小麦抗白粉病基因Pm43定位区段内RGA分析

利用普通小麦测序草图可从基因组范围内对小麦单条染色体上的某个区段进行分析。Pm43是作物遗传与分子改良山西省重点实验室在小麦2D染色体长臂上定位的一个抗白粉病基因。利用信息学方法分析Pm43所在物理图谱、遗传图谱和基因组图谱上的位置,可为其精细定位乃至候选基因的确定提供参考。试验采用Pm43两侧标记序列进行比对,将Pm43定位于染色体C-2DL3-0.49区间的79~99 c M内,所在基因组区段为2DL_9835990~2DL_9823315。利用目前已克隆小麦抗病基因的保守基序作为探针,从目标区段内检索出89条包含抗病基因类似物(Resistance gene analogues,RGA)序列的scaffold,其中,36条scaffold被诊断出含有SSR位点,之后针对SSR位点开发分子标记。利用携带有Pm43的普通小麦材料CH5025、感白粉病材料台长29以及CH5025×台长29的F2作图群体的抗感池DNA,对开发的SSR标记进行连锁性检测,共筛选出4个多态性标记,从而将目标区段进一步确定在标记PK_9908430和NBS_9908778之间。最后经聚类分析,筛选出与已克隆Pm基因同源性较高的1个PK序列和1个NBS序列,且在粗山羊草2D染色体和水稻第4染色体中均存在与这2个序列同源的RGA表达序列。     

小麦全基因组NBS类R基因分析及2AL染色体NBS-SSR特异标记开发

NBS (nucleotide binding site)类基因是植物界中最重要的一类抗病基因。用信息学方法从普通小麦(Triticum aestivum L.)全基因组中分离出2406条含有NBS结构的完整蛋白序列,每条包含48~2272个氨基酸。根据NBS结构域两端是否连接CC或LRR结构域,将TaNBS分为N、CN、NL和CNL4类。对TaNBS所在scaffold序列的SSR位点进行诊断,从1203条scaffold序列上发现2177个SSR位点,以二碱基重复位点最多,占73.5%。针对小麦2AL染色体上的51个SSR位点开发标记,缺体-四体和双端体验证结果表明,有39个标记(76.5%)为2AL特异标记,其中24个特异标记在抗白粉病材料Khapli (2AL上携带Pm4a)和感病材料Chancellor间存在多态性。利用近等基因系Khapli/8*Cc筛选出3个可能与Pm4a连锁的NBS-SSR标记,分别是Sxaas_2AL22、Sxaas_2AL39和Sxaas_2AL46。本研究开发的与抗病序列紧密连锁的特异SSR标记可用于2AL染色体上抗病新基因的检测以及已有抗病基因的候选序列筛选。     

普通小麦品种Brock抗白粉病基因分子标记定位

为明确利用Brock转育成的小麦抗白粉病品系3B529(京411*7//农大015/Brock, F₆)抗性的遗传基础,将高感白粉病小麦品系薛早和3B529杂交,获得F₁代、F₂分离群体和F_2:3家系。抗病性鉴定和遗传分析结果表明,3B529对E09小种的抗性受1对显性基因控制,暂被定名为MlBrock。利用BSA和分子标记分析,获得了与MlBrock连锁的3个SSR标记Xcfd81、Xcfd78、Xgwm159和2个SCAR标记SCAR203和SCAR112,根据SSR和SCAR标记在中国春缺体四体、双端体和缺失系的定位结果,将MlBrock定位在小麦染色体臂5DS Bin 0~0.63区间上。MlBrock与Xcfd81和SCAR203共分离,与SCAR112的遗传距离为0.5 cM。这些分子标记的建立有利于今后Brock抗白粉病基因分子标记辅助选择和基因聚合。综合抗白粉病基因MlBrock的染色体定位和抗谱分析结果,推测MlBrock很可能是Pm2基因。     

青海大黄油菜Brsc1基因的精细定位及图谱整合

大黄油菜是源于青海湟源的地方品种,种皮颜色鲜黄,其大黄油菜的黄籽性状受1对隐性基因(Brsc1)控制,该基因被定位于白菜A9染色体上一段1.7 Mb的区间内。为了更好地利用这一黄籽资源,对Brsc1基因进一步精细定位。利用青海大黄油菜和褐籽白菜型油菜09A-126构建BC4及F2分离群体。利用白菜同源区段内已公布的SSR标记,同时利用该区段序列信息开发新的SSR引物,共获得6个与目标基因紧密连锁的标记(Br ID10711、Br A5~Br A9),其中Br A5与目标基因共分离,Br A9为一侧最近标记,它与目标基因之间的遗传图距为0.69 c M。至此,Brsc1基因进一步被限定于标记Y06和Br A9之间约1.2 Mb的区间内。利用本研究中获得的标记检测F2群体中3种类型单株,鉴定出一个共显性标记Br A8。将本研究中获得的SSR标记与前人研究结果进行整合,加密了Brsc1基因所在区间的标记密度。同时,特异片段与拟南芥基因组进行序列比对的结果表明,共有5个标记与拟南芥的第1染色体有较好的共线性关系,暗示Brsc1基因的同源基因可能位于拟南芥的第1染色体上。本研究中获得的标记将为Brsc1基因的克隆及利用Brsc1基因进行黄籽油菜的分子辅助育种提供有利条件。     

小麦品种汶农14抗白粉病基因的染色体定位

汶农14是近年山东省和国家审定一个半冬性小麦品种。采用来自不同地区的52个小麦白粉菌菌株对汶农14进行抗性鉴定,并利用分子标记分析定位了其抗白粉病基因。汶农14对43个菌株(82.7%)表现抗性反应型,对9个菌株表现感病反应型。这些菌株对汶农14的毒力谱与已知抗白粉病基因Pm2相似,但汶农14对11个菌株的反应与携带Pm2的Ulka/8*Cc不同。此外,利用26个菌株的鉴定结果表明,汶农14与携带Pm46的Tabasco相比,与3个菌株的反应型表现不同。汶农14在成株期对白粉病混合菌株表现高抗。利用汶农14×邯4564的F₂和F_2:3群体进行遗传分析,发现汶农14对E09菌株的抗性受1对显性基因控制,暂命名为PmW14。分子标记分析显示,PmW14与Xcfd8、Xcfd81和SCAR203连锁,遗传距离分别为7.5、1.8和7.7 cM。由于这些分子标记被定位于小麦5DS染色体的5DS-1-0-0.63区间,且与Pm2基因紧密连锁,因此推测,PmW14可能与Pm2位于相同的基因座。     

水稻线粒体SCAR分子标记的开发与应用

为了开发一套能够简单快捷地筛选水稻细胞质的分子标记,本研究选用了5个配子体雄性不育系W15A、W20A、W34A、W46A和YA为材料,采用线粒体基因组DNA超纯提取和多态性扩增(random amplified polymorphic DNA,RAPD)的方法,开发了一套水稻线粒体基因组分子标记(sequence characterized amplified region,SCAR),该套分子标记共37个标记,其中16个定位水稻线粒体基因组,12个为无同源序列,其他的能在叶绿体、核以及细菌中找到同源。用这套分子标记筛选20份含红莲型不育基因orf H79的水稻材料和35个野生稻材料,均呈现出很好的细胞质多态性。因此,这些标记为水稻线粒体基因组多态性鉴定、新型不育细胞质的筛选提供一个快速的方法,同时也可用于不育系品种鉴定和水稻细胞质进化研究。     

河南省小麦品种白粉病抗性基因的分子鉴定及分子标记辅助育种

对河南省50年来大面积推广的30个小麦品种进行白粉病抗性鉴定,同时利用与Pm2,Pm4,Pm8,Pm13,Pm21及Pm24紧密连锁的PCR标记对这些主推品种进行抗白粉病基因鉴定。其中仅有3个品种抗白粉病,20世纪80年代以前的品种几乎没有上述抗白粉病基因,80年代以后利用Pm8较多,Pm2,Pm4和Pm24也得到了一定的应用,而Pm13和Pm21没有在这些品种中得到使用。同时利用这些标记对2005年在河南省收获面积大于6.67万hm2的14个品种进行鉴定。采用回交育种及分子标记技术相结合将Pm13,Pm21,Pm30和Pm33等抗性基因导入了大面积生产应用的小麦品种郑麦9023之中,获得了抗白粉病的郑麦9023近等基因系,育成郑麦9023抗白粉病的多系品种。采用杂交育种与分子标记技术相结合,育成郑麦835、郑麦863等白粉病抗性基因聚合的新品种和含有Pm21的抗病品种郑麦883。     

从野生二粒小麦导入普通小麦的抗白粉病基因MlWE18分子标记定位

野生二粒小麦(Triticum turgidumvar. dicoccoides)是小麦抗白粉病遗传改良的重要基因资源。利用野生二粒小麦WE18与普通小麦品种(系)连续多次杂交和自交,育成对白粉病菌生理小种E09高度抵抗的小麦新品系3D249(京双27//燕大1817/WE18/3/温麦4,F₇)。利用高感白粉病品系薛早和3D249组配杂交组合,获得杂种F₁代、F₂分离群体和F₃代家系,进行苗期白粉病抗性鉴定和遗传分析。结果表明,小麦品系3D249对E09小种的抗性受显性单基因控制,暂命名该基因为MlWE18。利用集群分离分析法(BSA)和分子标记分析,发现4个简单重复序列(SSR)标记(Xwmc525、Xwmc273、Xcfa2040和Xcfa2240)、1个EST-STS标记(Xmag1759)和1个EST-STS序列标记(XE13-2)与抗白粉病基因MlWE18连锁,在遗传连锁图谱上的顺序为Xwmc525–Xcfa2040–Xwmc273–XE13-2–Xmag1759–MlWE18–Xcfa2240。SSR标记的染色体缺失系物理定位结果表明,抗白粉病基因MlWE18位于小麦7A染色体长臂末端的Bin 7AL 16–0.85–1.00。与已知定位于该染色体区域的Pm基因遗传连锁图谱比较表明,MlWE18与抗白粉病基因Pm1、MlIW72、PmU、Mlm2033和Mlm80均位于7AL相同染色体区段。     

小麦抗白粉病基因聚合体DH材料的分子标记鉴定

小麦白粉病是由Blumeria graminis f.sp. tritici引起的世界性病害,利用分子标记辅助选择进行抗白粉病基因累加,可延长品种抗病性寿命,有利于提高育种效率。本研究利用Pm4b的STS-PCR标记,Pm13和PmV的SCAR-PCR标记,以及与Pm12共分离的同功酶标记（α-Amy-1）对来自小麦与玉米杂交产生的双单倍体材料的7个株系和9个穗系的4     

39份外引小麦种质抗病基因的分子标记检测及其抗病性评价

为了初步明确39份外引小麦种质中条锈病和白粉病抗性基因组成,利用共分离或紧密连锁的分子标记对抗条锈病基因Yr5、Yr10、Yr18和抗白粉病基因Pm4、Pm13、Pm21进行检测,同时结合田间鉴定,对外引种质的抗病性进行评价。结果表明,携带Yr18基因的种质有6份,对条锈病菌表现近免疫至中抗,抗性表现稳定; Yr5连锁标记S1320阳性的种质有21份,Yr10连锁标记SC200阳性的种质有2份,但标记阳性的种质中抗病性表现不一致,可能跟载体品种的遗传背景有关,利用这些分子标记进行辅助育种时,要结合接种鉴定结果综合判断。Pm4基因基本丧失白粉病抗性,携带该基因的7份种质中仅有1份抗病。在39份种质中,均未检测到抗白粉病基因Pm13和Pm21。此外,有2份种质澳阿优1号和bermude兼具条锈病和白粉病抗性,综合抗病性好,在育种中可以合理利用。为小麦抗病育种亲本选择和种质资源的合理利用提供了参考依据。     

分子标记辅助选择小麦抗白粉病基因Pm2、Pm4a、Pm21 的聚合体

利用与小麦抗白粉病基因Pm2、Pm4a和Pm21紧密连锁的PCR标记,对含有Pm2、Pm4a和Pm21的小麦品系复合杂交后代经3轮分子标记选择,得到了一批聚合有Pm2+Pm4a+Pm21 3个基因的抗病植株,以及若干株Pm2+Pm21、Pm4a+Pm21和Pm2+Pm4a 2个基因聚合的植株。同时,还对中选植株进行抗病性人工接种鉴定。结果表明,含有Pm21的聚合体与Pm21基因单独存在时抗性相当,均对白粉病免疫,聚合体Pm2+Pm4a的抗性好于Pm2或Pm4a单独存在时的抗性。为降低分子标记选择成本,将检测Pm4a和Pm21的2种PCR放在一个反应体系中进行,扩增产物经1次电泳,可同时检测出Pm4a和Pm21,不同引物之间没有明显交叉扩增现象。     

小麦合成种M53抗白粉病基因的RAPD和SSR标记

运用RAPD和SSR技术，采用分离群体分组分析法(BSA)进行了小麦合成种M53抗白粉病基因连锁的分子标记研究。结果表明，M53的抗白粉病基因由显性单基因控制，RAPD标记OPL09-1700与抗病基因连锁，遗传距离为16.8cM。SSR标记Xgwm205也与抗白粉病基因连锁，遗传距离为9.3cM，通过SSR标记将该基因定位于5DS，标记与基因间的排列顺序     

Microsatellite mapping of powdery mildew resistance allele <Emphasis Type="Italic">Pm5d</Emphasis> from common wheat line IGV1-455

The powdery mildew resistance allele Pm5d in the backcross-derived wheat lines IGV1-455 (CI10904/7*Prins) and IGV1-556 (CI10904/7*Starke) shows a wide spectrum of resistance and virulent pathotypes have not yet been detected in Germany. Although this allele may be distinguished from the other documented Pm5 alleles by employing a differential set of Blumeria graminis tritici isolates, the use of linked molecular markers could enhance selection, especially for gene pyramiding. Pm5d was genetically mapped relative to six microsatellite markers in the distal part of chromosome 7BL using 82 F₃ families of the cross Chinese Spring × IGV1-455. Microsatellite-based deletion line mapping placed Pm5d in the terminal 14% of chromosome 7BL. The closely linked microsatellite markers Xgwm577 and Xwmc581 showed useful variation for distinguishing the different Pm5 alleles except the ones originating from Chinese wheat germplasm. Their use, however, would be limited to particular crosses because they are not functional markers. The occurrence of resistance genes closely linked to the Pm5 locus is discussed. Ghazaleh Nematollahi and Volker Mohler equally contributed to this work.     

A high-resolution map of the rice blast resistance gene Pi15 constructed by sequence-ready markers

The gene Pi15 for resistance of rice to Magnaporthe grisea was previously mapped to a ≈0.7-cM region on chromosome 9. To further define the chromosomal region of the Pi15 locus, a contig spanning the locus was constructed, in silico , through bioinformatics analysis using a reference sequence of the cultivar 'Nipponbare'. One simple sequence repeat marker adopted from the International Rice Microsatellite Initiative and six candidate resistance gene (CRG) markers, developed from gene annotation of the reference sequence of the contig, were used for linkage analysis in a mapping population consisting of 504 extremely susceptible F₂ plants. The Pi15 locus was delimited to a ≈0.5-cM region flanked by the markers CRG5 and CRG2 and co-segregated with the markers BAPi15₇₈₂, CRG3 and CRG4, which was physically converted to a 44-kb interval.     

Development and identification of wheat–Haynaldia villosa T6DL.6VS chromosome translocation lines conferring resistance to powdery mildew

Three lines conferring resistance to powdery mildew, Pm97033, Pm97034 and Pm97035, were developed from the cross of Triticum durum-Haynaldia villosa amphidiploid TH3 and wheat cv.'Wan7107' via backcrosses, immature embryo and anther culture. Genomic in situ hybridization analysis showed that these lines were disomic translocation lines. Cytogenetic analysis indicated that the F1 plants of crosses between the three translocation lines and 'Wan7107' and crosses between the three translocation lines and substitution line 6V(6D) formed 21 bivalents at meiotic metaphase I. Aneuploid analysis with 'Chinese Spring' double ditelocentric stocks indicated that the translocated chromosomes were related to chromosome 6D. Biochemical and restriction fragment-length polymorphism (RFLP) analyses showed that the translocation lines lacked a specific band of 6VL of H. villosa compared with the substitution and addition lines but possessed specific markers on the short arm of the 6V chromosome of H. villosa. The three translocation lines lacked specific biochemical loci and RFLP markers located on chromosome 6DS. The results confirmed that Pm97033, Pm97034 and Pm97035 were T6DL.6VS translocation lines.     

Identification of AFLP fragments linked to one recessive genic male sterility (RGMS) in rapeseed (Brassica napus L.) and conversion to SCAR markers for marker-aided selection

117AB is a recessive genic male sterility (RGMS) line in which the sterility is controlled by a duplicate recessive gene named ms, located at two separate loci. In the RGMS line, the genotype of the sterile plant (117A) is msmsmsms, and that of the fertile plant (117B) is Msmsmsms. The present study was aimed to identify DNA markers linked to the ms locus by amplified fragment length polymorphism (AFLP). From the survey of 512 AFLP primer combinations, 6 AFLP fragments (y1, k1, k2, k3, k4, k5) were identified as being tightly linked to the Ms locus. The genetic distances between the markers and the Ms locus were all less than 8 cM, among which two fragments, designated as k2 and k3, co-segregated with the target gene in the tested population. Fragment k2 was successfully converted into a sequence characterized amplified region (SCAR) marker. The markers detected could be valuable in marker-assisted breeding of RGMS in Brassica napus.     

Molecular detection of rye (Secale cereale L.) chromatin in wheat line 07jian126 (Triticum aestivum L.) and its association to wheat powdery mildew resistance

Powdery mildew (Pm), caused by Blumeria graminis f. sp. tritici (Bgt), is one of the most serious diseases for common wheat in many regions around the world. Seeking for new resistance source is urgently required to meet the challenge of the rapid loss of resistance due to the co-evolution of the pathogen’s virulence. Wheat line 07jian126 (Triticum aestivum L.) is highly resistant to the Pm disease prevailing in Sichuan province of China. Previous study showed that a SSR marker Xbarc183 was linked to the Pm resistance in 07jian126, which might be controlled by a single dominant gene, designated as Pm07J126. In this study, two additional F₂ populations were used to confirm the linkage between Pm07J126 and Xbarc183. Furthermore, rye chromatin was detected in 07jian126 by molecular analysis of a rye-specific SCAR marker O5 which co-segregated with Pm07J126. This result indicated that Pm07J126 might originate from rye. The reaction patterns to 21 Bgt isolates and molecular marker analysis implied that Pm07J126 might be different from the known rye-derived Pm genes Pm7, Pm8, Pm17 and PmJZHM2RL. Chromosome observation, molecular marker, and A-PAGE analysis suggested that 07jian126 might be a rye introgression line and neither contain 1RS translocation nor secalins gene. Consequently, 07jian126 could be considered as a valuable resource for Pm resistance development of wheat. Besides, the molecular markers Xbarc183 and O5 are useful in marker-assisted selection of Pm07J126 in wheat breeding programs.     

Molecular marker-facilitated pyramiding of different genes for powdery mildew resistance in wheat

Breeding durable resistance to pathogens and pests is a major task for modern plant breeders and pyramiding different resistance genes into a genotype is one way of achieving this. Three powdery mildew resistance gene combinations, Pm2+Pm4a, Pm2+Pm21, Pm4a+Pm21 were successfully integrated into an elite wheat cultivar ‘Yang047′. Double homozygotes were selected from a small F₂ population with the help of molecular markers. As the parents were near‐isogenic lines (NILs) of ‘Yang158′, the progenies showed good uniformity in morphological and other non‐resistance agronomic traits. The present work illustrates the bright prospects for the utilization of molecular markers in breeding for host resistance.