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。     

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...     

小麦基因组的一种简易提取方法

摘要：【研究目的】在综合分析多种提取小麦DNA方法的基础上,改良发展了一种快速简便高效的小麦基因组DNA提取方法。【方法】以春小麦Thatcher和以Thatcher为背景的近等基因系TcLr19,TcLr20, TcLr28为材料,改进后的方法提取的基因组DNA用抗叶锈基因Lr20的STS标记、Lr19和Lr28的SCAR标记、Lr28的SSR 标记进行PCR扩增,【结果】各分子标记都扩增出条带清晰正确的目的片段【结论】这表明该方法能够获得适用于STS、SCAR、SSR标记PCR扩增的高质量的DNA,而且该DNA简易提取方法加快了DNA提取速度、降低了污染源和成本,为大批量提取DNA提供了技术支撑。     

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。     

中国小麦育成品种和农家种中慢锈基因Lr34/Yr18的分子检测

Lr34/Yr18是重要的慢叶锈和慢条锈基因, 携带该连锁基因的小麦品种被广泛种植于世界许多国家。利用STS标记csLV34对慢叶锈和慢条锈基因Lr34/Yr18进行分子检测的结果表明, 我国231份育成品种(系)中仅有14份材料携带Lr34/Yr18基因, 占6.1%。不同麦区分布频率不同, 其中北部冬麦区为零, 黄淮冬麦区、长江中下游冬麦区、西南冬麦区和西北春麦区分别为3.0%、21.4%、16.7%和33.3%。在422份农家种中, 359份含有Lr34/Yr18基因, 占85.1%。Lr34/Yr18基因在不同麦区的分布频率也存在差异, 北部冬麦区、黄淮冬麦区、长江中下游冬麦区、西南冬麦区、南部冬麦区和西北春麦区分别为89.6%、77.4%、93.1%、93.8%、96.6%和61.1%。csLV34标记扩增产物为150 bp和229 bp的片段, 能有效鉴别品种是否携带Lr34/Yr18基因, 是一个重复性好、准确率高的分子标记, 可用于小麦Lr34/Yr18基因的鉴定与选择。     

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。     

中国小麦育成品种和农家种中慢锈基因Lr34/Yr18的分子检测

Lr34/Yr18是重要的慢叶锈和慢条锈基因, 携带该连锁基因的小麦品种被广泛种植于世界许多国家。利用STS标记csLV34对慢叶锈和慢条锈基因Lr34/Yr18进行分子检测的结果表明, 我国231份育成品种(系)中仅有14份材料携带Lr34/Yr18基因, 占6.1%。不同麦区分布频率不同, 其中北部冬麦区为零, 黄淮冬麦区、长江中下游冬麦区、西南冬麦区和西北春麦区分别为3.0%、21.4%、16.7%和33.3%。在422份农家种中, 359份含有Lr34/Yr18基因, 占85.1%。Lr34/Yr18基因在不同麦区的分布频率也存在差异, 北部冬麦区、黄淮冬麦区、长江中下游冬麦区、西南冬麦区、南部冬麦区和西北春麦区分别为89.6%、77.4%、93.1%、93.8%、96.6%和61.1%。csLV34标记扩增产物为150 bp和229 bp的片段, 能有效鉴别品种是否携带Lr34/Yr18基因, 是一个重复性好、准确率高的分子标记, 可用于小麦Lr34/Yr18基因的鉴定与选择。     

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。     

贵州小麦品种(系)中慢锈基因Lr34/Yr18的分子检测

小麦慢锈病是危害小麦生产的重要病害。为了鉴定258份贵州小麦品种(系)中慢锈基因Lr34/Yr18的组成,筛选含慢锈抗性基因Lr34/Yr18的种质资源,本研究利用STS标记csLV 34结合毛细管电泳技术对258份小麦品种(系)中慢锈抗性基因Lr34/Yr18的等位变异进行了分子检测。结果表明:毛细管电泳谱带清晰易读,可根据扩增片段分子量直接判断目标片段有无。STS标记csLV 34可在含有Lr34/Yr18基因的材料中扩增出150 bp片段,部分不含Lr34/Yrl8的材料则扩增出229 bp片段,余下大部分不含Lr34/Yrl8的材料没有扩增出150 bp和229 bp的片段;258份小麦品种(系)中有5份材料扩增出150 bp片段,可能含有Lr34/Yr18基因,占供试材料的1.9%。筛选的这些慢锈抗性种质资源可为今后贵州小麦的慢锈抗病品种选育提供参考。     

122份小麦品种(系)抗叶锈病基因Lr26、Lr34、Lr38分子标记检测

运用抗叶锈病基因Lr 26、Lr 34、Lr 38的特异性分子标记,对122份小麦品种(系)进行了检测,以明确各品种(系)的抗叶锈性.结果表明:122份供试材料中,含有Lr 26的有33个,含有Lr 34的有3个,含有Lr 38的有37个;同时含有Lr 26和Lr 38的有30个,同时含有Lr 26、Lr 34和Lr 38的有3个.本研究结果可为小麦的抗叶锈育种提供理论参考.     

SCAR marker tagged to the alien leaf rust resistance gene Lr19 uniquely marking the Agropyron elongatum-derived gene Lr24 in wheat: a revision

A sequence characterized amplified region (SCAR) marker tagged to an Agropyron elongatum‐derived leaf rust resistance (Lr) gene Lr19 was validated on 18 known alien Lr gener in near‐isogenic lines (NILs) in the variety ‘Thatcher’, along with three wheat cultivers carrying Lr24 and two carrying Lr19. The marker was expressed only in the Lr24 lines confirming that the marker tagged the geneLr24. The monomorphic expression of the SCAR marker in 10NIL pairs for Lr19 and Lr24 revealed that each NIL pair possessed the same gene, Lr24. The donor parents used in the NIL pairs for Lr19 (‘Sunstar*6/C80‐1′) and Lr24 (‘TR380‐14*7/3Ag#14′) amplified the same fragment. Nonsegregation for leaf rust in the F₂ population of the cross between the above donor parents confirmed the presence of the same gene in the two parents. Apparently, a genuine parent stock of ‘Sunstar*6/C80‐1’ was not involved in the development of the NIL pairs for Lr19 due to an improper maintence bredding protocol either at source or destination which went undetected in the absence of signs of virulence for either gene in the region.     

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.     

Identification of a molecular marker linked to an Agropyron elongatum-derived gene Lr19 for leaf rust resistance in wheat

The leaf rust resistance gene Lr19, transferred from Agropyron elongatum into wheat (Triticum aestivum L.) imparts resistance to all pathotypes of leaf rust (Puccinia recondita f.sp. tritici) in South‐east Asia. A segregating F₂ population from a cross between the leaf rust resistant parent ‘HW 2046’ carrying Lr19 and a susceptible parent ‘Agra Local’ was screened in the phytotron against a virulent pathotype 77‐5 of leaf rust with the objective of identifying the molecular markers linked to Lr19. The gene was first tagged with a randomly amplified polymorphic DNA (RAPD) marker S73₇₂₈. The RAPD marker linked to the gene Lr19 which mapped at 6.4 ± 0.035 cM distance, was converted to a sequence characterized amplified region (SCAR) marker. The SCAR marker (SCS73₇₁₉) was specific to Lr19 and was not amplified in the near‐isogenic lines (NILs) carrying other equally effective alien genes Lr9, Lr28 and Lr32 enabling breeders to pyramid Lr19 with these genes.     

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.     

小麦抗病品系5R625抗叶锈病基因的分子鉴定

小麦品系5R625苗期和田间均对小麦叶锈病有良好抗性,但其所携带的抗病基因还不清楚。利用36个携带已知抗叶锈病基因的对照品系和15个中国小麦叶锈菌小种对5R625携带的抗病基因进行了苗期人工接种鉴定和基因推导,结果 5R625对这15个叶锈菌生理小种的侵染型与Lr9、Lr19、Lr24、Lr28、Lr39、Lr47、Lr51、Lr53相同。利用5R625和感病品种郑州5389的杂交后代F1、F2和F2:3群体对5R625的抗病性进行了遗传分析,苗期和成株期的分析结果均表明5R625对小麦叶锈菌的抗性由1个显性基因控制。进一步利用F2:3家系和分子标记方法将该基因定位在3DL染色体上。与5R625携带的抗病基因连锁的5个分子标记中,STS标记24-16和SCAR标记OP-J09此前已经被证明与已知抗叶锈病基因Lr24共分离,因此,推测5R625携带的抗病基因与Lr24可能为同一基因。     

小麦新品种“山农20”抗病基因的分子检测

山农20是2011年和2012年分别通过国家黄淮南、北片审定的小麦高产多抗新品种,在国家区试抗病性鉴定和生产中都表现出良好的抗黄淮麦区主要病害的特性。本研究利用与小麦抗白粉病、条锈病、叶锈病、纹枯病基因和抗赤霉病主效QTL紧密连锁的SSR、SCAR、STS等标记对该品种进行了分子检测,发现山农20含有6个抗白粉病基因(Pm12、Pm24、Pm30、Pm31、Pm35和Pm36),6个抗条锈病基因(Yr5、Yr9、Yr15、Yr24、Yr26和YrTp1),2个抗叶锈病基因(Lr21和Lr26),1个抗纹枯病基因(Ses1),但未检测到抗赤霉病主效QTL。分子检测结果部分解释了山农20的优良抗病性,也为利用分子标记辅助选择培育抗病稳产小麦新品种提供参考。     

Evaluation of leaf rust resistance genes Lr1 , Lr9 , Lr24 , Lr47 and their introgression into common wheat cultivars by marker-assisted selection

Epidemiological field controls in different Italian locations and seedling evaluations of the ‘Thatcher’ near-isogenic lines (NILs) carrying the leaf rust resistance genes Lr1, Lr9, Lr24 and Lr47 were conducted during 5 years of testing. These genes confirmed their effectiveness in both field and greenhouse conditions. Moreover a backcross program was carried out by using as recurrent parents the susceptible high-quality common wheat cvs ‘Bolero’, ‘Colfiorito’, ‘Serio’ and ‘Spada’ and the ‘Thatcher’ NILs carrying the above mentioned genes as donor parents. The progenies of different cross combinations were selected by both resistance tests and marker assisted selection using molecular markers (STS, SCAR, CAPS) closely linked to Lr genes: a complete cosegregation was observed between the resistance genes used and the corresponding molecular markers.     

Molecular mapping of Aegilops speltoides derived leaf rust resistance gene Lr28 in wheat

In a segregating homozygous F₂ population of bread wheat involving a leaf rust resistance gene Lr28 derived from Aegilops speltoides, six randomly amplified polymorphic DNA (RAPD) markers, three each in coupling and repulsion phase were identified as linked to Lr28, mapped to a region spanning 32 cM including the locus. The F₂ and F₃ populations were studied in the phytotron challenged with the most virulent pathotype 77-5 of leaf rust. A coupling phase linked RAPD marker S464₇₂₁ and a repulsion phase linked RAPD marker S326₅₅₀ flanked the gene Lr28 by a distance of 2.4± 0.016 cM on either side. The flanking markers genetically worked as co-dominant markers when analyzed together after separate amplification in the F₂ population by distinguishing the homozygotes from the heterozygotes and increased the efficiency of marker assisted selection by reducing the false positives and negatives. One of the three RAPD markers, S421₆₄₀ was converted to locus specific SCAR marker SCS421₆₄₀ which was further truncated by designing primers internal from both ends of the original RAPD amplicon to eliminate a non-specific amplification of nearly same size. The truncated polymorphic sequence characterized amplified region marker (TPSCAR) SCS421₅₇₀ was 70 bp smaller, but resulted in a single band polymorphism specific to Lr28 resistance. The TPSCAR marker was validated for its specificity to the gene Lr28 in nine different genetic backgrounds and on 43 of the 50 Lr genes of both native and alien origin, suggesting the utility of the SCAR markers in pyramiding leaf rust resistance genes in wheat.     

Development of a PCR assay and marker-assisted transfer of leaf rust resistance gene <Emphasis Type="Italic">Lr58</Emphasis> into adapted winter wheats

Leaf rust resistance gene Lr58 derived from Aegilops triuncialis L. was transferred to the hard red winter wheat (HRWW) cultivars Jagger and Overley by standard backcrossing and marker-assisted selection (MAS). A co-dominant PCR-based sequence tagged site (STS) marker was developed based on the sequence information of the RFLP marker (XksuH16) diagnostically detecting the alien segment in T2BS·2BL-2^tL(0.95). STS marker Xncw-Lr58-1 was used to select backcross F₁ plants with rust resistance. The co-dominant marker polymorphism detected by primer pair NCW-Lr58-1 efficiently identified the homozygous BC₃F₂ plants with rust resistance gene Lr58. The STS marker Xncw-Lr58-1 showed consistent diagnostic polymorphism between the resistant source and the wheat cultivars selected by the US Wheat Coordinated Agricultural Project. The utility and compatibility of the STS marker in MAS programs involving robust genotyping platforms was demonstrated in both agarose-based and capillary-based platforms. Screening backcross derivatives carrying Lr58 with various rust races at seedling stage suggested the transferred rust resistance in adapted winter wheats is stable in both cultivar backgrounds. Lr58 in adapted winter wheat backgrounds could be used in combination with other resistance genes in wheat rust resistance breeding.