簇毛麦抗白粉病基因向四川小麦转移的分子标记育种研究

利用小麦 6VS/6AL易位系的抗白粉病基因Pm2 1的SCAR标记 ,对不同簇毛麦来源的小麦抗白粉病材料及其杂交后代进行了分子鉴定和标记辅助选择研究 ,结果表明利用该标记可以鉴定小麦遗传背景下的簇毛麦 6VS染色体臂 ,而且引物对小麦背景中的多年生簇毛麦也同样得到扩增。对簇毛麦染色体易位系 6VS/6DL与四川小麦品种杂交的后代群体 ,可以利用SCAR标记进行辅助抗性选择。本文还对四川小麦分子标记辅助育种的策略进行了讨论     

Characterization of RAPD Markers, and the RFLP Marker Linked to Powdery Mildew Resistance Gene Derived from Different Accessions of H. villosa

The analysis was carried out on performance of the resistance gene from Haynaldia villosa accession of the former Soviet Union to different isolates ofBluemerie graminis. Polymorphisms were revealed between 6D/6V substitution line Pm930640 and its pedigree parents using five RAPD markers of OPAN031700, OPAI01700, OPAL03750, OPAD07480 and OPAG 15580 screened out from 120 random 10-mers primers. Three RAPD markers of OPAN03, OPAI01 and OPAL03 were linked with the resistance gene by analysis of F2 population of ChancellorxPm930640. Analysis of 29 wheat lines including part of lines conferring the known genes from Pro1 to Pro20 respectively, lines conferring resistance gene from two H. villosa accessions and the related wheat parents, were analyzed and the results showed that these markers not only linked to the gene resistant to powdery mildew from H. villosa, but also detected different genetic backgrounds. OPAL03750 can be used as the marker to distinguish the different resistant lines from two H. villosa accessions because it was only observed in the materials from H. villosa of the former Soviet Union. RFLP analysis also showed the polymorphisms between two H. villosa accessions and their derived resistant lines.     

小麦抗白粉病基因Pm4的STS标记

根据与小麦抗白粉病基因Pm4a共分离的RFLP探针BCD1231的序列设计引物,以含小麦抗白粉病基因Pm4b的小麦近等基因系VPM/7*百农3217、抗病亲本VPM和轮回亲本百农3217为材料进行PCR扩增,结果在抗病池与VPM中扩增出一条约为410bp大小的特异带STS410,而在百农3217中无此特异PCR扩增带.进一步对144株VPM/7*百农3217的F2分离群体进行遗传连锁分析,估算出该PCR标记STS410与抗病基因Pm4b的遗传距离为3.0cM.用设计的这对STS-PCR引物对8个感病品种和17个含Pm基因的抗病亲本进行PCR扩增,结果发现:STS410只出现在含有Pm4基因的材料中.因此,PCR标记STS410可方便地用于小麦抗白粉病基因Pm4的分子标记辅助选择育种.     

小麦抗白粉病基因Pm13和Pm21特异标记的鉴定和应用

为给小麦生产提供抗谱更广、抗性较持久的育种材料,促进累加基因在小麦抗白粉病育种中的应用.利用与抗白粉病基因Pm13和Pm21相连锁的特异标记,对含有抗白粉病基因Pm13、Pm21的抗病亲本材料和它们杂交的F1代单株,以及在育种中使用的50个小麦品种(系)进行了分子检测.结果表明,与抗白粉病基因Pm13、Pm21连锁的特异标记可以在含有相应基因的材料中分别扩增出1条大小约564 bp和140bp的特异带,而在含有Pm13和Pm21抗白粉病基因的4个F1植株中也检测到这2条特异带;在50个小麦品种(系)中,有10个小麦品种(系)具有Pm21抗白粉病基因,而所有品种(系)均没有扩增出Pm13抗白粉病基因的标记带;结合田间抗白粉病性鉴定,所有具有抗病基因Pm13或Pm21连锁标记的小麦品种(系)都表现高抗小麦白粉病.说明,与抗白粉病基因Pm13和Pm21相连锁的特异标记可以有效应用于小麦抗白粉病育种中,分子标记是检测抗病基因累加体、辅助育种的有效手段.     

小麦抗白粉病基因Pm4的STS标记

根据与小麦抗白粉病基因Pm4a共分离的RFLP探针BCD1231的序列设计引物,以含小麦抗白粉病基因Pm4b的小麦近等基因系VPM／7百农3217、抗病亲本VPM和轮回亲本百农3217为材料进行PCR扩增,结果在抗病池与VPM中扩增出一条约为410bp大小的特异带STS410,而在百农3217中无此特异PCR扩增带。进一步对144株VPM／7^*百农3217的F2分离群体进行遗传连锁分析,估算出该PCR标记STS410与抗病基因Pm4b的遗传距离为3.0cM。用设计这对STS－PCR引物对8个感病品种和17个含Pm基因的抗病亲本进行PCR扩增,结果发现：STS410只出现在含有Pm4基因的材料中。因此,PCR标记STS410可方便地用于小麦抗白粉病基因Pm4的分子标记辅助选择育种。     

簇毛麦抗白粉病基因的导入及AFLP标记

小麦白粉病是小麦的主要病害之一。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标记,确定了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和7个连锁不太紧密的标记,为簇毛麦抗白粉病基因的鉴定和利用奠定了基础。     

簇毛麦抗白粉病基因的导人及AFLP标记

小麦白粉病是小麦的主要病害之一。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标记,确定了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和7个连锁不太紧密的标记,为簇毛麦抗白粉病基因的鉴定和利用奠定了基础。     

贵农21白粉病抗性基因的RAPD分子标记

应用RAPD技术对贵农21的白粉病抗性基因进行了分子标记，结果找到了贵农21中的两个白粉病抗性基因的分子标记，其中有一个抗病性基因相同于P38中的Pm21，来自于簇毛麦，其分子标记就是P38的RAPD标记OPH171265和OPH171400，并已转化为SCAR标记；另外一个抗病性基因的RAPD的分子标记为OPO151200，其白粉病抗性基因来自于硬粒小麦。     

小麦抗白粉病和条锈病基因的分子标记辅助鉴定

【目的】选育小麦新种质N95175和新品种远丰175,并检测其是否含有来源于小麦-簇毛麦易位系92R149的抗白粉病基因或抗条锈病基因。【方法】利用92R149/咸87(30)//小偃6号杂交组合选育N95175和远丰175,并以N95175、远丰175及其亲本92R149、咸87(30)和小偃6号为材料,利用与抗白粉病基因Pm21共分离的SCAR标记及与抗条锈病基因Yr26紧密连锁的SSR标记Xgwm11和Xgwm18,对N95175和远丰175所携带的抗病基因进行分子标记辅助鉴定。【结果】从N95175中扩增出与92R149相同的SCAR标记特异条带,而在2个感病亲本咸87(30)、小偃6号和远丰175中没有扩增出该条带。N95175和远丰175的扩增产物与抗条锈病亲本92R149相同,与2个感病亲本不同。【结论】导入N95175的抗白粉病基因为Pm21,导入N95175和远丰175中的抗条锈病基因为Yr26。     

小麦品种Bogatka抗白粉病基因的分子标记定位

为明确小麦品种Bogatka抗白粉病性状的遗传基础,利用感病亲本薛早和Bogatka以及其杂交所得"薛早/Bogatka"F1与薛早回交得到的BC1群体,进行遗传分析和分子标记定位。结果表明,Bogatka中含有1个显性抗白粉病基因,暂命名为MlBogatka。进一步利用BSA法对BC1分离群体进行分子标记检测,得到与MlBogatka基因连锁的分子标记STSBCD135、Xgwm501和Xwmc332,并构建遗传连锁图。根据这些分子标记的染色体定位信息,该基因位于小麦2B染色体长臂。综合对该基因的标记定位和Pm6基因特异分子标记检测结果,推测该基因可能是Pm6或与Pm6位点紧密连锁的抗白粉病基因。本研究结果为Bogatka在小麦抗白粉病育种中的利用提供了依据。     

Characterization of RAPD Markers, and the RFLP Marker Linked to Powdery Mildew Resistance Gene Derived from Different Accessions of H. villosa

The analysis was carried out on performance of the resistance gene from Haynaldia villosa accession of the former Soviet Union to different isolates of Bluemerie graminis. Polymorphisms were revealed between 6D/6V substitution line Pm930640and its pedigree parents using five RAPD markers of OPAN031700, OPAI017oo, OPAL03750, OPAD07480 and OPAG1558oscreened out from 120 random 10-mers primers. Three RAPD markers of OPAN03, OPAI01 and OPAL03 were linked with the resistance gene by analysis of F2 population of Chancellor×Pm930640. Analysis of 29 wheat lines including part of lines conferring the known genes from Pm1 to Pm20 respectively, lines conferring resistance gene from two H. villosaaccessions and the related wheat parents, were analyzed and the results showed that these markers not only linked to thegene resistant to powdery mildew from H. villosa, but also detected different genetic backgrounds. OPAL03750 can beused as the marker to distinguish the different resistant lines from two H. villosa accessions because it was only observedin the materials from H. villosa of the former Soviet Union. RFLP analysis also showed the polymorphisms between twoH. villosa accessions and their derived resistant lines.     

分子标记选择小麦抗白粉病基因Pm4b、Pm13和Pm21聚合体

 培育多个抗病基因聚合的小麦品种是提高其抗病广谱性和持久性的有效途径之一。利用小麦抗白粉病基因Pm4、Pm13、Pm2 1的特异PCR标记 ,对含有Pm4b、Pm13、Pm2 1的小麦品系复合杂交F2 代 4 0个植株进行检测 ,从中选择到Pm4b +Pm13+Pm2 13个基因聚合的抗病植株 11个 ,检测、选择到Pm4b +Pm13、Pm4b +Pm2 1、Pm13+Pm2 12个基因聚合的抗病植株 19个 ,为持久、广谱抗病小麦育种奠定了基础。研究还表明 ,3个独立的显性抗病基因在F2 代分离群体中的分离比例基本符合孟德尔独立分配定律 ,在小麦背景下的遗传稳定性 ,与该基因供体和普通小麦的亲缘关系密切相关 ,亲缘关系越远 ,丢失的概率越大。因此 ,在育种过程中对外源基因鉴定和跟踪非常必要     

小麦抗白粉病基因Pm17在亲本和F_2代抗感集群中的RAPD分析

实验应用ＲＡＰＤ技术,采用集群分离分析方法对小麦白粉病感病品系Ｃｈａｎｃｅｌｏｒ和具有抗性基因Ｐｍ１７的抗病品系Ａｍｉｇｏ及其Ｆ２代抗感集群基因组进行分析,旨在鉴定与抗白粉病基因连锁的分子标记。对３８０个随机引物进行筛选,其结果为：①有４３个引物的扩增片段在亲本间出现多态性;②有两个引物ＯＰＣ０１和ＯＰＤ０３的扩增片段在亲代和Ｆ２代抗感集群中出现一致多态性。     

小麦抗白粉病基因Pm4b的RGA分析

为克隆抗性基因和发展Pm4b的特异分子标记奠定基础。利用10对RGA引物,对小麦抗白粉病基因的一些载体品种(系)进行扩增,将引物对R11F/R11R从Pm4b基因的载体品种VPM中扩增出的稳定多态性条带回收、克隆、测序,获得与小麦Pm4b基因的相关抗病基因的同源片段,并对不同的小麦Pm基因载体品系作了检测分析。该稳定多态性条带全长1 321 bp。序列分析表明这个片段属于RGA类序列。用该标记检测小麦不同Pm基因载体品种(系),发现该多态性片段仅出现在Pm4b基因载体品种中。该研究可为分离抗性基因和发展Pm4b的特异分子标记奠定基础。     

人工合成小麦对白粉病的抗性评价

本研究利用我国不同地区的20个小麦白粉菌菌株对28份合成小麦材料进行抗病性评价,并利用与基因连锁的分子标记检测这些合成小麦中Pm2抗白粉病基因。在供试的合成小麦材料中,C7、C14和C2分别抗14～16个菌株,另有6份材料能抗10个以上菌株,C16和C20不抗任何菌株。来自相同杂交组合的材料抗病性表现有很大不同。根据分子检测的结果,C9和C19的扩增片段与Pm2基因相同,但是抗谱分析表明这两个合成小麦对白粉菌的反应型与Pm2基因不同。     

Molecular detection of the powdery mildew resistance genes in winter wheats DH51302 and Shimai 26

Resistance to powdery mildew is an important trait of interest in many wheat breeding programs. The information on genes conferring resistance to powdery mildew in wheat cultivars is useful in parental selection. Winter wheat breeding line DH51302 derived from Liangxing 99 and cultivar Shimai 26 derived from Jimai 22 showed identical infection patterns against 13 isolates of Blumeria graminis f. sp. tritici(Bgt) that causes wheat powdery mildew. DH51302 and Shimai 26 were crossed to a powdery mildew susceptible cultivar Zhongzuo 9504 and the F_(2:3) families were used in molecular localization of the resistance genes. Fourteen polymorphic markers, which were linked to Pm52 from Liangxing 99, were used to establish the genetic linkage maps for the resistance genes Pm DH51302 and Pm SM26 in DH51302 and Shimai 26, respectively. These genes were placed in the same genetic interval where Pm52 resides. Analysis of gene-linked molecular markers indicated that Pm DH51302 and Pm SM26 differed from other powdery mildew resistance genes on chromosome arm 2 BL, such as Pm6, Pm33, Pm51, Ml Zec1, Ml AB10, and Pm64. Based on the results of reaction patterns to different Bgt isolates and molecular marker localization, together with the pedigree information, DH51302 and Shimai 26 carried the same gene, Pm52, which confers their resistance to powdery mildew.     

Identification of Co-Segregating RAPD Marker Linked to Powdery Mildew Resistance Gene Pm 18 in Wheat

The Pm18 gene of wheat confers resistance to the powdery mildew which is oneof the most serious diseases in many regions of the world. In this study, bulked segregant analysis (BSA) was used to develop randomly amplified polymorphic DNA (RAPD) markers linked to Pml8 gene. Three hundred and twenty decamer primers were screened and one of them was identified as RAPD marker (S411600) linked to Pml8. Using the F2 mapping population from the cross Pml8 × Chancellor, the marker S411600 was shown to co-segregate with the gene Pml8. This marker can be conveniently used for marker-assisted selection in wheat breeding programs for the identification or pyramiding of Pml8 with other resistance genes.     

基于基因特异性标记分析Pm21在中国冬小麦品种（系）中的分布

【目的】来自小麦-簇毛麦6VS/6AL易位系的抗病基因Pm21对小麦白粉病具有持久和广谱抗性,开发该基因的特异性标记,分析其在全国冬麦区中的应用情况,为Pm21的合理布局及分子标记辅助选择育种提供理论依据和技术支撑。【方法】根据已克隆的与Pm21抗性途径紧密相关的丝氨酸/苏氨酸蛋白激酶基因Stpk-V的序列（GenBank登录号为HQ864471.1）,提取其蛋白序列并利用Pfam软件分析其保守结构域起止位点,在其保守结构域外设计开发特异序列标记WS-1;构建Pm21载体品种92R137和感病品种Avcoet S（AvS）的F2群体,以小麦白粉病菌E09对该群体每个单株进行苗期抗白粉病表型鉴定,同时利用WS-1对F2群体进行分子检测,分析检测表型与抗病表型,以验证WS-1标记的准确性;利用WS-1标记对来自中国不同冬麦区的662份小麦品种（系）进行分子标记检测,分析Pm21在不同麦区小麦品种（系）的分布情况,并将检测到含有Pm21的品种（系）在田间进行抗白粉病鉴定;选取WS-1已检测到及没有检测到Pm21的品种（系）各50份,利用曹爱忠等开发的标记NAU/xibao15902进行PCR扩增,进一步证明WS-1的准确性。【结果】（1）开发的Pm21特异标记WS-1为显性标记,含Pm21的小麦材料在8%非变性聚丙烯酰胺凝胶中扩增出一条大小为949 bp的片段,而不含该基因的小麦材料中无该片段。（2）在包含377个株系的F2群体中,286个单株为抗病,91个单株为感病,抗感比符合3﹕1的分离比例,表明在该群体中Pm21表现为显性单基因,WS-1对F2群体的每个株系的检测结果与抗/感表型完全一致。（3）供试的662份小麦材料中,49份携带Pm21,平均分布频率为7.4%,其中,西南冬麦区中检测到33份,占该区参鉴品种（系）数的34.4%,而北部冬麦区、黄淮冬麦区和长江中下游冬麦区,分别检测到4份、9份和3份,各占该区参鉴品种（系）数的5.3%、3.1%和1.5%。【结论】开发的Pm21特异性标记WS-1可以作为该基因的检测标记,也可应用到今后的基因聚合育种中;该基因在不同麦区分布相差很大,其中,西南冬麦区四川、贵州省的小麦品种（系）中Pm21使用频率过高,有促进病原菌定向选择的风险,在当前小麦育种中应给以重视。