Characterization of wheat monogenic lines with known Sr genes and wheat cultivars for resistance to three new races of Puccinia graminis f. sp. tritici in China

Wheat stem rust, caused by Puccinia graminis f. sp. tritici(Pgt), is a potentially devastating fungal disease of wheat worldwide. The present study was to evaluate the resistance of 42 wheat monogenic lines with known stem rust resistance(Sr) genes and 69 wheat cultivars to three new Pgt races(34C0MRGQM, 34C3MKGQM, and 34C6MTGSM)identified from aeciospores at the seedling and adult-plant stages. The phenotyping results revealed that monogenic lines harboring resistance genes Sr9e, Sr17, Sr21, Sr22, Sr26, Sr30, Sr31, Sr33, Sr35, Sr36, Sr37, Sr38, Sr47, SrTmp,and SrTt3 were effectively resistant to all three Pgt races at the seedling and adult-plant stages. In contrast, monogenic lines containing Sr5, Sr6, Sr7b, Sr9a, Sr9d, Sr9f, Sr9g, Sr9b, Sr16, Sr24, Sr28, and Sr39 were highly susceptible to these races at both seedling and adult-plant stages. The other lines with Sr8a, Sr10, Sr11, Sr13, Sr14, Sr15, Sr18, Sr20,Sr19, Sr23, Sr25, Sr27, Sr29, Sr32, and Sr34, displayed variable levels of resistance to one or two of the tested races.Seedling infection types(ITs) and adult-plant infection responses(IRs) indicated that 41(59.4%) of the wheat cultivars showed high resistance to all the three races. Molecular marker analysis showed that four wheat culitvars likely carried Sr2, 20 wheat culitvars likely carried Sr31, 9 wheat culitvars likely carried Sr38, and none of the cultivars carried Sr24,Sr25, and Sr26. Our results provide a scientific basis for rational utilization of the tested Sr genes and wheat cultivars against these novel Pgt races.

Virulence and Diversity of Blumeria graminis f.sp.tritici Populations in China

Wheat powdery mildew, caused by Blumeria graminis f. sp. tritici, is an important disease in China. To characterize the virulence and diversity of the pathogen, 1 082 isolates were obtained from 8 major wheat-growing regions during the spring growing season in 2011. The virulence test was performed by inoculation on detached leaves of 22 differential lines with known Pm genes. Frequencies of virulence on these genotypes ranged from 0 to 97.4%. None of the 1 082 isolates was compatible to Pm21 and less than 20.0%were virulent to the genotype carrying Pm13. In contrast, the virulence frequencies of each population was more than 50.0%to differentials carrying Pm1a, Pm3b, Pm3c, Pm3f, Pm5a, Pm6 and Pm8. In total, 1 028 pathotypes were detected, of which 984 were unique. Phenotypic diversity indices revealed a high level of diversity within populations. Genetic distance between different populations correlated signiifcantly with geographical distance （R2=0.494, P 0.001）. In addition, isolates from Xinjiang appear to form a separate group. Signiifcant positive or negative associations between alleles at pairs of virulence loci were detected in 57 allele pairs to Pm genes. Virulence and diversity of the 8 populations suggested that varieties with effective resistance gene combinations should be developed at a regional level.

Population Genetic Analysis of Blumeria graminis f. sp. tritici in Qinghai Province, China

To gain more precise information about molecular genetic variation for wild populations of Blumeria graminis f. sp. tritici from Qinghai Province, China, 38 single-colony isolates were purified from samples collected from Haidong District, Xining City and Hainan Tibetan Autonomous Prefecture in 2010. The virulence of 21 isolates among them was tested at seedling stage on 34 wheat cultivars（lines） carrying known powdery mildew（Pm） resistant genes. The results showed that V1 a, V3 a, V3 c, V3 e, V5 a, V6, V7, V8 and V19 had high virulence frequencies（〉75%）, indicating a wide distribution; and V1 c, V5 b, V12, V13, V16, V21, VXBD, V2＋6, V2＋Mld and V4＋8, with less distribution, appeared to be lower in frequencies（0-20%）. The Nei＇s gene diversity（H）, Shannon＇s information index（I） and the percentage of polymorphic loci（P） were 0.23, 0.35 and 67.65%, respectively, which revealed a virulent diversity. The results from single nucleotide polymorphisms（SNPs） of 38 isolates showed that three housekeeping genes were found to contain a total of 9 SNP sites. 10 haplotypes（H1-H10） were inferred from the concatenated sequences, with 1 haplotype（H1） comprising of over 55% of Qinghai population. Phylogenic analysis did not show obvious geographical subdivision between the isolates. A multilocus haplotype network presented a radial structure, with H1 in the central as an inferred ancestor. Using analysis of molecular variance（AMOVA）, we found 1.63% of the total variation was among populations and 98.37% within populations, with a low fixations index（FST=0.01634, P〈0.05）. This revealed a relatively high genetic diversity but a low genetic divergence in Qinghai population. Moreover, the molecular data on gene flow（Nm=6.32） confirmed the migration of pathogen populations among areas in Qinghai Province.

Development of SSR Markers for a Phytopathogenic Fungus,Blumeria graminis f.sp.tritici,Using a FIASCO Protocol

Simple sequence repeats （SSR） have been widely used as molecular markers due to their abundance and high polymorphism, However, up to now, the SSR markers had not been developed in the obligate biotrophic phytopathogenic fungus, Blumeria graminis f.sp. tritici. From （AC）10 and （AG）10 enriched genomic libraries for Bgt, 25 primer pairs were designed using the FIASCO （fast isolation by AFLP of sequences containing repeats） protocol. Five primer pairs exhibited polymorphism with allelic diversity from two to seven alleles and produced 29 alleles in a survey of 90 isolates collected from six provinces （cities） in China, while the others displayed monomorphic. Levels of observed heterozygosity ranged from 0.000-0.044 （mean 0.025） and expected heterozygosity ranged from 0.297-0.816 （mean 0.538）. These molecular markers provide a novel source to genetic diversity assays and to genetic and physical mapping ofBgt. SSR markers of Bgt need to be further explored.

Population Diversity of Puccinia graminis is Sustained Through Sexual Cycle on Alternate Hosts

A high degree of virulence diversity has been maintained in the population of Puccinia graminis f. sp. tritici （Pgt） in northwestern United States. Although Berberis vulgaris is present in the region and Pgt has been isolated from aecial infections on B. vulgaris, the population is too diverse to be explained by the limited presence of B. vulgaris alone. Since 2008, we have isolated P. graminis from aecial infections on fruits of Mahonia repens and Mahonia aquifolium from northwestern United States. These two native woody shrub species, widely distributed in western North America, were once classified as resistant to P. graminis based on artificial inoculations. By isolating P. graminis from aecia, we established that M. repens and M. aquifolium along with B. vulgaris （albeit infrequent） serve as the alternate hosts ofP. graminis in the region. The isolates of P. graminis from Mahonia of North America had diverse virulence patterns and most of the isolates could be differentiated on Morocco, Line E, Chinese Spring, Little Club, LMPG-6, Rusty, and other genotypes that are considered to be universally susceptible to most Pgt isolates. This discovery explained the persistence of virulence diversity of Pgt observed in isolates derived from uredinia on cereal crops in the region. In addition to cereal crops, uredinial stage of the P. graminis population is sustained by wild grasses, especially Elymus glaucus, a native grass sharing the same habitat with the rusted Mahonia spp. Although virulence to some important stem rust resistance genes was observed in some isolates derived from Mahonia of North America when tested against single stem rust resistance gene stocks, the overall virulence is very limited in these isolates. This is likely a result of limited selection pressure on the rust population. In contrast to northwestern United Sates, the Pgt population in east of the Rocky Mountains of North America has declined steadily with a single race, QFCSC, being predominant in the last decade. This decline is likely due to a combination of factors, of which a lack of sexual recombination in the region is perhaps the most important one.

Identification of QTL for adult-plant resistance to powdery mildew in Chinese wheat landrace Pingyuan 50

Powdery mildew caused by Blumeria graminis f. sp. tritici is one of the major wheat diseases worldwide. The Chinese wheat landrace Pingyuan 50 has shown adult-plant resistance(APR)to powdery mildew in the field for over 60 years. To dissect the genetic basis of APR to powdery mildew in this cultivar, a mapping population of 137 double haploid(DH) lines derived from Pingyuan 50/Mingxian 169 was evaluated in replicated field trials for two years in Beijing(2009–2010 and 2010–2011) and one year in Anyang(2009–2010). A total of 540 polymorphic SSR markers were genotyped on the entire population for construction of a linkage map and QTL analysis. Three QTL were mapped on chromosomes 2BS(QPm.caas-2BS.2), 3BS(QPm.caas-3BS),and 5AL(QPm.caas-5AL) with the resistance alleles contributed by Pingyuan 50 explaining 5.3%,10.2%, and 9.1% of the phenotypic variances, respectively, and one QTL on chromosome 3BL(QPm.caas-3BL) derived from Mingxian 169 accounting for 18.1% of the phenotypic variance.QPm.caas-3BS, QPm.caas-3BL, and QPm.caas-5AL appear to be new powdery mildew APR loci.QPm.caas-2BS.2 and QPm.caas-5AL are possibly pleiotropic or closely linked resistance loci to stripe rust resistance QTL. Pingyuan 50 could be a potential genetic resource to facilitate breeding for improved APR to both powdery mildew and stripe rust.

Cytological and genetic analyses of a wheat-rye 2RL ditelosomic addition line with adult plant resistance to powdery mildew

Rye(Secale cereale genome RR),a close relative of common wheat,possesses valuable resistance genes for wheat improvement.Due to the co-evolution of pathogen virulence and host resistance,some resistance genes derived from rye have lost effectiveness.Development and identification of new,effective resistance genes from rye is thus required.In the current study,wheat-rye line WR56 was produced through distant hybridization,embryo rescue culture,chromosome doubling and backcrossing.WR56 was then proved to be a wheat-rye 2 RL ditelosomic addition line using GISH(genomic in situ hybridization),mc-FISH(multicolor fluorescence in situ hybridization),ND-FISH(non-denaturing FISH),mc-GISH(multicolor GISH)and rye chromosome arm-specific marker analysis.WR56 exhibited a high level of adult plant resistance to powdery mildew caused by Blumeria graminis f.sp.tritici(Bgt).This resistance was carried by the added 2 RL telosomes and presumed to be different from Pm7 which is also located on chromosome arm 2 RL but confers resistance at the seedling and adult stages.WR56 will be a promising bridging parent for transfer of the resistance to a more stable wheat breeding line.A newly developed2 RL-specific KASP(kompetitive allele specific PCR)marker should expedite that work.

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 F2:3 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.

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 。

若干小麦抗白粉病品系的有效抗病基因的测定

采用具有不同毒性基因的白粉菌菌株进行苗期接种,通过与已知抗病基因材料抗谱的相似性比较,对17个抗白粉病育种品系的有效抗病基因进行了分析,其中3个阿拉拉特小麦(T.araraticum)杂种后代品系、4个V.P.M系统的杂种后代品系及另外1个杂交组合的后代品系含主效抗病基因Pm2;4个具有V.P.M或C39血缘的品系及另外1个品系含主效抗病基因Pm4b;1个品系含Pm2+6;2个普通小麦-黑麦6D/6R代换系的抗谱相似,且不同于所有已知抗病基因材料;此外,含Pm21的普通小麦-簇毛麦6AL/6VS易位系对所有供试菌株免疫。

两个抗小麦白粉病新基因的遗传分析与染色体定位

YU25是从八倍体小偃麦TAI7047与小麦栽培品种川麦107杂交后代中选育出的对白粉病免疫的小麦育种新材料。以感白粉病小麦品种MY11与YU25杂交和回交的后代F1、F2、BC1F1和BC2F1为材料,采用四川省当前流行的小麦白粉病优势生理小种人工接种,对YU25的白粉病抗性进行了遗传分析。结果表明,YU25含有2对表现免疫反应和高抗反应的显性抗病基因,暂命名为PmE(免疫)和PmYU25(高抗)。用294对小麦微卫星引物和221个F2植株,对这2个基因进行连锁分析,发现小麦微卫星标记Xgwm-297-7B与PmE基因的遗传距离为13.0 cM,而Xgwm-210-2D与PmYU25基因的遗传距离为16.6 cM,因此将PmE和PmYU25分别定位在7BS和2DL上。根据系谱和基因位点分析,推断PmE和PmYU25均为起源于中间偃麦草、不同于已知的抗小麦白粉病基因的2个新基因。小麦育种新材料YU25含有可能来源于小麦-中间偃麦草的染色体多重易位,其细胞学基础和在实际育种中的应用值得进一步研究。

2007年我国部分麦区小麦白粉菌对三唑酮的抗药性监测

采用拌种离体叶段法测定了我国9个省(市)的112个小麦白粉菌株对三唑酮的抗性。结果表明,供试菌株的平均EC50为57.25mg/L,平均抗性水平为27.39倍,其中86.61%供试菌株已经产生抗性,抗药性比敏感菌株高出10～40倍的菌株占47.32%,四川、山东、甘肃和贵州等地菌株的抗性高于其他地区。此结果可为三唑类杀菌剂在我国的推广应用和制定合理的抗药性治理措施提供依据。

小麦白粉病菌对喹氧灵和苯菌酮的敏感基线及药剂的生物活性

采用离体叶段法,分别测定了从河北、河南、湖北、陕西和四川5省分离的53个小麦白粉病菌单孢菌株对苯菌酮和喹氧灵的敏感性,并分析了白粉病菌对三唑酮和苯菌酮以及喹氧灵之间的交互抗性。结果表明:小麦白粉病菌群体对苯菌酮和喹氧灵的平均EC50值分别为(0.001 9±0.000 6)和(0.013 1±0.002 0)mg/L,苯菌酮比喹氧灵具有更高的抑菌活性;小麦白粉病菌对三唑酮和苯菌酮与喹氧灵之间均不存在交互抗性(R2值分别为0.102 6和0.491 9);室内盆栽试验结果显示:接种前1 d和接种后1 d施药,苯菌酮和喹氧灵对小麦白粉病的保护与治疗作用防效分别为92.21%、84.25%和82.43%、70.25%,表明这2种药剂不仅具有优异的保护作用,同时还具有较好的治疗作用。

小麦白粉病抗性基因在山西省的有效性评价

为明确小麦白粉病抗性基因在山西省的有效性，在温室条件下，通过闭囊壳释放子囊孢子侵染麦苗发病，利用28个已知抗病基因品种（系）对分离自山西省不同生态区的166个小麦白粉病菌株进行了毒性基因频率测定。结果表明，毒性基因V4a、V4b、V13、V20、V21、VXBD、V2＋6、V4＋8、V2＋Mzi、V4b＋Mli、V5＋6、V1＋2＋9的出现频率为3．6％～29．7％，其对应抗性基因Pm4a、Pm4b、Pm13、Pm20、Pm21、PmXBD、Pm2＋6、Pm4＋8、Pm2＋Mli、Pm4b＋Mli、Pm5＋6、Pm1＋2＋9为目前山西省小麦白粉病菌的有效抗病基因，可供转育利用；毒性基因V1、化、V3a、V3d、V3f、V6、VI7、V19、V2＋Ta的出现频率为38．6％～79．4％，其对应抗性基因Pm1、Pm2、Pm3a、Pm3d、Pm3f、Pm6、Pml7、Pml、Pm2＋Ta利用价值已下降；毒性基因V3b、V3c、V3e、V5、V7、V8的出现频率为92．5％～98．7％，其对应抗性基因Pm3b、Pm3c、Pm3e、Pm5、Pm7、Pm8单独使用无利用价值。对各抗性基因在山西省小麦三个生态区的有效性差异分析认为，各生态区应针对毒性基因的变化动态结合农艺性状选择选用合适的抗性基因进行转育。

湖北麦区小麦白粉病菌毒性结构分析

对湖北省不同年度(2001、2003年和2007年)、同一年度(2007年)不同生态区间的小麦白粉病菌毒性基因频率进行了比较。结果表明,毒性基因V2、V4 b、V13、V20、V21、V2+MLD、V2+6、V4+8和V4 b+Mli在测定的3个年度中毒性频率均低于25%,而V1、V3 b、V3 c、V3 d、V3 e、V5、V8、V17和V193个年度的毒性频率均高于50%;白粉病菌毒性结构年度之间和生态区之间均基本相似,但个别毒性基因年度及生态区间存在一定的差异;V2、V3 a、V3 b、V3 c、V3 e、V5、V6、V7、V8和V20在十堰以及武昌2个生态区毒性频率年度间均有明显的上升趋势,而V4 b、V5(MLi)、V13、V21、V5+6则表现为下降的趋势;2007年仅在十堰生态区检测到V2+Mld和V2+6菌株。

BTH对小麦产生白粉病抗性的诱导作用

用化学诱抗剂BTH(benzothiadiazole)分别处理幼苗期和成株期小麦后,再接种小麦白粉病菌,以研究BTH诱发小麦对白粉病产生系统性抗性的能力.结果表明,用BTH处理幼苗期小麦后,小麦白粉病的病情指数较对照显著降低,BTH诱发小麦幼苗对白粉病产生抗性的最佳浓度为0.20～0.25 mmol/L,最佳时间间隔应大于6 d.对于成株期小麦,在分蘖中期和后期以0.20 mmol/L BTH喷雾,对小麦白粉病的防效为60.82%,较对照增产16.00%.说明BTH可以诱导小麦对白粉病产生系统获得抗性,并可用于田间白粉病防治.

小麦对秆锈菌新小种Ug99的抗性研究进展

为了及时了解国际小麦育种的发展动态,介绍了国外在小麦秆锈病新小种Ug99抗性方面的研究进展,主要包括成立国际锈病研究协作网,负责全球范围的小麦锈病研究;基本明确了Ug99的可能传播路线;在肯尼亚对17个国家和组织的4 157份小麦品种进行了抗性鉴定,337份表现抗病,为抗病育种提供了重要亲本;明确了现有抗性基因的利用价值,建议利用Sr2、Sr24、Sr26、Sr36、SrTmp和来自1A/1R易位的未知抗性基因,采用持久抗性与主效基因相结合的策略,通过分子标记辅助选择提高品种选育效率。最后提出了国内技术储备的主要内容。

小麦白粉病菌诱导的TaWRKY34基因的鉴定与分析

WRKY转录因子在植物抗病防卫反应中发挥重要作用。利用cDNA宏阵列(macroarray)和RT-PCR相结合的方法,从小麦全长cDNA文库的WRKY转录因子中筛选出一个应答小麦白粉病菌胁迫的TaWRKY34转录因子,该基因编码464个氨基酸。染色体定位分析表明,该转录因子位于小麦第一同源群染色体的短臂上,并且只在细胞核中表达。其蛋白序列与拟南芥、大麦和葡萄抗病相关WRKY转录因子的亲缘关系较近,与其中的3个WRKY基因具有相似的表达模式。TaWRKY34在Pm16/北京8377抗白粉病近等基因系中,对小麦白粉病菌、水杨酸和茉莉酸诱导的表达模式存在差异。TaWRKY34可能与小麦对白粉菌的抗性有关。

河南两地市小麦白粉病菌的分子鉴定和进化分析

通过对河南省周口市和商丘市小麦白粉病菌进行分子鉴定及分析,以期为小麦白粉病菌的防治和系统进化奠定基础。对小麦白粉病菌进行了扫描电镜的显微形态观察、核糖体DNA转录间隔区(ITS)序列测定及进化树分析。结果显示:周口和商丘的小麦白粉病菌均属于禾本科布氏白粉菌;系统进化发现,商丘的小麦白粉病菌与地域相距较远的英国、法国、美国的小麦白粉病菌的同源性极高,而与地域很近的周口小麦白粉病菌同源性却不高,推测小麦白粉病菌的进化可能与其自身的小种进化有关。

小麦白粉病菌闭囊壳萌发及其对三唑酮敏感基线的建立

在没有甾醇脱甲基抑制剂 (DMI)类药剂用药历史的湖北利川山区麦田采集小麦白粉病菌闭囊壳 ,带回室内萌发 ,从每样本单孢子堆中分离得到 6 1个野生菌株。采用叶段法测得每个菌株的EC50 ,建立起小麦白粉病菌对三唑酮的相对敏感基线EC50 =(0 10 90± 0 0 0 48) μg/ml。