易位染色体长臂的1/2。对易位系进行的双端二体侧交分析证交易位所涉及的小麦染色体分别为1A和4B。连续3年单花滴注法进行的田间赤霉病抗性接种鉴定结果表明,普通小麦-大赖草异源异位系MAU618(MS142-3)对赤霉病的抗性与抗病对照品种苏麦3号相仿,易位系MAU601（MS101-4）对赤霉病抗性低于苏麦3号,但明显高于感病亲本中国春。     

利用减数分裂期成株电离辐射选育小麦—大赖草易位系的研究

采用６００１１２５Ｒ剂量的６０Ｃｏγ射线对小麦（ＴｒｉｔｉｃｕｍａｅｓｔｉｖｕｍＬ．）大赖草（Ｌｅｙｍｕｓｒａｃｅｍｏｓｕｓ（Ｌａｍ．）Ｔｚｖｅｌ．）Ｌｒ．７单体异附加系在减数分裂期进行成株辐射处理,经过Ｍ１代根尖细胞有丝分裂中期（ＲＴＣ,Ｍ期）染色体ＧｉｅｍｓａＣ分带粗筛,Ｍ２代ＲＴＣＭ期染色体Ｃ分带和荧光原位杂交鉴定,选育出２个小麦大赖草Ｌｒ．７异易位系。其中Ｔ０２易位系是由Ｌｒ．７染色体绝大部分与一小片段小麦染色体接在一起组成的易位类型（ＴＬｒ．７·Ｌｒ．７Ｗ）;Ｔ０８的易位染色体由小麦４ＡＬ和大赖草Ｌｒ．７某臂组成。     

家猪减数分裂粗线期二价体与有丝分裂中期染色体的比较研究

以性成熟公猪睾丸和外周血为材料,采用长低渗、高氯仿卡诺固定液固定和外周血细胞培养制备减数分裂粗线期二价体和有丝分裂中期染色体,通过对二价体和有丝分裂中期染色体分裂指数和长度的比较研究,发现二价体的分裂指数和长度分别是有丝分裂中期染色体的5倍和3．42倍（1．87～5．98）;同时以12号染色体为例,比较了二价体上的染色粒结构带与有丝分裂中期染色体G－带,表明染色粒结构带比中期染色体G－带纹丰富,而与早中期G－带带织吻合。     

用顺序GISH-FISH 技术鉴定小麦-中间偃麦草小片段易位系

利用顺序基因组-重复序列原位杂交技术对1个来自中3不育系和普通小麦恢75杂种后代稳定株系H96276-2的染色体组成进行了分析。以中间偃麦草（Agropyronintermedium）基因组DNA为探针的荧光原位杂交结果表明,H96276-2的体细胞中有42条染色体,包括20对小麦染色体和1对小麦-中间偃麦草易位染色体,中间偃麦草染色体的易位片段位于1对小麦染色体的端部。进而用重复序列探针pSc119进行第2次荧光原位杂交,证明H96276-2中的中间偃麦草染色体易位片段位于小麦2B染色体的短臂上。     

用分子标记定位源于中间偃麦草的小麦抗黄矮病基因

利用生物素标记的中间偃麦草基因组总DNA作为探针 ,对以L1为抗源的抗黄矮病小麦新品系H960 642的有丝分裂中期染色体进行原位杂交 ,结果表明 :H960 642是纯合的小麦 中间偃麦草易位系 ,携有抗黄矮病基因的中间偃麦草染色体片段易位到小麦染色体端部 .采用小麦第 7部分同源群上的 8个RFLP探针进行Southern分析 ,结果表明 :H960 642的小麦 7D染色体长臂末端片段被中间偃麦草染色体 7X长臂末端片段所取代 ,即该染色体为T7DS·7DL -7XL ,易位断点位于Xpsr680和Xpsr965之间 ,距着丝点的遗传距离约 90～ 99cM .筛选出了与 7X上抗黄矮病基因紧密连锁的RFLP标记psr680和psr687.将该抗黄矮病基因定位于 7XL端部、RFLP遗传图Xpsr680与Xpsr687位点附近 .Ep 1同工酶分析结果佐证了RFLP分析结果 .     

小麦等离子束处理的生物学效应研究

本文以早熟品种冀麦31号和晚熟品系88—4284萌动种子为材料,报道了等离子束处理对小麦种子萌发的影响和细胞学效应。观察表明,等离子束长时间处理抑制种子发芽。在根尖细胞有丝分裂中期观察到染色体断裂和断片,冀麦31号染色体断裂的频率为0.49—1.34％,88—4284为0.21—2.14％。随着处理时间延长,染色体断裂频率逐渐提高。有丝分裂后期和末期出现大量的落后染色单体和染色体桥。在花粉母细胞减数分裂中期Ⅰ,经过等离子束处理的材料出现环状单价体。后期Ⅰ观察到染色体倒位造成的染色体桥和断片。在四分体期还有微核出现。     

Molecular cytogenetic analysis of durum wheat x tritordeum hybrids.

Southern and in situ hybridization were used to examine the chromosome constitution, genomic relationships, repetitive DNA sequences, and nuclear architecture in durum wheat x tritordeum hybrids (2n = 5x = 35), where tritordeum is the fertile amphiploid (2n = 6x = 42) between Hordeum chilense and durum wheat. Using in situ hybridization, H. chilense total genomic DNA hybridized strongly to the H. chilense chromosomes and weakly to the wheat chromosomes, which showed some strongly labelled bands. pHcKB6, a cloned repetitive sequence isolated from H. chilense, enabled the unequivocal identification of each H. chilense chromosome at metaphase. Analysis of chromosome disposition in prophase nuclei, using the same probes, showed that the chromosomes of H. chilense origin were in individual domains with only limited intermixing with chromosomes of wheat origin. Six major sites of 18S-26S rDNA genes were detected on the chromosomes of the hybrids. Hybridization to Southern transfers of restriction enzyme digests using genomic DNA showed some variants of tandem repeats, perhaps owing to methylation. Both techniques gave complementary information, extending that available from phenotypic, chromosome morphology, or isozyme analysis, and perhaps are useful for following chromosomes or chromosome segments during further crossing of the lines in plant breeding programs.     

Confocal analysis of chromosome behavior in wheat x maize zygotes.

Herein, we profile the first embryonic mitosis in a hybrid of wheat and maize by using a whole-mount genomic in situ hybridization method and immunofluorescence staining with a tubulin-specific antibody. We have successfully captured the dynamics of each set of parental chromosomes in the first zygotic division of the hybrid embryo 24-28 h after crossing. During the first zygotic metaphase, although both sets of parental chromosomes congressed into the equatorial plate of the zygote, the maize chromosomes tended to lag in comparison with the wheat chromosomes. During anaphase, each parental chromosome separated into its sister chromosomes; however, some of the maize chromosomes lagged around the metaphase plate as segregants. The maize sister chromosomes that did move toward the pole showed delayed and asymmetric movement as compared with the wheat ones. Immunological staining of tubulin revealed a bipolar spindle structure in the first zygotic metaphase. The kinetochores of the maize chromosomes that lagged around the metaphase plate did not attach to the spindle microtubules. These results suggest that factors on the kinetochores of maize chromosomes that are required to control chromosome movement are deficient in the zygotic cell cycle.     

The metaphase chromosome: functional inertia and ability related to the presence of sequences of single-stranded DNA

Isolated metaphase chromosomes from KB cells were used as template in an in vitro DNA synthesis assay. In these conditions, no synthesis was noticed, confirming template inactivity of isolated metaphase chromosomes. DNA synthesis was noticed after a pretreatment with either methanol-acetic acid or RNase A. Analysis of in vitro synthesized polydeoxyribonucleotides showed two fractions of 4 S and 7-8 S. These results suggest the presence in metaphase chromosome of single stranded DNA sequences. Such sequences are shown in DNA extracted from chromosomes. They would preexist in this organelle and would be unmasked by the treatments that restore template activity of metaphase chromosome.     

Genomic in situ hybridization to identify alien chromosomes and chromosome segments in wheat

Summary Genomic in situ hybridization was used to identify alien chromatin in chromosome spreads of wheat, Triticum aestivum L., lines incorporating chromosomes from Leymus multicaulis (Kar. and Kir.) Tzvelev and Thinopyrum bessarabicum (Savul. and Rayss) Löve, and chromosome arms from Hordeum chilense Roem. and Schult, H. vulgare L. and Secale cereale L. Total genomic DNA from the introgressed alien species was used as a probe, together with excess amounts of unlabelled blocking DNA from wheat, for DNA:DNA in-situ hybridization. The method labelled the alien chromatin yellow-green, while the wheat chromosomes showed only the orange-red fluorescence of the DNA counterstain. Nuclei were screened from seedling root-tips (including those from half-grains) and anther wall tissue. The genomic probing method identified alien chromosomes and chromosome arms and allowed counting in nuclei at all stages of the cell cycle, so complete metaphases were not needed. At prophase or interphase, two labelled domains were visible in most nuclei from disomic lines, while only one labelled domain was visible in monosomic lines. At metaphase, direct visualization of the morphology of the alien chromosome or chromosome segment was possible and allowed identification of the relationship of the alien chromatin to the wheat chromosomes. The genomic in-situ hybridization method is fast, sensitive, accurate and informative. Hence it is likely to be of great value for both cytogenetic analysis and in plant breeding programmes.     

Proteome analysis of human metaphase chromosomes

DNA is packaged as chromatin in the interphase nucleus. During mitosis, chromatin fibers are highly condensed to form metaphase chromosomes, which ensure equal segregation of replicated chromosomal DNA into the daughter cells. Despite >1 century of research on metaphase chromosomes, information regarding the higher order structure of metaphase chromosomes is limited, and it is still not clear which proteins are involved in further folding of the chromatin fiber into metaphase chromosomes. To obtain a global view of the chromosomal proteins, we performed proteome analyses on three types of isolated human metaphase chromosomes. We first show the results from comparative proteome analyses of two types of isolated human metaphase chromosomes that have been frequently used in biochemical and morphological analyses. 209 proteins were quantitatively identified and classified into six groups on the basis of their known interphase localization. Furthermore, a list of 107 proteins was obtained from the proteome analyses of highly purified metaphase chromosomes, the majority of which are essential for chromosome structure and function. Based on the information obtained on these proteins and on their localizations during mitosis as assessed by immunostaining, we present a four-layer model of metaphase chromosomes. According to this model, the chromosomal proteins have been newly classified into each of four groups: chromosome coating proteins, chromosome peripheral proteins, chromosome structural proteins, and chromosome fibrous proteins. This analysis represents the first compositional view of human metaphase chromosomes and provides a protein framework for future research on this topic.     

Flow Karyotyping of Wheat Addition Line “T240” with a Haynaldia villosa 6VS Telosome

To construct a chromosome-specific DNA library of chromosome 6VS from Haynaldia villosa, a wheat alien chromosome addition line T240 with a 6VS chromosome arm and its parental common wheat cv. CA921 were used to optimize protocols for preparing chromosome suspension amenable to flow sorting of the 6VS chromosome. Our results showed that root tips incubated sequentially in Hogland’s solution containing 1.25 mM hydroxyurea (DNA synthesis inhibitor) for 18 h, a hydroxyurea-free period for 2 h, and 1 μM trifluralin for 4 h (metaphase blocking reagent) increased the metaphase index (MI) by up to 62 % . Many metaphase chromosomes were released to form a chromosome suspension when root tips fixed in 2 % paraformaldehyde were treated in a homogenizer at 9,500 rpm for 10–15 s. Most of the released chromosomes had intact morphology. The background solution of chromosome suspension was clear and relatively few of cell debris and chromosome clumps. Univariate flow karyotypes were established with chromosome suspension flow through FACS Vantage 2000 SE flow cytometer. The flow karyotype of CA921 consisted of four chromosome peaks, whereas that for T240 had a fifth peak. This fifth peak in T240 contained the telosome, which was further confirmed to be 6VS through fluorescence in situ hybridization.     

Molecular Cytogenetic Identification of Triticum aestivum-Secale cereale Substitution and Addition Lines

Two substitution lines, designated as 930498 and 930483, and one addition line, designated as 930029, via Fo immature embryo culture of Triticum aestivum x octoploid triticale ( x Triti-cosecale Wittmack) were identified. Fluorescence in situ hybridization (FISH) using total genomic DNA of rye ( Secale cereale L. ) as probe corroborated the existence of rye chromosomes, further confirmed through chromosome paring at meiotic metaphase 1, C-banding and glutenin SDS- PAGE. The results demonstrated that the two substitution lines are ID/IR, and the addition line is also IR addition. Rye chromosomes that are distinct to the red-colored wheat chromosomes appear yellow-green at mitotic metaphase after FISH.     

Sequential chromosome banding and in situ hybridization analysis.

Different combinations of chromosome N- or C-banding with in situ hybridization (ISH) or genomic in situ hybridization (GISH) were sequentially performed on metaphase chromosomes of wheat. A modified N-banding-ISH/GISH sequential procedure gave best results. Similarly, a modified C-banding - ISH/GISH procedure also gave satisfactory results. The variation of the hot acid treatment in the standard chromosome N- or C-banding procedures was the major factor affecting the resolution of the subsequent ISH and GISH. By the sequential chromosome banding - ISH/GISH analysis, multicopy DNA sequences and the breakpoints of wheat-alien translocations were directly allocated to specific chromosomes of wheat. The sequential chromosome banding- ISH/GISH technique should be widely applicable in genome mapping, especially in cytogenetic and molecular mapping of heterochromatic and euchromatic regions of plant and animal chromosomes.     

Homoeology of rye chromosome arms to wheat

Summary Cytological markers such as diagnostic C-bands, telocentrics, and translocations were used to identify the arms of rye chromosomes associated with wheat chromosomes at metaphase I in ph1b mutant wheat × rye hybrids. Arm homoeologies of rye chromosomes to wheat were established from the results of metaphase I pairing combined with available data on the chromosomal location of homoeoloci series in wheat and rye. Only arms 1RS, 1RL, 2RL, 3RS, and 5RS showed normal homoeologous relationships to wheat. The remaining arms of rye appeared to be involved in chromosome rearrangements that occurred during the evolution of the genus Secale. We conclude that a pericentric inversion in chromosome 4R, a reciprocal translocation between 3RL and 6RL, and a multiple translocation involving 4RL, 5RL, 6RS, and 7RS are present in rye relative to wheat.     

DNA content of wheat monosomics at interphase estimated by flow cytometry

 Two complete, independently maintained sets of 21 monosomic wheat lines derived from cv. ‘Chinese Spring’ were analyzed for their DNA content at the G1 stage with flow cytometry. The DNA content of individual chromosomes was estimated by subtracting the value of a monosomic line from that of euploid wheat. Our data show that the estimated 2C DNA of individual wheat chromosomes in 21 monosomics at the G1 stage ranges from about 0.58 pg in chromosome 1D to approximately 1.12 pg in chromosome 3A. The A genome (2C=6.15 pg) seems to contain more DNA than the B (2C=6.09 pg) and D (2C=5.05 pg) genomes. Analysis of variance showed significant differences (α=0.01) in DNA content both among homoeologous groups and among genomes. Our estimates of interphase DNA content of wheat chromosomes from monosomic lines were poorly correlated to the chromosome sizes at metaphase (r=0.622, P≤0.01). This poor correlation might be due to differential coiling among chromosomes during cell division, possible bias of fluorochrome binding to heterochromatin, or heterogeneity among monosomic lines. Finally, flow cytometry may aid but cannot replace cytological checks in aneuploid maintenance. Received: 21 January 1997 / Accepted: 23 June 1997