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－重复序列荧光原位杂交技术对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（Ａｇｒｏｐｙｒｏｎ ｉｎｔｅｒｍｅｄｉｕｍ）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ｐ     

单体异附加系花药培养创制小麦- 中间偃麦草纯合易位系

利用单体异附加系花药培养细胞工程途径,诱导小麦与中间偃麦草发生染色体易位,通过细胞学分析、荧光原位杂交(F ISH)和SSR鉴定出纯合易位系.研究结果表明,经单体异附加系花药培养创制出1个小麦-中间偃麦草纯合易位系99-803;其花粉母细胞(PM C s)减数分裂中期I染色体构型为18.42个环状二价体 2.57个棒状二价体 0.01个单价体;中间偃麦草的7A i-1染色体与小麦7A或7B染色体发生了非罗伯逊易位,且中间偃麦草易位片段较小;通过该途径获得纯合易位系的频率约为2%.以上结果表明,单体异附加系花药培养是一条向小麦转移异源染色体小片段(基因)的快速高效途径.     

小麦-中间偃麦草双体异附加系的鉴定

利用形态学、细胞学、A-PADE和RAPD方法,对5个小麦-中间偃麦草(Thinopyrum intermedium)双体异附加系Line 1、Line 4、Line 10、Line 14和Line 15进行了鉴定。细胞学鉴定结果表明,它们根尖细胞染色体数目为2n=44,花粉母细胞减数分裂中期Ⅰ(PMCMⅠ)染色体构型为2n=22 Ⅱ,具有高度的细胞学稳定性;形态学鉴定和A-PADE电泳分析证明,Line 1和Line 15可能附加了中间偃麦草第7部分同源群的染色体,Line 10和Line 14可能附加了中间偃麦草第1部分同源群的染色体,Line4则可能同时存在多种染色体变异;RAPD分析表明,在供试的100个随机引物中,有5个引物S21、S29、S57、S121和S152能够在亲本中间偃麦草和双体异附加系中稳定扩增出特异带型,并可作为异附加系所附加染色体的特异RAPD标记。     

小麦—中间偃麦草衍生材料农艺性状、HMW-GS及GISH鉴定

本研究分析了143个小麦—中间偃麦草种质材料的农艺性状、高分子量麦谷蛋白亚基及部分代表性材料的染色体构成,旨在为小麦育种中广泛有效地利用这些种质提供有用信息。结果表明,小麦—中间偃麦草种质主要农艺性状变异丰富,其在穗长、小穗数和分蘖数性状上明显优于主栽品种,分别有142(99.3%)、125(87.4%)和62(43.4%)个小麦—中间偃麦草材料的穗长、分蘖数和小穗数大于主栽品种的平均值。供试材料在Glu-1的3个基因位点上共检测到12个等位变异,形成15种亚基组合类型,以(2*,7+8,5+10)为主,占所有材料的25.7%;Glu-A1(1和2*)、Glu-B1(7+8)和Glu-D1(5+10)位点的优质亚基比例分别达到了68.4%、68.4%和52.0%,有102(71.3%)个材料在Glu-1的2或3个位点同时具有优质亚基;有17个材料的优质亚基组合为(2*,7+8,5+10)或(1,7+8,5+10),且在穗长、小穗数和分蘖数性状上均优于主栽品种。进一步对30个代表性材料GISH分析发现,8个为八倍体小偃麦,其他为非整倍体。研究结果表明这些材料可以作为改良普通小麦的有益基因资源。     

抗白粉病耐盐小麦-中间偃麦草附加系‘山农120211’的鉴定

以中间偃麦草(Thinopyrum intermedium,2n=42)与普通小麦‘烟农15’杂交,从其杂种后代中选育出一个细胞学稳定的二体异附加系‘山农120211’,该研究对其细胞学和主要性状特点进行了鉴定。白粉病抗性鉴定结果表明,‘山农120211’成株期对白粉病的田间抗性为免疫,苗期对白粉病菌种E09表现为免疫。以耐盐品种‘山融3号’为对照进行苗期耐盐性鉴定表明,‘山农120211’耐盐级别为2级(较强)。细胞学鉴定表明:‘山农120211’根尖细胞染色体数目为2n=44,PMC MI染色体构型为2n=22Ⅱ,具有高度的细胞学稳定性。以拟鹅观草基因组DNA为探针,‘烟农15’DNA为封阻,在‘山农120211’的根尖有丝分裂细胞中检测到2条染色体具有明显的杂交信号,确定其为二体异附加系。利用该实验室筛选的71对E组染色体特异分子标记,对‘山农120211’分析显示,标记BE494262在中间偃麦草和‘山农120211’中可以稳定扩增出1条440bp特异带,而‘烟农15’中缺少此带,BE494262可作为‘山农120211’中附加中间偃麦草染色体的特异标记。利用二倍体长穗偃麦草和一套中国春-长穗偃麦草异附加系(1Ee~7Ee),进一步将BE494262定位在2Ee染色体,确定‘山农120211’所附加的中间偃麦草染色体为2Ee染色体。     

中间偃麦草的GISH分析

以染色体组为E^eE^e的二倍体长穗偃麦草（Thinopyrum elongatum,2n=2x=14)、染色体组为E^bE^b的二倍体比萨偃麦草（Th.bessarabicum,2n=2x=14）、染色体组为StStStSt的四倍体拟鹅冠草（Pseudoroegneiria strigosa,2n=4x=28)的总基因组DNA为探针,对中间偃麦草（Th.intermedium)进行GISH分析。结果表明,中间偃麦草是由2个亲缘关系较近的染色体组、1个亲缘关系较远的染色体组构成;中间偃麦草所含的亲缘关系较近的染色体组分别与二倍长穗偃麦草染色体组E^e、比萨偃麦草染色体组E^b、以及1个亲缘关系较远的染色体组与拟鹅冠草染色体组St基本相似,但不完全一样,因此,中间偃麦草的染色体组用E^etE^etE^btStSt表示。     

一个小麦-中间偃麦草异代换系的形态学和细胞学鉴定

中间偃麦草含有丰富的优良基因,在小麦的遗传改良中具有重要利用价值。对从中间偃麦草与小麦品种烟农15杂种后代(BC2F4)中选育的小麦种质系山农0095进行形态学和细胞学鉴定,结果表明：山农0095株高78cm,穗长17．3cm,旗叶长36．3cm,旗叶宽3．03cm,茎杆粗壮,繁茂性好,既长又宽的旗叶、长圆锥型穗是其显著的形态学特征;其根尖细胞染色体数日为2n=42,花粉母细胞减数分裂中期Ⅰ(PMC M Ⅰ)染色体构型为2n=21Ⅱ;它与普通小麦的杂种FⅠPMC M Ⅰ绝大多数细胞出现2个单价体,没有观察到多价体,平均染色体构型为2n=20．08Ⅱ 1．84Ⅰ。以上结果表明,山农0095是一个小麦-中间偃麦草的双体异代换系。     

一个多抗小麦—中间偃麦草新种质的选育和细胞分子生物学鉴定

经过多年田间和温室接种抗病性鉴定,从(77-5433×中5)杂交组合花药培养后代中选育出一个兼抗大麦黄矮病、条锈、叶锈和秆锈4种小麦主要病害的新种质遗4212。遗4212的体细胞染色体数为42,在减数分裂中期Ⅰ,在几乎所有的花粉母细胞中都可以观察到21个二价体,这说明遗4212是一个在遗传上业已稳定的整倍体材料。对(遗4212×77-5433)F_1代花粉母细胞的观察表明,遗4212可能是含1对外源中间偃麦草染色体的代换系或具较大中间偃麦草染色体片段的易位系。用基因组原位杂交(genomic in situ hybridization,GISH)对遗4212的有丝分裂中期相、减数分裂后期Ⅰ相和(遗4212×77-5433)F_1代花粉母细胞减数分裂中期Ⅰ、后期Ⅰ进行了检测,确证遗4212含1对外源中间偃麦草染色体。这些结果表明,遗4212是一个小麦一中间偃麦草代换系,其抗病性来自其携带的1对中间偃麦草。     

小麦-中间偃麦草双体异附加系的选育和鉴定

在小麦-中间偃麦草59个杂交后代种质系中,筛选出6个小麦-中间偃麦草双体异附加系(line0605,line0607,line0609,line0610,line0611,line0625),并对其进行了形态学、白粉病抗性、细胞学和RAPD鉴定。形态学结果表明:6个双体异附加系农艺性状较好地结合了双亲的优良特点;细胞学结果表明:6个双体异附加系具有高度的细胞学稳定性,花粉母细胞减数分裂中期Ⅰ(PMCMI)的染色体构型为2n=22Ⅱ;RAPD分析表明:在供试的209个随机引物中有5个引物分别能在6个异附加系中稳定地扩增出不同的特异带型,可以作为各个异附加系所附加染色体的特异分子标记;白粉病抗性鉴定结果表明:line0605表现免疫,line0610和line0625表现高抗,line0607表现中抗,line0609和line0611表现中感。     

抗黄矮病小麦-中间偃麦草易位系基因组可转化人工染色体文库的构建及初步筛选

利用抗黄矮病小麦 -中间偃麦草易位系HW6 4 2的细胞核DNA构建了一个可转化人工染色体 (transformation competentartificialchromosome,TAC)文库 ,文库由 2 .3× 10 6 克隆构成 ,重组率为 90 .4 8% ,平均插入片段大小为 2 2kb左右 ,约覆盖普通小麦单倍体基因组 2 .5倍 ,在该文库中分离得到单拷贝DNA序列的几率约是 95 .77%。文库保存在 2 4块 96孔板中 ,每个孔中约含有 10 0 0个不同的重组克隆 ,可以采用PooledPCR的方法对文库进行筛选。用来源于小麦的简单重复序列 (simplesequencerepeat,SSR)引物wms37扩增中间偃麦草、抗病易位系及感病材料 ,得到一条与抗性共分离的特异条带 ,约 4 5 0bp。将此特异标记条带转化为SCAR(sequencecharacterizedamplifiedregion)标记 ,用于筛选HW6 4 2基因组TAC文库 ,得到 12个阳性克隆。对阳性克隆进行了PCR Southern验证 ,以中间偃麦草基因组总DNA为探针与限制酶HindⅢ消化后的阳性克隆杂交 ,其中 10个阳性克隆分别有 1～ 6条杂交带 ,结果表明 ,这 10个阳性克隆可作为抗黄矮病相关基因筛选的候选克隆     

小偃麦异附加系TAI-13中来自中间偃麦草的低分子量麦谷蛋白亚基基因的分子克隆

通过分析小麦(TriticumaestivumL.)-中间偃麦草(Agropyronintermedium(Host)Beav)异附加系TAI-Ⅰ系列的麦谷蛋白SDS-PAGE电泳图谱和基因组DNA的PCR扩增产物,发现在异附加系TAI-13中附加的中间偃麦草染色体上具有编码高分子量和低分子量麦谷蛋白亚基基因的位点,属于第一同源群。随后,采用RT-PCR方法,从TAI-13的未成熟子粒中克隆了4个来自中间偃麦草的低分子量麦谷蛋白亚基基因。序列分析表明,13003、13006和13054是包括信号肽编码序列的全长基因,而13514没有信号肽编码序列。根据由核苷酸序列推导的蛋白质分子的N-末端氨基酸序列,这4个基因编码的麦谷蛋白亚基可分为3种类型,即Ai-M型(由基因13514编码,命名为LAi1)、Ai-Q型(由基因13006和13045编码,分别命名为LAi2和LAi3)和Ai-I型(由基因13003编码,命名为LAi4)。通过与小麦的低分子量麦谷蛋白亚基分子比较,发现Ai-M和Ai-Q是两种未见报道的新的低分子量麦谷蛋白亚基类型,而Ai-I型与小麦的I型亚基相似。氨基酸序列分析发现,基因13514编码的蛋白质亚基分子LAi1有较长的重复区(26个重复模块)和较多的半胱氨酸残基(9个),推测其可形成3个分子间二硫键,可能对增强面团的强度和粘弹性有正面效应。     

小偃麦异附加系TAI-13中来自中间偃麦草的低分子量麦谷蛋白亚基基因的分子克隆

通过分析小麦(Triticum aestivum L.)-中间偃麦草(Agropyron intermedium(Host)Beav)异附加系TA1-Ⅰ系列的麦谷蛋白SDS-PAGE电泳图谱和基因组DNA的PCR扩增产物,发现在异附加系TAI-13中附加的中间偃麦草染色体上具有编码高分子量和低分子量麦谷蛋白亚基基因的位点,属于第一同源群.随后,采用RT-PCR方法,从TAI-13的未成熟子粒中克隆了4个来自中间偃麦草的低分子量麦谷蛋白亚基基因.序列分析表明,13003、13006和13054是包括信号肽编码序列的全长基因,而13514没有信号肽编码序列.根据由核苷酸序列推导的蛋白质分子的N-末端氨基酸序列,这4个基因编码的麦谷蛋白亚基可分为3种类型,即Ai-M型(由基因13514编码,命名为LAi1)、Ai-Q型(由基因13006和13045编码,分别命名为LAi2和LAi3)和Ai-Ⅰ型(由基因13003编码,命名为LAi4).通过与小麦的低分子量麦谷蛋白亚基分子比较,发现Ai-M和Ai-Q是两种未见报道的新的低分子量麦谷蛋白亚基类型,而Ai-Ⅰ型与小麦的Ⅰ型亚基相似.氨基酸序列分析发现,基因13514编码的蛋白质亚基分子LAi1有较长的重复区(26个重复模块)和较多的半胱氨酸残基(9个),推测其可形成3个分子间二硫键,可能对增强面团的强度和粘弹性有正面效应.     

Characterization of six wheat x Thinopyrum intermedium derivatives by GISH, RFLP, and multicolor GISH.

Restriction fragment length polymorphism (RFLP) analysis and multicolor genomic in situ hybridization (GISH) are useful tools to precisely characterize genetic stocks derived from crosses of wheat (Triticum aestivum) with Thinopyrum intermedium and Thinopyrum elongatum. The wheat x Th. intermedium derived stocks designated Z1, Z2, Z3, Z4, Z5, and Z6 were initially screened by multicolor GISH using Aegilops speltoides genomic DNA for blocking and various combinations of genomic DNA from Th. intermedium, Triticum urartu, and Aegilops tauschii for probes. The probing (GISH) results indicated that lines Z1 and Z3 were alien disomic addition lines with chromosome numbers of 2n = 44. Z2 was a substitution line in which chromosome 2D was substituted by a pair of Th. intermedium chromosomes; this was confirmed by RFLP and muticolour GISH. Z4 (2n = 44) contained two pairs of wheat--Th. intermedium translocated chromosomes; one pair involved A-genome chromosomes, the other involved D- and A- genome chromosomes. Z5 (2n = 44) contained one pair of wheat--Th. intermedium translocated chromosomes involving the D- and A-genome chromosomes of wheat. Z6 (2n = 44) contained one pair of chromosomes derived from Th. intermedium plus another pair of translocated chromosomes involving B-genome chromosomes of wheat Line Z2 was of special interest because it has some resistance to infection by Fusarium graminearum.     

一个小麦/黑麦小片段染色体易位系的创制和鉴定

从来源于组合中国春×M27(1R/1D代换系)的8个花粉植株中,分离获得1个小麦/黑麦小片段染色体易位系WER-1-2。C-分带和荧光原位杂交结果显示,衍生出易位系WER-1-2的原始麦穗含有1条完整的1R染色体和1条长臂端部发生缺失的1R染色体,所缺失部分易位到1条小麦染色体上。推断该易位是在花药离体培养过程中经异常有丝分裂产生的,而非异常减数分裂的产物。表明花药培养是一条实现异源染色体小片段(基因)向小麦转移的快速而有效的途径。     

Genomic in situ hybridization (GISH) analyses of Thinopyrum intermedium, its partial amphiploid Zhong 5, and disease-resistant derivatives in wheat

Genomic in situhybridization (GISH) to root-tip cells at mitotic metaphase, using genomic DNA probes from Thinopyrum intermedium and Pseudoroegneria strigosa, was used to examine the genomic constitution of Th. intermedium, the 56-chromosome partial amphiploid to wheat called Zhong 5 and disease-resistant derivatives of Zhong 5, in a wheat background. Evidence from GISH indicated that Th. intermedium contained seven pairs of St, seven J^S and 21 J chromosomes; three pairs of Th. intermedium chromosomes with satellites in their short arms belonging to the St, J, J genomes and homoeologous groups 1, 1, and 5 respectively. GISH results using different materials and different probes showed that seven pairs of added Th. intermedium chromosomes in Zhong 5 included three pairs of St chromosomes, two pairs of J^S chromosomes and two pairs of St-J^S reciprocal tanslocation chromosomes. A pair of chromosomes, which substituted a pair of wheat chromosomes in Yi 4212 and in HG 295 and was added to 21 pairs of wheat chromosomes in the disomic additions Z1, Z2 and Z6, conferred BYDV-resistance and was identical to a pair of St-J^S tanslocation chromosomes (StJ^S) in Zhong 5. The StJ^S chromosome had a special GISH signal pattern and could be easily distinguished from other added chromosomes in Zhong 5; it has not yet been possible to locate the BYDV-resistant gene(s) of this translocated chromosome either in the St chromosome portion belonging to homoeologous group 2 or in the J^S chromosome portion whose homoeologous group relationship is still uncertain. Among 22 chromosome pairs in disomic addition line Z3, the added chromosome pair had satellites and belonged to the St genome and homoeologous group 1. Disomic addition line Z4 carried a pair of added chromosomes which was composed of a group-7 J^S chromosome translocated with a wheat chromosome; this chromosome was different to 7 Ai-1, but was identical to 7 Ai-2. The leaf rust and stem rust resistance genes were located in the distal region of the long arm, whereas the stripe rust resistance gene(s) was located in the short arm or in the proximal region of the long arm of 7 Ai-2. A pair of J^S-wheat translocation chromosomes, which originated from the WJ^S chromosomes in Z4, was added to the disomic addition line Z5; the added chromosomes of Z5 carried leaf and stem rust resistance but not stripe rust resistance; Z5 is a potentially useful source for rust resistance genes in wheat breeding and for cloning these novel rust-resistant genes. GISH analysis using the St genome as a probe has proved advantageous in identifying alien Th. intermedium in wheat. Received: 17 May 1999 / Accepted: 22 June 1999     

用克隆池PCR法筛选小麦-簇毛麦易位系6VS/6AL基因组TAC文库获得抗病基因候选克隆

用根据抗病基因保守区设计的一对简并性引物,从小麦－簇毛麦易位系6VS／6AL cDNA中PCR扩增获得一个具有抗病基因核苷酸结合位点（Nucleotide binding site,NBS）结构特点的DNA片段克隆N7。从小麦－簇毛麦易位系6VS／6AL基因组TAC（Transformation-competent artificial chromosome,TAC）文库的22块96孔板提取所有2112个克隆池（每个池含约1000个克隆）的质粒,再根据N7的核苷酸序列设计一对特异引物,用克隆池PCR（pooled PCR)法经分级筛选从文库中获得一个阳性克隆。以N7为探针,通过Southern杂交证实了该TAC克隆为真正含有抗病候选基因的克隆。研究结果表明克隆池PCR法对克隆数目巨大的基因组文库的筛选很有效。     

应用基因组原位杂交鉴定蓝粒小麦及其诱变后代

应用基因组原位杂交技术（GISH）对普通小麦（Triticum aestivumL.)和长穗偃麦草[Agropyron elongatum(Host)Beauv,2n=10x=70]杂交后选育出的蓝粒小麦蓝-58及其诱变后代的染色体组成进行了鉴定。结果表明,GISH可方便地检测到小麦遗传背景中的长穗偃麦草染色体或易位的片段。如前人报道,蓝-58（2n=42)是一个具有2条长穗偃麦草4E染色体的异代换系（4E/4D）。LW004可能是一个具有两对相互易位染色体的纯合系,其田间表现磷高效特性,LW43-3-4为41条染色体的蓝单体（40W 1’4E）,种子颜色为浅蓝色,通过此法还检测出一些染色体结构发生很大变异的材料如4E的单端体（40W 1‘4E),种子颜色为浅蓝色,通过此法还检测出一些染色结构发生很大变异的材料如4E的单端体（40W 1‘t4E)以及组型为39W 1‘4E 1‘t4E的个体,此项研究结果更为直观地表明控制蓝粒体状的基因的确在来自长穗偃麦草的染色体上。同时说明有效的突变方法与灵活方便的检测手段的有机结合在染色体工程材料的创制和染色体工程育种中起着至关重要的作用。     

Pathogenicity of three RPV isolates of Barley Yellow Dwarf Virus on barley, wheat and wheat alien addition lines

Barleys with and without the Yd₂ resistance factor, wheat alien addition stocks with other barley yellow dwarf virus (BYDV) resistance factors and true wheats were challenged with three Australian isolates of BYDV-RPV. Yd₂ resistance was effective against two of the BYDV-RPV isolates and inoculated barleys which carry Yd₂ did not develop BYD symptoms and shoot growth was not affected. However, barleys with Yd₂ were susceptible to the third BYDV-RPV isolate. All barley lines inoculated with the third virus isolate developed typical BYD symptoms (yellowing), shoot growth was reduced compared to uninfected controls and virus titres determined by ELISA were high and similar in barleys with and without Yd₂. In contrast, resistances from Thinopyrum intermedium and Agropyron pulcherrimum in wheat backgrounds were effective against all three BYDV-RPV isolates. Shoot growth of inoculated plants with either of these resistance factors did not differ from uninfected controls and virus titres determined by ELISA were very low.     

Characterization of B chromosomes in Lilium hybrids through GISH and FISH

Supernumerary (B) chromosomes and small aberrant chromosomes were detected in Lilium hybrids and characterized through genomic in situ hybridization (GISH) and florescence in situ hybridization (FISH). Two small, supernumerary or B chromosomes were detected as extra chromosomes in a tetraploid plant derived from chromosome doubling of a hybrid (2n = 2x = 24) between a cultivar of the Longiflorum (L) and the Trumpet (T) group. When this tetraploid LLTT hybrid was crossed with a triploid LLO hybrid (O = Oriental), the B chromosome was transmitted to 73.4 % of the progenies. Based on GISH and FISH characterization, it was shown that the B chromosome consisted of two identical arms, with 5S rDNA hybridizing to the majority of it, which were flanked by normal telomeres, suggesting that this is an isochromosome. In another population, which is a backcross progeny between a F1 hybrid of Longiflorum × Asiatic (LA) and its Asiatic parent, the former produced functional 2n gametes which resulted in a triploid LAA progeny (2n = 3x = 36), in which three exceptional plants possessed 35 normal chromosomes and a small aberrant chromosome instead of the expected normal number of 36. In all three cases, the small aberrant chromosomes were isochromosomes which had obviously originated during the first backcross generation. These three chromosomes showed normal telomeres and mitosis. In addition, one of the new generated chromosomes possessed two 45S rDNA sites in the proximal positions. These new arisen isochromosomes were proposed to originate from centric breakage and fusion of two short arms of the missing chromosome in three genotypes, respectively, based on the comparison of arm lengths as well as rDNA loci. Their relevance to the origin of Bs is discussed.