小麦抗白粉病基因Pm21的分子鉴定和标记辅助选择

利用小麦抗白粉病基因Ｐｍ２１的ＲＡＰＤ标记、ＳＣＡＲ标记和荧光源位杂交技术对小麦抗病育种材料中的抗白粉病Ｐｍ２１基因进行了分子鉴定和标记辅助选择。     

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.     

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。     

Transmission of 6VS Chromosome in Wheat- Haynaldia villosa Translocation Lines and Genetic Stability of Pm21 Carried by 6VS

Fa plants from Yang94-138 ×92R149 were analyzed using species-specific probe pHv62 for Haynaldia villosa (L.) Schur and RFLP probe Psrll3 located on the short ann of homoeologous group six for wheat ( Triticum aestivum L. ). The results showed that the transmission rate of 6VS in the F2 was 69.5 %, which was close to the expected value of 75%. 147 F2 plants from the above Fl plant were tested for their resistance in seedling stage to 6 pathogenic strains of powdery mildew. It was observed that Pm21 genes on 6VS inherited dominantly and expressed well when transferred to wheat－"Yangmai 158" genotype.     

一个小麦丝氨酸-苏氨酸蛋白激酶基因的克隆和分析

用mRNA差异显示技术在含有抗白粉病基因Pm21的小麦(Tri ticum aestivum L.) -簇毛麦(Haynaldia villosa) 6VS /6AL易位系92R137中分离与抗白粉病相关的基因,获得一个命名为TaPK1的全长cDNA克隆.序列分析表明,它与大豆(Glycine max (L.) Merr.)蛋白激酶基因GmPK6高度同源.经推测,TaPK1 编码416个氨基酸的多肽,属丝氨酸-苏氨酸蛋白激酶家族,并具酪氨酸激酶特性.TaPK1是从小麦中分离的新基因.     

Development and molecular cytogenetic analysis of wheat-Haynaldia villosa 6VS/6AL translocation lines specifying resistance to powdery mildew

Several Triticum aestivum L.-Haynaldia villosa disomic 6VS/6AL translocation lines with powdery mildew resistance were developed from the hybridization between common wheat cultivar Yangmai 5 and alien substitution line 6V(6A). Mitotic and meiotic C-banding analysis, aneuploid analysis with double ditelosomic stocks, in situ hybridization, as well as the phenotypic assessment of powdery mildew resistance, were used to characterize these lines. The same translocated chromosome, with breakpoints near the centromere, appears to be present in all the lines, despite variation among the lines in their morphology and agronomic characteristics. The resistance gene, conferred by H. villosa and designated as Pm21, is a new and promising source of powdery mildew resistance in wheat breeding.This research was supported by grants from the National High-Tech R and D Program and the National Science and Technology Commission     

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。     

小麦抗白粉病基因Pm23的RAPD标记

小麦抗白粉病基因Pm23对世界上很多麦区流行的白粉病表现高抗或免疫.本研究以Pm23和Chancellor为抗感亲本,用集群分离分析法对抗性基因Pm23进行了RAPD分析,从320个十碱基随机引物中筛选到一个与Pm23紧密连锁的相引相标记OPE051100. 对F2分离群体进行RAPD分析表明,该标记与Pm23基因之间的连锁距离为10.65±3.25 cM.该标记可以有效用于小麦育种分子标记辅助选择中.     

Suppressed recombination rate in 6VS/6AL translocation region carrying the Pm21 locus introgressed from Haynaldia villosa into hexaploid wheat

Pm21 is an effective gene for powdery mildew resistance transferred from Haynaldia villosa into common wheat cultivars. No virulence against this gene has been detected so far. A set of 42 powdery mildew isolates collected in Israel and tested in the current study also revealed no virulence against this gene. Pm21 was previously reported to be located on the short arm of 6VS/6AL translocation chromosome. We constructed a high-density genetic map of chromosome 6A, consisting of 28 PCR markers and the Pm21 gene. A comparison with previously published genetic maps of wheat chromosome 6A revealed that the recombination rate in the 6VS/6AL translocation region was poor. We assume that suppressed recombination caused by the alien H. villosa genetic material is the most reasonable explanation for the tight genetic linkage and the inadequacy between the Pm21 genetic map and the Pm21 physical map of 6A. A large number of sequence-tag sites (STS) and simple sequence repeat markers, which co-segregate with or are closely linked to the Pm21 gene, and the conversion of three resistance gene analog markers into new STS markers, provide a reliable and easy-to-use molecular tool for marker-assisted selection of Pm21 in wheat breeding programs. An additional gene, Pm31, previously reported to be derived from Triticum dicoccoides, was mapped into a similar genomic location to Pm21. Screening of the parental lines and the mapping population with Pm21 diagnostic markers clearly confirmed that the donor line of Pm31 is H. villosa and not T. dicoccoides. Therefore, we conclude that Pm21 and Pm31 refer to the same gene, derived from H. villosa, and that the designation of Pm31 as a new Pm gene was erroneous.     

从小麦-簇毛麦易位系TAC文库中筛选Hv-S/TPK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5160 bp(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。     

由簇毛麦V染色体引起的小麦-簇毛麦染色体代换系、易位系中线粒体蛋白质组的变化(简报)

线粒体是需氧生物中的一种半自主性细胞器.在能量代谢中,它起了一个关键的作用,柠檬酸循环、电子传递和氧化磷酸化过程均在线粒体内完成.线粒体具有高度的生物化学和遗传上的独立性.含有DNA和核糖核蛋白体.负责合成生物体内2%-5%的蛋白质[1].线粒体DNA具有自身复制能力,控制着众多的遗传性状.在植物中广泛存在的细胞质雄性不育则被认为是由线粒体基因组控制的性状[2].     

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。     

小麦抗白粉病基因Pm6的RAPD标记

从提莫菲维小麦转移到普通小麦中的小麦白粉病抗性基因Ｐｍ６是小麦白粉病（Ｅｒｙｓｉｐｈｅ ｈｒａｍｉｎｉｓ ｆ ｓｐ．ｔｒｉｔｉｃｉ）的有效抗性基因。用７００个随机引物对Ｐｍ６近等基因系进行ＲＡＰＤ分析,发现引物ＯＰＶ２０可在抗病近等基因系中产生大小为２ｋｂ的稳定的多态片段。用该引物检测１０个其他携Ｐｍ６的渐渗系材料,均可稳定扩增出该２ｋｂ的多态片段。理一步用ＯＰＶ２０对Ｐｍ６Ｆ２（ＩＧＶ１－４６３     

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     

小麦白粉病抗性基因的导入及AFLP分析

本研究以簇毛麦为抗源,采用杂交与辐射、组织培养相结合的方法,将簇毛麦的抗白粉病基因导入小麦,选育出高产、抗白粉病的小麦新品种和农艺性状较好、抗白粉病的小麦新种质。经AFLP分析,确定4个抗白粉病种质均为含有一段簇毛麦DNA的易位系。并得到3个可能与抗性基因紧密连锁的标记。     

抗白粉病小偃麦异代换系的细胞学和RAPD鉴定

利用细胞学和RAPD方法,对从长穗偃麦草与普通小麦复合杂交后代中选育的抗白粉病小麦种质系山农87074－526和山农87074－551进行了鉴定。结果表明,两种质系的根尖细胞染色体数目均为2n=42,花粉母细胞减数分裂中期I（PMC MI）染色体构型为2n=21Ⅱ;二者杂交F1 PMC MI染色体构型亦为2n=21Ⅱ,两种质系分别与小麦中国春的杂种F1 PMC MI染色体构型均为2n=20Ⅱ 2I,说明两种质系为相同的双体异代系。在苗期和成株期两种质系对白粉病15号菌种均表现免疫,其白粉病抗性为显性,并且来自长穗偃麦草,抗白粉病基因位于它们所含的偃麦草染色体上。从80个随机引物中,筛选出2个引物OPE13和OPH15能在两种质系中稳定地扩增出长穗偃麦草亲本的特异DNA片段。     

一个小麦丝氨酸—苏氨酸蛋白激酶基因的克隆和分析

用mRNA差异显示技术在含有抗白粉病基因Pm2 1的小麦 (TriticumaestivumL .)_簇毛麦 (Haynaldiavillosa)6VS/ 6AL易位系 92R137中分离与抗白粉病相关的基因 ,获得一个命名为TaPK1的全长cDNA克隆。序列分析表明 ,它与大豆 (Glycinemax (L .)Merr.)蛋白激酶基因GmPK6高度同源。经推测 ,TaPK1编码 416个氨基酸的多肽 ,属丝氨酸_苏氨酸蛋白激酶家族 ,并具酪氨酸激酶特性。TaPK1是从小麦中分离的新基因。     

普通小麦99-2439 中的白粉病抗性遗传

普通冬小麦品系99-2439在郑州连续4年对田间白粉菌(Blumeria graminis sp. tritici)表现高抗,但其抗性基因来源不清。通过染色体C-分带和1RS染色体特异性SCAR标记鉴定, 表明它是一个小麦-黑麦(Triticum aestivum - Secale cereale)1BL/1RS异易位系。通过对中国春×99-2439杂交F2代分离群 体抗性鉴定和1RS染色体臂检测结果分析, 证明该抗病基因不在1RS染色体臂上。用单孢小麦白粉菌分离株对其抗性遗传进行研究, 结果表明, 99-2439的白粉病抗性由一对小种专化、隐性抗病基因控制。由于携带Pm5a的Hope/8Cc对中国的21个小麦白粉菌分离菌株均高度感病, 而99-2439高抗混和白粉菌和5个单孢分离菌株, 所以, 99-2439所携带的抗白粉病基因不同于Pm5a。     

小麦品种复壮30中与抗白粉病基因连锁的一个RAPD标记

小麦品种复壮30中与抗白粉病基因连锁的一个RAPD标记@王立新$北京市农业科学院植物细胞工程实验室!北京100089小麦;;抗白粉病;;基因;;RAPD标记