从小麦-簇毛麦易位系TAC文库中筛选Hv-SITPK基因

本实验室已经通过基因芯片技术筛选到一个白粉菌诱导后上调表达的抗病相关基因Hv-S／TPK,并获得了它的全长cDNA序列。利用Hv-S／TPK的特异引物筛选小麦．簇毛麦6VS／6AL易位系基因组可转化人工染色体（Transformation-competent artificial chromsome,TAC）文库,获得了阳性TAC单克隆,并进一步获得了含有Hv-S／TPK cDNA序列的5160bp（GenBank Accession No．EU153366）的亚克隆。对亚克隆的序列分析结果表明,Hv-S/TPK基因在起始密码子和终止密码子之间有3个内含子和4个外显子,4个外显子序列与簇毛麦上已得到的Hv-S／TPK的cDNA序列100％同源。对起始密码子上游序列分析结果表明,该基因的调控序列中,含有W-Box、OCS-element等与抗病相关的元件。以TAC克隆为探针与小麦．簇毛麦6VS／6AL易位系有丝分裂中期染色体进行荧光原位杂交（Fluorescence in situ hybridization,FISH）,结果表明含有Hv-S／TPK基因的TAC克隆来自于簇毛麦。     

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。     

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。     

小麦硫代硫酸硫转移酶类似基因的克隆与定位

小麦-簇毛麦6VS/6AL易位系92R137含有抗白粉病基因Pm21。为了研究该易位系的抗病机理,应用mRNA差异显示和快速扩增cDNA未端（Rapid Amplification of cDNAEnd,RACE)技术对在白粉菌诱导后表达增强的基因进行了克隆,分离到1个命名为TaTST的全长cDNA序列。Northern杂交分析表明,TaTST基因在白粉菌诱导后表达明显增强,24h达到峰值,氨基酸序列同源性分析表明,TaTST与Datisca glomerata的硫代硫酸硫转移酶基因（rho-danese,EC,2.8.1.1)序列有64%相同,80%相似,用中国春缺体/四体系和端体系Southern杂交和基因特异性引物扩增（gene specific primer-PCR)将TaTST基因定位在小麦6B染色体短臂上,Southern杂交表明,该基因为单拷贝基因,由于在杨麦5号和6VS/6AL易位系间存在明显多态,可以推测在6VS上有TaTST的同源基因,TaTST是从小麦中分离的新基因。白粉菌诱导后的表达变化提示;TaTST与小麦抗白粉病反应有关。     

簇毛麦NBS类R基因相关序列的分子标记开发及其染色体定位

小麦近缘种簇毛麦携带许多尚未克隆的抗病(R)基因。NBS-LRR类型的R基因占已克隆植物R基因的绝大多数,因此,本研究根据NBS-LRR类型R基因的保守序列设计引物,从簇毛麦基因组DNA和cDNA中扩增获得23条相关序列。基于其中5条抗病基因同源序列(RGAs)H-56/d6、H-66/b2和CDS40设计引物,对小麦、簇毛麦、硬-簇双二倍体及其杂种以及已知携带个别簇毛麦染色体或染色体臂的小麦材料进一步进行PCR扩增,结果表明:3对引物均可对簇毛麦、硬-簇双二倍体进行特异扩增;同时,源于序列H-66/b2的引物可对1VL和6VL染色体臂进行特异扩增;源于序列CDS40的引物可在同时携带1VL和2VS或同时携带2VS和4V的小麦材料以及具有6VL的小麦材料中特异扩增,而H-56/d6的引物在携带1VL、2VS、4V和6V染色体臂或染色体的小麦背景中都不能获得目的片段的扩增。这些结果不仅为外源染色体臂在小麦背景中的追踪与鉴定提供了新的分子标记,而且这些标记还与外源染色体或染色体臂上的抗病基因或抗病基因同源物紧密连锁或共分离。     

簇毛麦5VS染色体臂特异分子标记开发与遗传效应分析

簇毛麦(Dasypyrum villosum(L.)P.Candargy 2n=14,VV)是小麦改良重要的三级基因源.簇毛麦5VS染色体臂上携带抗白粉病基因Pm55、抗条锈病基因Yr5V和籽粒硬度基因Dina/Dinb等优异基因.已创制的小麦-簇毛麦T5VS.5AL和T5VS.5DL易位系为小麦抗病和品质改良提供了优...     

小麦-簇毛麦6VS/6AL易位染色体对小麦农艺性状的影响

利用3类试验材料,即由不同生态类型的推广品种与小麦-簇毛麦6VS/6AL易位系经过杂交回交选育的高代品系(种),3份涉及6VS/6AL的高代分离品系以及5个以小麦-簇毛麦6VS/6AL易位系作杂交亲本的F2群体,对含有与不含有6VS/6AL易位染色体材料的产量、株高、穗长、德粒数、穗粒重和千粒重等农艺性状进行方差分析.结果表明,6VS/6AL易位染色体对后代的小穗数、穗粒数、穗粒重和产量等农艺性状没有表现出明显的影响,对穗长和千粒重表现出一定的正向效应.多数6VS/6AL衍生品系的株高与亲本相比有所增加,但在同一组合的不同品系之间表现出一定的差异,在育种过程中通过选择可改变增高趋势.6VS/6AL易位系对白粉病免疫,并且遗传稳定,对小麦的抗病育种是很有潜力的抗源亲本.     

小麦—簇毛表6VS／6AL易位系可转化人工染色体（TAC）文库的构建

可转化人工染色体（Ｔｒａｎｓｆｏｒｍａｔｉｏｎ ｃｏｍｐｅｔｅｎｔ Ａｒｔｉｆｉｃｉａｌ Ｃｈｒｏｍｏｓｏｍｅ,ＴＡＣ）是具有克隆和转移大片段基因能力的新型载体,是植被基因克隆和转化的有效工具。为了克隆泪科抗白粉病基因和其它基因,本研究用ＴＣＡ载体ｐＹＬＴＡＣ１７构建了带有抗白粉病基因Ｐｍ２１的小麦＝簇毛麦６ＶＳ／６ＡＬ易位系的基因组ＤＮＡ文库。该文库包含２１０万个克隆平均插入征段３５ｌｂ,相当于     

Identification, mapping, and application of polymorphic DNA associated with resistance gene Pm21 of wheat.

A new powdery mildew resistance gene designated Pm21, from Haynaldia villosa, a relative of wheat, has been identified and incorporated into wheat through an alien translocation line. Cytogenetic and biochemical analyses showed that chromosome arms 6VS and 6AL were involved in this translocation. Random amplified polymorphic DNA (RAPD) analysis was performed on recipient wheat cultivar Yangmai 5, the translocation line, and H. villosa with 180 random primers. Eight of the 180 primers amplified polymorphic DNA in the translocation line, and the same results were obtained in four replications. Furthermore, RAPD analysis was reported for substitution line 6V, seven addition lines (1V-7V), and the F1, as well as F2 plants of (translocation line x 'Yangmai 5'), using two of the eight random primers. One RAPD marker, specific to chromosome arm 6VS, OPH17-1900, could be used as a molecular marker for the detection of gene Pm21 in breeding materials with powdery mildew resistance introduced from H. villosa. Key words : RAPD analysis, 6VS-specific marker, Pm21, Erysiphe graminis f.sp. tritici, Triticum aestivum - Haynaldia villosa translocation.     

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.     

基因组分析与小麦抗病育种

系统总结了南京农业大学细胞遗传研究所近 2 0多年来利用基因组分析方法培育从簇毛麦 (Haynaldiavil losaSch .)、大赖草 (LeymusracemosusLam .)、鹅观草 (RoegneriakamojiC .Koch)和纤毛鹅观草 (R .ciliaris (Trin .)Nevs ki)导入白粉病和赤霉病抗性的小麦种质的研究进展。利用染色体C_分带、基因组原位杂交、分子标记 (特别是RFLP)等技术与非整倍体分析相结合对所创制的种质进行了系统分析与鉴定。还对所培育的小麦种质在育种实践和理论研究中的潜在价值及相关问题进行了讨论     

基因组分析与小麦抗病育种

系统总结了南京农业大学细胞遗传研究所近20多年来利用基因组分析方法培育从簇毛麦(Haynaldia villosa Sch.)、大赖草(Leymus racemosus Lam.)、鹅观草(Roegneria kamoji C. Koch)和纤毛鹅观草(R. ciliaris (Trin.) Nevski)导入白粉病和赤霉病抗性的小麦种质的研究进展.利用染色体C-分带、基因组原位杂交、分子标记(特别是RFLP)等技术与非整倍体分析相结合对所创制的种质进行了系统分析与鉴定.还对所培育的小麦种质在育种实践和理论研究中的潜在价值及相关问题进行了讨论.     

簇毛麦端体6VS的显微切割及其专化DNA序列的克隆和分析

从簇毛麦(Haynaldia villosa (L.) Schur.)组合CA9211/RW15(6D/6V异代换系)幼胚培养SC2后代中,用原位杂交方法鉴定出T240-6为6VS端体异代换系. 以此为材料,采用微细玻璃针切割法及"单管反应"技术体系,对6VS进行切割分离及LA (Linker adaptor)-PCR扩增.扩增带在100～3 000 bp 之间,大部分集中在600～1 500 bp.利用32P标记的簇毛麦基因组为探针进行Southern杂交,证实扩增产物来源于簇毛麦.扩增产物纯化后,连接到pGEM-T载体上,构建了6VS DNA质粒文库.对文库的分析表明,文库大约有17 000个白色克隆;插入片段分布在100～1 500 bp,平均600 bp.点杂交结果表明,37%克隆有中度到强烈的杂交信号,证明含有中度或高度重复序列;63%克隆有较弱的信号或没有信号,证明为单/低拷贝序列克隆.从文库中获得8个簇毛麦特异克隆,对其中两个克隆pHVMK22和 pHVMK134进行了RFLP分析和序列分析,并利用该探针对小麦抗白粉病基因Pm21进行了检测.RFLP 结果表明,两个克隆一个为低拷贝序列克隆(pHVMK22),另一个为高度重复序列克隆,均为簇毛麦专化DNA序列.以pHVMK22为探针对抗、感病小麦(Triticum aestivum L.)品系的Southern杂交发现抗病品系有一条2 kb的特征带, 该探针可能作为检测抗病基因Pm21的探针.     

Radiation-induced translocations with reduced Haynaldia villosa chromatin at the Pm21 locus for powdery mildew resistance in wheat

Haynaldia villosa Schur. (syn. Dasypyrum villosum Candargy, 2n = 2x = 14, genome VV), a species related to wheat, is highly resistant to powdery mildew. The powdery mildew resistance gene Pm21 from H. villosa was introduced into common wheat by means of a translocation line T6VS·6AL, where the 6VS chromosome arm of H. villosa was joined at the centromere with wheat chromosome arm 6AL. To develop small alien translocations, especially interstitial translocations of small alien chromosome segments, we irradiated mature female gametes of a T6VS·6AL translocation line with gamma rays. More than 20 new translocations and deletions of 6V chromatin were obtained and subsequently used to map Pm21. Pm21 was located in a small region (FL 0.45–0.58) by genomic in situ hybridization, molecular marker analysis, and powdery mildew response. Two homozygous translocation lines with small H. villosa chromosome fragments carrying Pm21 were identified by fluorescence in situ hybridization and molecular marker analysis: an interstitial translocation in which a small fragment of 6VS is inserted into chromosome 4B and a terminal translocation with a small fragment of 6VS inserted into 1A. These small alien translocations are being transferred into an adapted elite wheat background by backcrossing to allow their easy use in breeding programs.