小麦抗叶锈基因Lr24、Lr35的STS、SCAR标记在近等基因系(NILs)上的特异性验证

用一套分别含有不同抗叶锈基因的53个以Thatcher为遗传背景的近等基因系（near-isogeniclines,NILs）对已报道的分别与抗叶锈基因Lr24和Lr35连锁的STS、SCAR进行特异性验证。结果对于与Lr24连锁的STS标记,在53个NILs中只在TcLr24亲本中扩增出片段大小与报道相同的310bp的条带,在TcLr35中也扩增出了一条片段,但片段大小不同于310bp约为270bp。对于与Lr35连锁的SCAR标记,只在TcLr35亲本中扩增出片段大小为900bp的条带,与报道片段大小一致。验证结果表明与抗病基因Lr24和Lr35连锁的STS、SCAR分子标记在NILs中特异性都较好,进一步证明了这两个分子标记可方便地用于小麦抗叶锈基因Lr24、Lr35的分子标记辅助选择育种。     

33个小麦品种(系)抗叶锈基因Lr19分子检测

由小麦叶锈菌(Puccinia triticinia)引起的小麦叶锈病在世界各地产麦区均有发生。利用抗病品种是防治该病害最经济、安全、有效的方法。小麦抗叶锈病基因Lr19是一个十分有效的抗叶锈性基因,自1966年首次将该基因从长穗偃麦草(Agropyron elongatum)转到普通小麦中,至今仍是一个应用潜力很大的抗病基因。本研究利用以PCR为基础的STS技术对以春小麦Thatcher为背景的50个近等基因系材料和TcLr19与Thatcher杂交F2小麦进行检测,并对33个小麦品种进行分子标记分析鉴定,结果如下:(1)对以春小麦Thatcher为背景的50个近等基因系材料和TcLr19×ThatcherF2代小麦进行PCR-STS检测,扩增结果表明在50个近等基因系材料中仅有TcLr19中出现一条130bp的DNA条带,STSLr19130标记在其他49个近等基因系材料中未检测到Lr19基因;F2代中表现感病的植株没有130bp的DNA片段,表现抗病的植株有130bp的DNA条带;重复两次结果相同,进一步证明了与小麦抗叶锈基因Lr19共分离的STS标记的稳定可靠。(2)利用以PCR为基础的STSLr19...     

小麦抗叶锈病基因Lr24的一个新STS标记

来源于长穗偃麦草的基因Lr24对小麦叶锈病具有很高的抗性,本研究旨在开发用于Lr24基因分子标记辅助育种的新的分子标记。从定位于小麦3D染色体的22对SSR、EST-SSR引物中筛选出4对揭示TcLr24多态性的引物,用468株F2抗感群体对这4对引物进一步检测,得到1个与Lr24共分离的EST-SSR标记Xcwem17。对该标记进行测序,并设计了STS引物。用该STS引物及已知的Lr24SCAR引物对试验群体进行验证,两对引物在该F2群体中均表现共分离,且Xcwem17可在TcLr24单基因系和已知含Lr24的农家品种泰山1号中可扩增出180bp单一条带,感病对照及其余7个近等基因系无扩增。该EST-SSR标记可直接用于分子标记辅助选择。     

小麦抗叶锈病基因Lr19的SRAP标记

以Thatcher和23个以Thatcher为遗传背景的小麦抗叶锈病近等基因系及TcLr19与Thatcher杂交的F2植株为材料,首次应用SRAP技术开展小麦抗叶锈病基因Lr19的SRAP分子标记研究,获得一个与小麦抗叶锈病基因连锁的分子标记,命名为M73,与目的基因的遗传连锁距离为2.6 cM,为分子辅助育种、构建密集的遗传图谱和最终实现Lr19基因的克隆奠定基础。     

小麦抗叶锈病基因Lr24的一个新STS标记

来源于长穗偃麦草的基因Lr24对小麦叶锈病具有很高的抗性, 本研究旨在开发用于Lr24基因分子标记辅助育种的新的分子标记。从定位于小麦3D染色体的22对SSR、EST-SSR引物中筛选出4对揭示TcLr24多态性的引物, 用468株F2抗感群体对这4对引物进一步检测, 得到1个与Lr24共分离的EST-SSR标记Xcwem17。对该标记进行测序, 并设计了STS引物。用该STS引物及已知的Lr24 SCAR引物对试验群体进行验证, 两对引物在该F2群体中均表现共分离, 且Xcwem17可在TcLr24单基因系和已知含Lr24的农家品种泰山1号中可扩增出180 bp单一条带, 感病对照及其余7个近等基因系无扩增。该EST-SSR标记可直接用于分子标记辅助选择。     

小麦STK类抗病基因同源序列的克隆与分析

根据丝氨酸/苏氨酸蛋白激酶类催化结构域Ⅰ和Ⅷ氨基酸保守序列设计简并引物,以TcLr19、TcLr35和感病对照Thatcher的cDNA为模板进行抗病基因同源序列的PCR扩增,得到了6条通读的抗病基因同源序列(RGAs)Lr19-RGA1、Lr19-RGA2、Lr19-RGA3、Lr35-RGA1、Lr35-RGA2和TC-RGA.在NCBI中用BLASTp比对发现,Lr19-RGA1、Lr19-RGA2、Lr19-RGA3、Lr35-RGA1和Lr35-RGA2编码的氨基酸序列具有丝氨酸/苏氨酸蛋白激酶(Serine-threonine kinase,STK)的催化结构域Ⅱ-Ⅷ.对序列分析还发现,它们与已克隆的STK类抗病基因有不同程度的相似性,为进一步克隆小麦抗叶锈病相关基因提供了依据.     

棉纤维DNA的提取及其在品种溯源中的尝试

【目的】针对我国进口棉花原料以及收储棉花的品种产地溯源监控技术体系缺乏的问题,探索了适用于棉纤维DNA的提取方法,旨在建立以棉纤维DNA为介质鉴定棉花品种真实性及产地溯源的体系。【方法】通过改进和优化提取方法,提取了开花后不同时间棉纤维及不同年份皮棉的DNA,以其作为模板,进行了常规聚合酶链式反应(Polymerase chain reaction,PCR)扩增;并选取13对简单重复序列(Simple sequence repeat,SSR)引物对鲁1127、石抗126和瑞杂816棉纤维DNA进行SSR-PCR扩增。【结果】此DNA提取法可提取出不同发育时期和不同年份的棉纤维DNA,随着纤维发育成熟及年代增加,所提取的DNA含量尽管有所降低,但仍能满足下游试验需求;所提取的棉纤维DNA可进行常规PCR扩增,且PCR扩增条带清晰;以13对棉花SSR引物对鲁1127、石抗126和瑞杂816棉纤维DNA进行SSR-PCR扩增,均可扩增出清晰的多态性谱带,且易于区分。【结论】该提取法适合提取棉花纤维的基因组DNA,且满足于常规PCR扩增和SSR分子标记。本研究证实基于棉纤维DNA分子标记用于棉花品种溯源切实可行,并有望为保障我国棉花产业安全提供技术支持。     

小麦抗叶锈病近等基因系TcLr41基因的差异表达

为了研究与小麦抗叶锈病相关基因的表达情况,从分子水平阐明小麦的抗病机制.以Thatcher和Thatcher为遗传背景的小麦抗叶锈病近等基因系TcLr41为材料,利用cDNA-AFLP技术,开展了与小麦抗叶锈病基因Lr41相关的差异表达基因研究.共选用180对引物组合对Lr41差异表达的基因进行了分析,获得了61对能够在小麦近等基因系TcLr41和感病对照Thatcher之间扩增出差异条带的多态性引物.获得5对能够扩增出5条对小麦抗叶锈病近等基因系TcLr41具有特异性条带的引物,分别是P-AC/M-CAA、P-AG/M-CAT、P-AG/M-CCC、P-CC/M-CCA、P-GA/M-CAA.将重复性好的4条差异片段进行回收、克隆、测序,经序列同源性检索发现了1条与小麦抗叶锈病基因Lr1编码蛋白同源性较高的序列,其他的3个序列功能未知,研究结果为探明TcLr41的抗病机制和进行该抗病基因的克隆奠定了基础.     

中国小麦LB0288中抗叶锈病基因的鉴定

明确中国小麦LB0288中所含的抗叶锈病基因,找到与其紧密连锁的DNA分子标记。将小麦LB0288和感病小麦品种Thatcher杂交,获得F1、F2代群体,用叶锈菌小FHTT分别对双亲及其杂交后代进行叶锈鉴定并进行标记分析。抗性鉴定结果表明F2代群体时呈现一对显性基因的抗感分离比例,经过亲本和抗感池间标记筛选以及F2代群体的标记检测,位于5DL的SSR标记barc144与抗病基因连锁,遗传距离为5.3 cM,同时Lr1的STS标记与之共分离,根据该基因的抗性特点和染色体位置推断为Lr1。此实验通过抗性鉴定、遗传分析和分子标记等手段确定LB0288中含有小麦抗叶锈病基因Lr1。     

黑麦重复序列在检测小麦品种中外源染色体的应用

本研究根据RAPD引物OPH20在黑麦中扩增出的特异序列pSc20H.2设计一对PCR引物pSc20ht-23/24,以来源于黑麦的小麦抗叶锈近等基因系材料TcLr45及感病对照Thatcher为亲本进行PCR扩增。并对42个小麦抗叶锈近等基因系及103个小麦品种材料进行检测。引物pSc20ht23/24在TcLr45中扩增出一条约750bp的条带,而在Thatcher中无扩增条带。对该特异片段回收、克隆测序为734bp。42个小麦抗叶锈近等基因系检测在TcLr26中扩增出与TcLr45相同的条带,而在同样来源于黑麦的小麦抗叶锈近等基因系TcLr25中未扩增出该条带;中国春-Imperial黑麦附加系1R-7R中除5R外均扩增出该条带;13个1B/1R易位系小麦品种也扩增出该条带;90个地方小麦品种中有16个扩增出该条带,6个品种经系谱分析具有黑麦遗传背景,表明该标记可用于检测小麦中含有的除黑麦5R染色体之外的外源染色质。     

小麦抗叶锈病基因Lr45的SSR分子标记

用定位于2A染色体的59对SSR、EST-SSR引物,对小麦抗叶锈病基因Lr45进行分子标记,共筛选出11对揭示TcLr45多态性的引物。用157株F₂抗感群体对这11对引物进一步检测,得到4个与Lr45共分离的SSR标记(Xgwm95、Xgwm47、Xgwm372和Xgwm122)。将经PAGE检测的标记Xgwm95的抗感差异带及Xgwm47的抗性片段进行克隆测序发现其中含有微卫星序列,且均为二核苷酸重复。Xgwm372和Xgwm122经琼脂糖凝胶电泳发现与Lr45的供体黑麦有共同的标记片段,可直接用于分子标记辅助选择。     

Identification of molecular markers linked to adult plant leaf rust resistance gene <Emphasis Type="Italic">Lr48</Emphasis> in wheat and detection of <Emphasis Type="Italic">Lr48</Emphasis> in the Thatcher near-isogenic line with gene <Emphasis Type="Italic">Lr25</Emphasis>

The recessive adult plant resistance (APR) gene Lr48 in wheat was tagged with flanking random amplified polymorphic DNA (RAPD) markers. Markers S336₇₇₅ in coupling and S3₄₅₀ in repulsion with Lr48 were identified in wheat line CSP44. Tests of these markers on available Thatcher near-isogenic lines (NILs) detected the likely presence of Lr48 in TcLr25. A test of allelism of APR involving the cross TcLr25 × CSP44 indicated that Lr48 was present in both lines. A separate experiment on inheritance of resistance in an F₂ population of TcLr25 × Agra Local confirmed the presence of a dominant seedling resistance gene (Lr25) and a recessive APR gene (Lr48) in TcLr25. This study demonstrated the value of molecular markers in identifying the presence of masked genes in genetic stocks where direct phenotyping failed to detect their presence.     

土耳其8个小麦品种农艺性状及抗叶锈性分析

为确定8个来自土耳其的普通小麦品种在我国的应用前景,对其进行全生育期农艺性状观察,并利用44个以Thatcher为背景的近等基因系(单基因系)作为已知基因的鉴别寄主,接种8个小麦叶锈菌致病型进行苗期抗叶锈基因推导,结合成株期抗病鉴定,初步明确了这些品种(系)的抗性和可能携带的抗病基因。利用20个与Lr基因紧密连锁或共分离的分子标记,对8个土耳其小麦品种进行抗叶锈病基因的进一步鉴定。推测YJ000900中可能含有Lr1、Lr3、Lr17、Lr20;YJ000906中可能含有Lr1、Lr17、Lr20;YJ000901、YJ000902、YJ000904、YJ000905、YJ000907中可能含有Lr1;8个材料中均不含Lr9、Lr19、Lr20、Lr21、Lr24、Lr26、Lr28、Lr29、Lr34、Lr35、Lr37、Lr38和Lr47基因。结果表明,来自土耳其的8个小麦材料具有较差的抗叶锈性、抗寒和抗倒伏能力,而且产量低,不适宜于大规模推广种植,也不能作为我国小麦抗叶锈的抗源使用。     

28个小麦微核心种质抗叶锈性分析

选取在成株期表现高、中、低抗叶锈的28个小麦微核心种质,利用39个以Thatcher为背景的近等基因系(或单基因系)作为已知基因的鉴别寄主,接种8个小麦叶锈菌致病型进行苗期抗叶锈基因推导,结合成株期抗病鉴定,初步明确了这些品种(系)的抗性和可能携带的抗病基因。利用19个与Lr基因紧密连锁或共分离的分子标记,对28个微核心种质进行抗叶锈病基因的进一步鉴定,推测新克旱9号可能含有Lr17、Lr2b、Lr14a和Lr33;兴义4号可能含有Lr26、Lr36和Lr37;紫皮可能含有Lr2b和Lr34;大白皮含有Lr1;毕红穗含有Lr1、Lr10和Lr34;中优9507含有Lr10;小白麦、红粒当年老、老麦、蝉不吱、苏麦3号和车锏子含有Lr1和Lr34;红花早可能含有Lr1、Lr34、Lr14a和Lr2b;江西早、泡子麦、三月黄、有芒扫谷旦、阜阳红、成都光头和酱麦可能含有Lr34;敦化春麦和甘肃96可能含有Lr28;欧柔可能含有Lr34、Lr16、Lr11、Lr3bg和Lr33;此外,新克旱9号、兴义4号、红花早、红粒当年老、欧柔、有芒扫谷旦、成都光头、甘肃96、小红皮、定兴寨、中优9507和红冬麦中可能含有未知抗病基因;在这28份种质中,不含Lr9、Lr19、Lr20、Lr21、Lr24、Lr29、Lr35、Lr38和Lr47基因。研究结果表明,测试的微核心种质中含有比较丰富的抗叶锈病基因,可为育种提供丰富的抗源。     

Genetics of stem rust resistance in the spring wheat cultivar Thatcher and the enhancement of stem rust resistance by <Emphasis Type="Italic">Lr34</Emphasis>

Three recombinant inbred line populations from the crosses RL6071/Thatcher, RL6071/RL6058 (Thatcher Lr34), and Thatcher/RL6058, were used to study the genetics of stem rust resistance in Thatcher and TcLr34. Segregation of stem rust response in each population was used to determine the number of genes conferring resistance, as well as the effect of the leaf rust resistance gene Lr34 on stem rust resistance. The relationship between resistance in seedling and adult plants was also examined, and an attempt was made to identify microsatellite markers linked to genes that were effective in adult plants. In field plot tests at least three additive resistance genes segregated in the RL6071/RL6058 population, whereas two resistance genes segregated in the RL6071/Thatcher population. The presence of the gene Lr34 permitted the expression of additional stem rust resistance in Thatcher-derived lines both at the seedling and adult plant stages. Seedling resistance to races TPMK and RKQQ was significantly associated with resistance in adult plants, whereas seedling resistance to races QCCD and QCCB may have made a minor contribution. The seedling resistance genes Sr16 and Sr12 may have contributed to resistance in adult plants. A molecular marker linked to resistance in adult plants was identified on chromosome 2BL.     

Development and Validation of SCAR Markers Co-Segregating with an Agropyron Elongatum Derived Leaf Rust Resistance Gene Lr24 in Wheat

Summary An Agropyron elongatum-derived leaf rust resistance gene Lr24 located on chromosome 3DL of wheat was tagged with six random amplified polymorphic DNA (RAPD) markers which co-segregated with the gene. The markers were identified in homozygous resistant F₂ plants taken from a population segregating for leaf rust resistance generated from a cross between two near-isogenic lines (NILs) differing only for Lr24. Phenotyping was done by inoculating the plants with pathotype 77-5 of Puccinia triticina. To enable gene-specific selection, three RAPD markers (S1302₆₀₉, S1326₆₁₅ and OPAB-1₃₈₈) were successfully converted to polymorphic sequence characterized amplified region (SCAR) markers, amplifying only the critical DNA fragments co-segregating with Lr24. The SCAR markers were validated for specificity to the gene Lr24 in wheat NILs possessing Lr24 in 10 additional genetic backgrounds including the Thatcher NIL, but not to 43 Thatcher NILs possessing designated leaf rust resistance genes other than Lr24. This indicated the potential usefulness of these SCAR markers in marker assisted selection (MAS) and for pyramiding leaf rust resistance genes in wheat.     

Identification of the leaf rust resistance genes Lr9, Lr26, Lr28, Lr34, and Lr35 in a collection of Iranian wheat genotypes using STS and SCAR markers

Brown rust or leaf rust is one of the most important diseases of wheat occurring almost in all wheat-producing regions and reduces crop yield. In order to produce resistant cultivars, it is necessary to identify resistance genes in different germplasms and combine them in (a) suitable stock(s). To identify the presence of the leaf rust resistance genes using STS and SCAR markers, 83 Iranian wheat genotypes, Lr near-isogenic lines in Thatcher (positive controls), and the cultivar Thatcher (negative control) were used. After growing plants in the greenhouse, DNA was extracted by SDS method. Following that, polymerse chain reaction was performed for the markers of the resistance genes Lr9, Lr26, Lr28, Lr34, and Lr35 which amplified 1,100, 1,100, 378, 150, and 900 bp bands, respectively. Based on the results, the resistance genes Lr9 and Lr35 were only present in the positive controls. The resistance gene Lr26 was only detected in four cultivars; Arta, Pishtaz, Shiroodi, and Falat, and the gene Lr34 was present in six cultivars (Akbari, Bam, Tajan, Khazar 1, Sistan and Niknezhad). The Lr28 primer amplified a band of the same size in all genotypes even the negative control and therefore the presence/absence of this gene could not be validated. These results indicate the necessity for designing a specific primer for Lr28. In general, only the genes Lr26 and Lr34 were present in some genotypes. The genes Lr9 and Lr35 were not present in this collection and as based on rust surveys, no virulence has been detected for Lr9 and Lr28, so they could be transferred to suitable lines from donor sources.