应用基因组原位杂交及RFLP标记鉴定小麦中的的大麦染色体

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 ｓｉｒｕ 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Ｂｅｔｚｅｓ２Ｈ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）。用已定位有小麦第２部分同源群     

荧光原位杂交分析小麦-簇毛麦杂种减数分裂与染色体易位

利用基因组ＤＮＡ荧光原位杂交技术详细地研究了小麦－簇毛麦杂种染色体的减数分裂和配对行为,结果表明,在中期Ⅰ,小麦和簇毛麦染色体多呈两个单价体,在０．３％的ＰＭＣ中小麦与簇毛麦染色体发生配对;在后期Ⅰ时,单价体错分裂频率为３２．７％－３７．５％,另有０．７％的小麦－簇毛麦染色体重组易位出现;后期Ⅱ时,断列染色体的频率为２０．５％－２２．４％,还发现有０．８２％－１．７２％的自发易位染色体形成,此外,     

麦类作物原位杂交影响因素的研究

为了更好地利用原位杂交技术进行麦类作物外源染色体的检测和基因定位,对麦类作物原位杂交的影响因素进行了研究。（１）利用缺口转译法对克隆的ＤＮＡ 片段进行生物素标记,即节省时间,又可以获得较高的标记效率;对麦类作物的基因组ＤＮＡ,宜采用随机寡核苷酸引物标记法进行生物素标记,适当延长标记时间可以提高标记效率。（２）共变性法比较适宜麦类作物的原位杂交,分别变性法如掌握不当易使染色体产生膨胀现象,变性温度过高也会使黑麦（Ｓｅｃａｌｅ ｃｅｒｅａｌ）染色体的轮廓模糊不清。（３）应根据麦类作物亲本之间的亲缘关系决定封阻ＤＮＡ 的使用浓度,并利用生物素标记的簇毛麦（Ｈａｙｎａｌｄｉａ ｖｉｌｌｏｓａ）基因组ＤＮＡ 为探针,从普通小麦（Ｔｒｉｔｉｃｕｍ ａｅｓｔｉｖｕｍ ）－簇毛麦双二倍体的染色体中识别了簇毛麦的染色体。（４）麦类作物的原位杂交受洗脱强度的影响很大,利用甲酰胺进行洗脱可以获得背景清晰的原位杂交带型。随着原位杂交技术分辨率的不断提高,该技术还将用于单拷贝基因的染色体定位     

利用AABBDDDD八倍体培育小麦-簇毛麦二体附加系的研究

对普通小麦（Triticumaestivum）-节节麦（Aegilopssquarrosa）八倍体（2n=8x=56,AABBDDDD）与硬粒小麦（Triticumdurum）-簇毛麦（Haynaldiavillosa）六倍体（2n=6x＝42,AABBVV）杂交后,将所得七倍体杂种（AABBDDV）进行连续自交,在F4代中利用C-分带鉴定出可能的簇毛麦6V二体附加系95－7和2V二体附加系26－7,其花粉母细胞染色体在减数分裂中期I的配对构型分别为0.14I＋20．42＋1．5和0．10I＋20．07＋1.82;进一步将95－7和26－7的基因组DNA用EcoRI酶切,分别用小麦族第6部分同源群短臂探针Psr113和第2部分同源群长臂探针BCD240进行Southern杂交,结果显示具有簇毛麦的特异杂交带,进一步确证了95－7和26－7分别是普通小麦-簇毛麦6V和2V二体附加系。     

导入小麦的外源染色体片段的准确鉴定及外源抗性基因的稳定性分析

用抗白粉病的普通小麦一簇毛麦６ＶＳ／６ＡＬ易位系与普通小麦品种扬麦５号、普通小麦一簇毛麦６Ｖ代换系和中国春６Ａ双端二体以及６Ｖ代换系和６Ａ双端二体配制了４个测交组合,分析了这４个杂交组合Ｆ１ＰＭＣ’ｓＭＩＣ－分带的减数分裂构型。在（６ＶＳ／６ＡＬ易位系×扬麦５号）和（６ＶＳ／６ＡＬ易位系×６Ｖ代换系）的Ｆ１ＰＭＣ’ｓＭＩ,分别观察到由易位染色体与６Ａ染色体和６Ｖ染色体配对形成的具有特定Ｃ－分带带型的棒状二价体,杂种中的棒状二价体数目高于各自亲本中的棒状二价体数。在（易位系×Ｃ．Ｓ．ｄ．ｄ．ｔ６Ａ）Ｆ１中,在８７．９％的ＰＭＣ中观察到由易位染色体长臂与６ＡＬ端体配对形成的异形二价体（ｔｌ”）。而在（６Ｖ代换系×Ｃ．Ｓ．ｄ．ｄ．ｔ６Ａ）Ｆ１中,９６．６８％的ＰＭＣ具有两个单价端体（ｔ’,ｔ’）。该结果进一步证实易位涉及簇毛麦染色体６ＶＳ和小麦染色体６ＡＬ,易位断点靠近着丝粒。在减数分裂中,易位染色体的正常配对和分离,保证了６ＶＳ上白粉病抗性基因Ｐｍ２１的正常传递,为这一新抗源在小麦育种中的应用奠定了细胞学基础。     

将大赖草种质转移给普通小麦的研究：Ⅶ.一个普通小麦—大赖草等臂？…

通过花粉母细胞减数分裂中期Ｉ染色体配对构型分析ＧｉｅｍｓａＣ－分带,从普通小麦－大赖草第７条染色体二体异附加系自交后代中选育并鉴定出了９３Ｇ５１－９和９３Ｇ５２－８２个等臂染色体异附加系,该异附加系在花粉母细胞减数分裂中期Ｉ,其等臂染色体自身两臂配对频率高,染色体易发生断裂,且又携带有较抗赤霉病的基因,是向小麦转移大赖草赤霉病抗性基因的有用中间材料。     

将大赖草种质转移给普通小麦的研究：VI.端体异附加系的选育与鉴定

利用根尖细胞有丝分裂中期染色体计数,花粉母细胞减数分裂中期１（ＰＭＣ ＭＩ）染色体构型分析仪及Ｃ－分带,从普通小麦中国春与大赖草（ＬｅｙｍｕｓｒａｃｅｍｏｓｕｓＬａｍ．）杂种回交后代中,选育出两个端二体异附加系９５Ｇ０９,９５Ｇ１１和一个添加了一对大赖草第１４号染色体和另一对端体的双重异附加系９５Ｇ３０２（２ｎ＝４４＋２ｔ）,它们的ＰＭＣ ＭＩ染色体配对构型分别为０．２１个单价体（其中０．１６个端     

四棱豆的核型和G-带带型研究

用改良ＡＳＧ法在四棱角Ｐｓｏｐｈｏｃａｒｐｕｓ ｔｅｔｒａｇｏｎｏｌｏｂｕｓ（Ｌ．）ＤＣ有丝分裂中期,染色体全长显示了密切邻近的多重Ｇ－带带纹,并进行了核型和Ｇ－带带型分析。核型公式为２ｎ＝１８＝４ｍ＋１４ｓｍ（２ＳＡＴ）,核型类型为２Ｂ。Ｇ－带带型分析表明,同源染色体的带纹数目、分布位置、染色深浅基本一致,可以较准确地进行配对;非同源染色体的带型有明显差异,可以准确区分。讨论了改良ＡＳＧ法在核型     

组织培养诱导的普通小麦─黑麦代换系和附加系分子细胞遗传学检测

通过组织培养从普通小麦（ＴｒｉｔｉｃｕｍａｅｓｔｉｖｕｍＬ．）与八倍体小黑麦（×ＴｒｉｔｉｃｏｓｅｃａｌｅＷｉｔｍａｃｋ）杂种Ｆ０幼胚再生植株后代中获得２个代换系９３０４９８、９３０４８３和１个附加系９３００２９。以黑麦（ＳｅｃａｌｅｃｅｒｅａｌｅＬ．）基因组ＤＮＡ为探针,采用荧光原位杂交（ＦＩＳＨ）证实了黑麦染色体的存在。在有丝分裂中期,经ＦＩＳＨ处理的黑麦染色体为黄绿色,明显区别于红色的小麦染色体。染色体配对、Ｃ分带、麦谷蛋白电泳分析,证明两个代换系为１Ｄ／１Ｒ代换,附加的也是黑麦１Ｒ染色体     

用基因组原位杂交与RFLP标记鉴定小麦——簇毛麦抗白粉病代换系

用荧光素标记的簇毛麦(Haynaldia villosa)基因组总DNA作探针,以普通小麦基因组总DNA作封阻,与花粉母细胞减数分裂中期Ⅰ制片的染色体进行原位杂交。结果表明,抗白粉病小麦品系GN22是普通小麦-簇毛麦二体代换系;用已定位在小麦第6部分同源群上的RFLP探针psr113、psr371进行Southern分析,进一步证明,小麦品系GN21、GN22是普通小麦-簇毛麦6A(6V)代换系;结合同工酶等电聚焦电泳分析,首次把簇毛麦编码的α-淀粉酶-1生化位点定位在簇毛麦6V染色体长臂上,暂命名为α-Amy-Ⅴ1。研究结果表明,原位杂交与RFLP技术相结合是全面、准确鉴定小麦外源染色体及其与小麦染色体部分同源关系的有效方法。     

Development of Wheat-alien Lines with Added Leymus racemosus Chromosomes and 6VS/6AL Translocation Chromosomes

By chromosome C-banding and bi-color fluorescence in situ hybridization (FISH) using digoxigenin-labelled total genomic DNA of Leymus racemosus (Lam.) Tzvel. and biotinylated total genomic DNA of Haynaldia villosa (L.) Schur as probes, three wheat-alien lines with L. racemosus Lr.7 addition and H.　villosa 6VS/6AL translocated chromosomes, and eight lines with L.　racemosus Lr.14 addition and H.villosa 6VS/6AL translocated chromosomes were respectively identified from DALr.7×T6VS/6AL (93G51-4×P64) and DALr.14×T6VS/6AL （94G15×P64）F2 or F3 hybrids. Fluorescein-isothiocyanate-conjugated avidin and rhodamine-conjugated sheep anti-digoxigenin Fab fragment were used in bi-color FISH detection. The chromosomes of L.racemosus and 6VS fragment of H.　villosa were simultaneously detected by their red and green fluorescence. Powdery mildew and scab resistance were also evaluated. The result showed that the obtained plants had high resistance to these two diseases. The potential usage of bi-color FISH in identifying chromatin of L.racemosus and H.villosa was discussed.     

筛选利用小麦微卫星标记追踪簇毛麦各条染色体

选用定位于普通小麦7个部分同源群上的276对微卫星引物对普通小麦中同春和簇毛麦的基因组DNA进行扩增分析,有148对引物可在两个物种间检测到多态性。利用上述显示多态性的引物进一步对7个中国春-簇毛麦二体附加系进行扩增分析,筛选出分别可用来追踪簇毛麦1V至7V染色体的引物wmc49（1BS）、wmc25（2BS）、gdm36（3DS）、gdml45（4AL）、wmc233（5DS）、wmc256（6AL）和gwm344（7BL）。此外还发现6DS上的微卫星引物gwm469可以用来追踪簇毛麦的2V染色体;2DS上的微卫星引物gdm107可用来追踪簇毛麦的6V染色体。进一步用涉及不同簇毛麦和小麦背景的小麦一簇毛麦染色体附加系、代换系和易位系进行扩增分析,这些微卫星标记也可用来鉴定簇毛麦的各条染色体。因此,这然簇毛麦染色体特异的微卫星标记可用来追踪普通小麦背景中的簇毛麦染色体。     

簇毛麦染色体组特异性RAPD标记的筛选、定位和应用

以普通小麦中国春、中国春－簇毛麦二体附加系以及不同来源的簇毛麦为材料,用100个10碱基随机引物进行RAPD扩增。引物OPF02能在不同来源的簇毛麦及所有中国春－簇毛麦二体附加系中扩增出一条长约750bp的片段OPF02 750。普通小麦和硬粒小麦不能扩增出该片段。因此,OPF02 750为分布于簇毛麦所有染色体上的一个簇毛麦染色体组特异片段。用引物OPF02对普通小麦－簇毛麦双二倍体、硬粒小麦－簇毛麦双二倍体以及几个普通小麦的簇毛麦二体代换系、二体附加系进行检测,发现NAU302已经丢失了其所附加的簇毛麦3V染色体。     

簇毛麦基因组特异性PCR标记的建立和应用

以普通小麦中国春、簇毛麦、中国春－簇毛麦二体附加系和代换系为材料进行RAPD分析,筛选出一个簇毛麦基因组特异性RAPD片段OPFO2757,该片段分布于簇毛麦所有染色体上。在对OPFO2757进行克隆、测序的基础上,设计一对PCR引物,建立了簇毛麦基因组特异性PCR标记。用这对PCR引物对不同普通小麦品种、不同硬粒小麦品种、不同居群的簇毛麦、中国春－簇毛麦二体附加系、中国春－簇毛麦二体代换系、普通小麦－簇毛麦双二倍体、硬粒小麦－簇毛麦双二倍体等材料进行扩增,凡具有簇毛麦染色体的材料都能扩增出一条长为677bp的DNA片段,而不具簇毛麦染色体的材料包括大麦、黑麦、长穗偃麦草、中间偃麦草等不能扩增出该片段。所以,该特异性PCR标记可用于快速跟踪检测小麦背景中的簇毛麦染色体。     

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。     

Identification of the parental chromosomes of the wheat-alien amphiploid Agrotana by genomic in situ hybridization.

Labelled total genomic DNA from four alien species, Thinopyrum ponticum (Host) Beauv. (2n = 70, genomes J1J1J1J2J2), Th. bessarabicum (Savul. &Rayss) Love (2n = 14, genome J), Th. elongatum (Host) Beauv. (2n = 14, genome E), and Haynaldia villosa (L.) Schur. (2n = 14, genome V), were used as probes in combination with blocking wheat DNA for in situ hybridization of the chromosomes of Agrotana, a wheat-alien hybrid (2n = 56) of unknown origin. The results showed that genomic DNA probes from Th. ponticum and Th. bessarabicum both clearly revealed 16 alien and 40 wheat chromosomes in Agrotana, indicating that the J genome present in these two species has a high degree of homology with the alien chromosomes in Agrotana. Biotinylated genomic DNA probe from Th. elongatum identified 10 chromosomes from Agrotana, while some regions of six other chromosomes yielded a weak or no signal. The probe from H. villosa produced no differential labelling of the chromosomes of Agrotana. The genomic formula of Agrotana was designated as AABBDDJJ. We suggest that the alien parent donor species of Agrotana is Th. ponticum rather than Th. bessarabicum. Genomic relationships of the three Thinopyrum species are discussed in relation to the distribution of GISH signals in the chromosomes of Agrotana.     

The genetic identity of alien chromosomes in potato breeding lines revealed by sequential GISH and FISH analyses using chromosome-specific cytogenetic DNA markers.

Genomic in situ hybridization (GISH) is one of the most popular and effective techniques for detecting alien chromatin introgressed into breeding lines; however, GISH analysis alone does not reveal the genetic identity of the alien chromosomes. We previously isolated a set of bacterial artificial chromosomes (BACs) specific to each of the 12 potato chromosomes. These BAC clones can be used as chromosome-specific cytogenetic DNA markers (CSCDMs) for potato chromosome identification. Here we demonstrate that GISH and fluorescence in situ hybridization (FISH), using CSCDMs, can be performed sequentially on the same chromosome preparations. Somatic metaphase chromosomes prepared using an enzymatic digestion and "flame-drying" procedure allows repeated probing up to five times without significant damage to chromosome morphology. The sequential GISH and FISH analyses reveal the genomic origin and genetic identity of the alien chromosomes in a single experiment and also determine whether an alien chromosome has been added to the genetic background of potato or is substituting for a homoeologous potato chromosome. The sequential GISH and FISH procedures should be widely applicable for germplasm characterization, especially in plant species with small-sized chromosomes.     

<Emphasis Type="Italic">Agropyron elongatum</Emphasis> chromatin localization on the wheat chromosomes in an introgression line

The introgressed small-chromosome segment of Agropyron elongatum (Host.) Neviski (Thinopyrum ponticum Podp.) in F₅ line II-1-3 of somatic hybrid between common wheat (Triticum aestivum L.) and A. elongatum was localized by sequential fluorescence in situ hybridization (FISH), genomic in situ hybridization (GISH) and karyotype data. Karyotype analysis offered basic data of arm ratios and relative lengths of 21 pairs of chromosomes in parent wheat Jinan177 and hybrid II-1–3. Using special high repetitive sequences pSc119.2 and pAs1 for FISH, the entire B- and D-genome chromosomes were detected. The FISH pattern of hybrid II-1-3 was the same as that of parent wheat. GISH using whole genomic DNA from A. elongatum as probe determined the alien chromatin. Sequential GISH and FISH, in combination with some of the karyotype data, localized the small chromosome segments of A. elongatum on the specific sites of wheat chromosomes 2AL, 1BL, 5BS, 1DL, 2DL and 6DS. FISH with probe OPF-03₁₂₉₆ from randomly amplified polymorphic DNA (RAPD) detected E-genome chromatin of A. elongatum, which existed in all of the small chromosome segments introgressed. Microsatellite primers characteristic for the chromosome arms above were used to check the localization and reveal the genetic identity. These methods are complementary and provide comprehensive information about the genomic constitution of the hybrid. The relationship between hybrid traits and alien chromatin was discussed.     

Mapping of a BYDV resistance gene from Thinopyrum intermedium in wheat background by molecular markers

The wheat line H960642 is a homozygous wheat-Thinopyrum intermedium translocation line with resistance to BYDV by genomicin situ hybridization (GISH) and RFLP analysis. The genomic DNA ofTh. intermedium was used as a probe, and common wheat genomic DNA as a blocking in GISH experiment. The results showed that the chromosome segments ofTh. intermedium were transferred to the distal end of a pair of wheat chromosomes. RFLP analysis indicated that the translocation line H960642 is a T7DS-7DL-7XL translocation by using 8 probes mapped on the homoeologous group 7 in wheat. The translocation breakpoint is located between Xpsr680 and Xpsr965 about 90–99 cM from the centromere. The RFLP markers psr680 and psr687 were closely linked with the BYDV resistance gene. The gene is located on the distal end of 7XL around Xpsr680 and Xpsr687. Project supported by the 863 program and the National Natural Science Foundation of China (Grant No. 39680027).     

Influence Factors of in Situ Hybridization in Triticeae

In order to increase the efficiency, accuracy, fidelity and reliability of in situ hybridization to identify the alien chromosomes and chromosome fragments in triticeae, major steps including probe labelling, chromosome denaturation, DNA concentration for blocking and post-hybridization washing in in situ hybridization were optimized. The results are as fel-lows. (1) The cloned repetitive DNA sequence could be biotin labelled more efficiently by nick translation than by random oligonucleotide labelling method: whereas the random oligonucleotide labelling is more suitable for genomic DNA probe and the labelling efficiency could be increased by prolonging the labelling time appropriately. (2) Denaturation of the biotinylated probe and chromosomes together in oven at 75 ℃ showed the satisfactory results of in situ hybridization, but the contour of treated rye chromosomes often became blurred when the temperature of denaturation was higher than 85℃. When 70% formamide (in 2 × SSC) was used to denature the chromosome DNA, rye chromosomes often swelled although the biotinylated signals could be detected. (3) The unlabeled DNA concentrations for blocking were tested in genomic in situ hybridization to detect the Haynaldia villosa chromosomes with biotin labelled H. villosa genomic DNA as probe. The best contrast between H. villosa and wheat chromosomes was obtained without using the blocking DNA (unlabeled wheat genomic DNA). (4) Post-hybridization washes were carried out in 50% formamide (in 2 × SSC) or in 2 × SSC at different temperature. When the post-hybridization washing temperature were increased gradually from room temperature to 42℃ in 50% formamide (in 2 × SSC). specific in situ hybridization signals on chromosome in triticeae were observed using both biotinylated repetitive DNA and genomic DNA as probe. With the improved resolution of this protocol, in situ hybridization would be widely applied to wheat breeding and genetics researches.