Identification of Molecular Markers for the Thinopyrum intermedium Chromosome 2Ai-2 with Resistance to Barley Yellow Dwarf Virus

The wheat_ Thinopyrum intermedium addition lines Z1,Z2 contain a pair of Th. intermedium chromosomes 2Ai_2 carrying the gene with resistance to barley yellow dwarf virus (BYDV). Genomic in situ hybridization (GISH) was used to analyze the chromosome constitution of Z1,Z2 by using genomic DNA probes from Th. intermedium and Pseudoroegneria strigosa . The results showed that the chromosome constitution of either Z1 or Z2 composes of 42 wheat chromosomes and two Th. intermedium chromosomes (2Ai_2). The 2Ai_2 chromosome is St_E intercalary translocation, in which the E genomic chromosome segment translocated into the middle region of the long arm of chromosome belonging to St genome. With the genomic DNA probe of Ps. strigosa , the GISH pattern specific to the 2Ai_2 chromosome may be used as a molecular cytogenetic marker. A detailed RFLP analysis on Z1, Z2 and their parents was carried out by using 12 probes on the wheat group 2 chromosomes. Twenty RFLP markers specific to the 2Ai_2 chromosome were identified. Two RAPD markers of OPR16 -350 and OPH09 -1580 , specific to the 2Ai_2 chromosome, were identified from 280 RAPD primers. These molecular markers could be used to assisted_select translocation lines with small segment of the 2Ai_2 chromosome and provide tools to localize the BYDV resistance.

Molecular diversity of barley yellow dwarf virus-PAV from China and the Czech Republic

Wheat yellow dwarf disease(BYD),caused by different species of barley/cereal yellow dwarf viruses(B/CYDVs),is one of the most serious cereal diseases in China and the Czech Republic.Because genetic diversity of the virus directly influences disease epidemiology,the molecular diversity and population structure of 24 Chinese isolates and 16 the Czech Republic isolates of BYDV-PAV from different regions in two countries were analyzed by sequencing their coat protein(CP)and readthrough protein(RTP)domain(RTD)genes and comparing the sequences with six CP and 16 RTP sequences of BYDVPAV isolates from the NCBI database based on nucleotide identity position,phylogenetic analysis and nucleotide diversity.Nucleotide identities between the Chinese and the Czech Republic isolates for the CP were 76.6–99.4%,73.9–89.1%for RTD(ORF5),respectively.The Chinese and the other country isolates showed 74.7–99.2%nucleotide identity for RTP(ORF3+ORF5).Phylogenetic analysis of CP sequences showed that 20 Chinese isolates clustered in the same clade,but the other four Chinese isolates clustered in another clade with the isolates from the Czech Republic and other counties.The population of BYDV-PAV in China had greater nucleotide variability and was more divergent than that in the Czech Republic.Geographical and ecological factors but not hosts might contribute to the population differences in the two countries.

Expression Comparisons of Pathogenesis-Related(PR) Genes in Wheat in Response to Infection/Infestation by Fusarium, Yellow dwarf virus(YDV) Aphid-Transmitted and Hessian Fly

Expression profiles of ten pathogenesis-related （PR） genes during plant defense against Fusarium, Yellow dwarf virus （YDV） aphid-transmitted and Hessian fly （Hf） were compared temporally in both resistant and susceptible genotypes following pathogen infection or insect infestation. Quantitative real-time PCR （qRT-PCR） revealed that PR1, PR2, PR3, PR5, PR6, PR8, PR9, and PR15 appeared to be induced or suppressed independently in response to Fusarium, YDV aphid-transmitted or Hf during the interactions. The PR gene（s） essential to defense against one organism may play little or no role in defense against another pathogen or pest, suggesting the alternative mechanisms may be involved in different interactions of wheat- Fusarium, wheat-YDV aphid-transmitted and wheat-Hf. However, strong up- or down-regulation of PRl2 and PR14 encoding low molecular membrane acting protein, defensin and lipid transfer protein （LTP）, respectively, had been detected after either pathogen infection or insect infestation, therefore showed broad responses to pathogens and insects. It was postulated that low molecular proteins such as defensins and LTPs might play a role in the early stages of pathogenesis in the signaling process that informs plants about the attack from biotic stresses. In addition, a synergistic action between different PR genes might exist in plants to defense certain pathogens and insects on the basis of comprehensive expression profiling of various pathogenesis-related genes revealed by qRT-PCR in this study.

Investigation of the mechanism of adult-stage resistance to barley yellow dwarf virus associated with awheat-Thinopyrum intermedium translocation

Barley yellow dwarf virus(BYDV)can infect wheat(Triticum aestivum L.),leading to yield loss.Among four BYDV strains(GAV,GPV,PAV,and RMV)identified in China,BYDV-GAV is the prevailing isolate.YW642,a wheat–Thinopyrum intermedium translocation line,is resistant to BYDV isolates at both seedling and adult stages.Zhong 8601 is the wheat recurrent parent of YW642 and is susceptible to BYDV.In this study,we investigated the adult-stage resistance mechanism of YW642,measured BYDV titer and hydrogen peroxide(H_2O_2) in adult-stage leaves of YW642 and Zhong 8601 inoculated with BYDV-GAV,and identified transcriptional differences between YW642 and Zhong 8601 using microarray-based comparative transcriptomics.Enzyme-linked immunosorbent assay and H_2O_2assay showed that both BYDV titer and H_2O_2 content were markedly lower in YW642 than in Zhong 8601 at 21,28,35,and 40 days post-inoculation(dpi).The transcriptomic comparison revealed that many types of genes were significantly up-regulated at 35 dpi in adult-stage leaves of YW642 compared to Zhong 8601.The important up-regulated genes associated with the adult-stage resistance encoded 15 resistance-like proteins,pathogenesis-related proteins(such as defensin and lipid transfer proteins),protein kinase homologs,transcription factors,reactive oxygen species scavenging-related proteins,and jasmonic acid and gibberellic acid biosynthesis enzymes.These results suggest that precise expression regulation of these proteins plays a crucial role in adult-stage resistance of YW642 against BYDV infection.

Effect of Natural Infection with Onion Yellow Dwarf Virus （OYDV） on Yield of Onion and Garlic Crops in Egypt

Present work highlights, first time in Egypt, the impact of OYDV infection on crops production. Plants grown in the field were individually labelled as either apparently healthy or diseased and were marked for harvesting. The percentage of yield loss due to viral infection was determined, on the basis of the average yield parameters of the healthy plant. The natural infection with OYDV in onion and garlic fields resulted in a substantial reduction in pseudo-stem length, number of leaves, plant weight and weight of bulb as well as number and weight of cloves. Results clearly indicate the impact of natural infection with OYDV on the crop yield of infected onion and garlic plants in comparison with the virus-free （uninfected） ones. The largest reduction in the yield of infected plants occurred in Chinese garlic （Seds40） followed by （Baladi） garlic and onion Giza 20. This information suggests that measures have to be taken to prevent natural infection with OYDV into and within onion and garlic fields in order to avoid yield loss.

Tobacco Yellow dwarf Virus完整基因组上串联重复序列分布

利用自编计算机程序提取并展示Tobacco Yellow dwarf Virus完整基因组上串联重复序列分布特性。借助MATLAB软件和借用最优完全子图算法,提取并展示NCBI数据库中Tobacco Yellow dwarf Virus完整基因组(NC_003822.1)上串联重复序列分布特性。

The complete nucleotide sequence and its organization of the genome of Barley yellow dwarf virus-GAV

The complete nucleotide sequence of genomic RNA of BYDV-GAV was determined. It comprised 5685 nucleotides and contained six open reading frames and four un-translated regions. The size and organization of BYDV-GAV genome were similar to those of BYDV PAV-aus. The nucleotide and deduced amino acid sequences of the six ORFs were aligned and compared with those of other luteoviruses. The results showed that there was a high degree of identity between BYDV-GAV and MAV-PS1 in all ORFs except ORF5 and ORF6, which had only 87.4% and 70.2% identities respectively. The reported genomic nucleotide sequence of MAV was shorter than that of BYDV-GAV, but the comparison of the genomic nucleotide sequences for MAV-PS1 and GAV showed 90.4% sequence identity for the same region of the genome. Ac-cording to the level of sequence similarities, BYDV-GAV should be closely related to BYDV-MAV.

Comparison of the potential rate of population increase of brown and green color morphs of Sitobion avenae （Homoptera： Aphididae） on barley infected and uninfected with Barley yellow dwarf virus

Life tables of brown and green color morphs of the English grain aphid, Sitobion avenae （Fabricius） reared on barley under laboratory conditions at 20 ＋ 1℃,65% ± 5% relative humidity and a photoperiod of 16 ： 8 h （L ： D） were compared. The plants were either： （i） infected with the Barley yellow dwarf virus （BYDV）; （ii） not infectedwith virus but previously infested with aphids; or （iii） healthy barley plants, which were not previously infested with aphids. Generally, both color morphs of S. avenae performedsignificantly better when fed on BYDV-infected plants than on plants that were virus free but had either not been or had been previously infested with aphids. Furthermore,when fed on BYDV-infected plants, green S. avenae developed significantly faster and had a significantly shorter reproductive period than the brown color morph. There wereno significant differences in this respect between the two color morphs ofS. avenae when they were reared on virus-free plants that either had been or not been previously infestedwith aphids. These results indicate that barley infected with BYDV is a more favorable host plant than uninfected barley for both the color morphs ofS. avenae tested, particularly the green color morph.

DEVELOPMENT OF COMMON WHEAT GERM-PLASM RESISTANT TO BARLEY YELLOW DWARF VIRUS BY BIOTECHNOLOGY

Barley yellow dwarf virus (BYDV) is one of the most serious wheat diseases in China. So far no resistance has been described in common wheat. A certain level of BYDV resistance was found in thirteen Triticeae species. Thinopyrum intermedium, two octoploids derived from TH. intermedium/wheat, Zhong 4 awnless and TAF46, and one disomic addition line, L1 derived from TAF46, showed good resistance to BYDV by enzyme linked immunosorbent assay (ELISA). Two wheat/TA. intermedium translocation lines, CPI 119880 and CPI 119899, showing good BYDV resistance were developed from L1 by using both CSph mutant and tissue culture. It is found that their BYDV resistance was controlled by a single dominant gene. Two cDNA probes pEleAcc3 and pPJN8 (E1-T1) were screened for detecting Th. intermedium DNA in wheat background. A specific band for the DNA of Th. intermedium and its derivatives was found in Southern hybridization. It is also possible to determine the size of the alien segment by comparing the relative density of the specific band. Therefore, this can be used as a marker to identify the BYDV resistance in wheat breeding program.

Obtained transgenic wheat expressing pac1 mediated by Agrobacterium is resistant against Barley yellow dwarf virus-GPV

In fission yeast (Schizosaccharomyces pombe), pac1 gene was cloned with 99.3% nucleo- tide sequence similarity with published pac1 in GenBank. In pET-5α expression system, the expres- sion product of cloned pac1 in E. coli showed activity to degrade the double-strand RNA. Harboring the binary vector pBI121, which contains pac1 gene, Agrobacterium tumefaciens strain LBA4404 was used to transform the wheat immature embryos pre- cultured 7―10 d. After preregeneration, regeneration and selection culture stage, totally 41 G418 resistant plants were obtained, in which 25 lines were proved to integrate with transgene and express transgene normally by PCR, Dot blot, RT-PCR and ELISA de- tection. Antivirus test carried out on 25 positive lines with high dose of Barley yellow dwarf virus-GPV re- vealed that 12 lines had resistance to BVDV-GPV in low level, another 12 lines had resistance to BVDV- GPV in middle level, and 1 line showed resistance to BVDV-GPV in high level. However, both low and middle level of resistance plants showed no symp- toms when infected by viruses at low dose, which suggested the dose-dependent effect of the resis- tance mediated by pac1 to BYDV-GPV.

Identification of a protein associated with circulative transmission of Barley yellow dwarf virus from cereal aphids, Schizaphis graminum and Sitobion avenae

Using 2-D electrophoresis and virus overlay assay, a 50-kDa protein (P50) exhibiting specific binding to purified virus particles of BYDV-GAV was found in the protein extracts from Schizaphis graminum and Sitobion avenae, two aphid species transmitting BYDV-GAV. P50 in the extracts of S. graminum was isolated by preparation electrophoresis and electro-eluted proteins from the gel slices for antiserum preparation. After feeding the antiserum through membrane, the transmission efficiencies of S. graminum and S. avenae for BYDV-GAV decreased significantly. It was suggested that P50 should be related with transmission pro- cess. Location of P50 was found at the plasma membrane surrounding the accessory salivary gland (ASG) in the head tissues of S. graminum by immunogold-labelling experiment. The ascertainment of the protein associated with virus transmission has a significance influence on further understanding the transmission mechanism and genetic engineering for resistant to vector transmission.

基于矮败小麦分子育种策略培育黄淮抗赤霉病小麦新品种轮选20

【目的】针对黄淮冬麦区抗赤霉病小麦品种短缺问题,开展产量与赤霉病抗性协同改良,为该麦区培育高产抗赤霉病小麦新种源。【方法】以苏麦3号为供体亲本(抗源),以矮败周麦16、周麦16、轮选136和轮选6号为受体亲本,利用骨干亲本矮败小麦结合Fhb1分子标记辅助选择和双单倍体(double haploid,DH)技术(简称矮败小麦分子育种策略)快速获得DH系,并对这些稳定品系进行株高、抽穗期和产量等主要农艺性状评价和赤霉病抗性鉴定。【结果】共获得51份半冬性、矮秆、白粒且携带Fhb1的DH系。在2个环境(2020河南和2020北京)下,51份DH系平均病小穗数分别为5.7和7.3,平均严重度分别为27.7%和35.2%,与中感对照淮麦20无显著差异;平均每公顷产量为8.3 t,略低于对照品种周麦18(8.5 t)。结合赤霉病抗性和主要农艺性状综合评价结果,选择DH116(轮选20)作为重点系进一步进行赤霉病抗性和农艺性状鉴定。单花滴注接种鉴定发现,轮选20在4个环境下均达到中抗-高抗对照抗性水平;每公顷产量在2个环境下均高于对照品种周麦18,每穗小穗数和千粒重显著高于对照(P<0.05),且比对照抽穗更早、植株更矮(P<0.05)。轮选20两年区试分别比对照百农207增产4.6%和1.7%,生产试验比对照增产3.5%;区试利用喷雾和单花滴注2种接种方法综合评价轮选20为中感-中抗赤霉病。分子标记检测发现,轮选20携带半矮秆基因Rht-D1b;在已知春化基因Vrn-A1、Vrn-B1和Vrn-D1位点均携带隐性等位基因。通过小麦660K SNP芯片分析显示,轮选20与4个亲本有64.7%SNP是一致的,亲本周麦16、轮选136、轮选6号和苏麦3号对轮选20的直接遗传贡献率分别为69.8%、12.6%、6.1%和11.5%。【结论】利用矮败小麦分子育种策略,实现了小麦高产和赤霉病抗性协同提高,培育出符合黄淮冬麦区生态类型的高产抗赤霉病小麦品种轮选20。

中熟半矮生鲜食黄肉桃新品种豫黄1号的选育

豫黄1号是以水蜜桃类型的鲜食黄肉桃品种黄水蜜为母本,以半矮生油桃新品种中油桃14号为父本通过杂交选育的半矮生黄肉鲜食桃新品种。该品种果面干净,茸毛稀少,离核,易剥皮。平均单果质量226.7 g,最大单果质量289 g。果肉黄色,软溶质,风味酸甜,香味浓郁,可溶性固形物含量(w,后同)12.1%。萌芽力和成枝力中等,树势中庸,为半矮生类型。大花型,5瓣,花粉多,育性强。在郑州地区果实7月中旬成熟,果实发育期125 d左右,适宜河南桃产区及类似生态条件地区栽培。

不同抗性青稞材料接种大麦黄矮病毒后防御酶活性变化研究

[目的]探究不同抗性青稞材料感染大麦黄矮病毒(Barley Yellow Dwarf Virus,BYDV)后防御酶活性的变化规律。[方法]以黄矮病高抗品系C280、高感品种康青3号为材料,测定接种BYDV后不同时间点叶片超氧化物歧化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT)、苯丙氨酸解氨酶(PAL)、多酚氧化酶(PPO)的活性。[结果]接种BYDV后,2个青稞材料的SOD、POD、CAT、PAL、PPO活性均显著高于未接种对照;SOD、PAL活性呈上升趋势,POD、CAT、PPO呈先上升后下降趋势,且抗病材料酶活性增幅高于感病材料;大部分时间点,抗病材料的5种防御酶活性均显著高于感病材料。[结论]SOD、POD、CAT、PAL、PPO活性变化可作为反映青稞黄矮病抗性的生理指标之一。

小麦黄矮病及其抗性育种研究进展

由大麦黄矮病毒(barley yellow dwarf viruses,BYDVs)侵染引起的小麦黄矮病(wheat yellow dwarf disease)是世界小麦上的主要病害之一,对小麦生产及粮食安全构成了严重威胁。本文综述了BYDVs的生物学特征、分类地位、致病机理及抗性育种等相关研究,并基于相关研究进展,提出了进一步扩大抗性基因挖掘、加快优异抗病基因克隆、加强抗病机理研究等方面的建议,旨在为我国小麦黄矮病抗性育种及科学防治提供一定的参考。

青稞种质资源对大麦黄矮病毒的抗性鉴定和生理分析

为明确青稞种质资源对大麦黄矮病毒(BYDV,barley yellow dwarf virus)的抗性水平,本研究采用田间人工接种法,对245份青稞材料进行了连续3年抗病性鉴定。供试材料中仅有08-1280表现为高抗黄矮病,6份材料(ZYM1289、北青6号、ZDM4409、甘青2号、藏青3000、ZYM1853)表现为抗病,中抗、感病、高感材料分别有47份、173份、18份。分子标记检测结果显示08-1280携带Yd2抗性基因。为了解大麦黄矮病毒侵染对青稞抗性生理指标的影响,分析了接种病毒后高抗材料08-1280、高感材料康青3号中总酚、脯氨酸、可溶性糖和可溶性蛋白含量的变化差异。接种后10 d,高抗材料总酚、脯氨酸含量增加幅度显著高于高感材料。接种后30 d时,高抗材料总酚含量增加幅度高于高感材料,可溶性糖含量增加幅度显著低于高感材料;与对照组相比,高抗材料可溶性蛋白显著下降,高感材料显著上升。本研究为青稞抗病品种培育和抗性机制研究提供了优异资源和理论参考。

乐都紫皮大蒜4种病毒多重RT-PCR检测技术的建立

【目的】建立乐都紫皮大蒜4种病毒的多重RT-PCR快速检测方法,为大蒜病毒病病原鉴定、脱毒效果验证等提供技术支撑。【方法】根据乐都紫皮大蒜RNA全长转录组测序结果,分别设计洋葱黄矮病毒(onion yellow dwarf virus,OYDV)、大蒜潜隐病毒(garlic latent virus,GLV)、大蒜D病毒(garlic virus D,GarVD)和大蒜X病毒(garlic virus X,GarVX)4种病毒的外壳蛋白特异性引物,筛选不同引物,优化引物用量、模板用量、退火温度等条件,并进行了多重RT-PCR的灵敏度检测和12份引进栽培大蒜种质资源的病毒检测。【结果】通过优化筛选,在一个PCR反应体系中实现了OYDV(1232 bp)、GLV(831 bp)、GarVD(506 bp)和GarVX(116 bp)4种病毒的多重检测,最优反应条件为退火温度55℃,模板用量2.5μL,混合引物用量1.0μL。多重RT-PCR的灵敏度略低于单一RT-PCR。对引进的12份栽培大蒜资源进行4种病毒的多重RT-PCR检测发现,有5份资源存在4种病毒,4份资源存在2种病毒,3份资源存在1种病毒。对7份资源中经多重PCR未检测到的病毒,使用单一RT-PCR进行检测和验证发现,除1份内蒙古资源(NMG)的1种病毒(GarVX)外,多重RT-PCR结果与单一RT-PCR检测结果一致。【结论】本研究建立的大蒜多重RT-PCR体系可靠性强,可用于大蒜病毒的检测与防控。

矮小黄鸡与三个正常型黄鸡正反交肉鸡性能比较分析

为确认dw基因在江村黄鸡改良与生产应用中的地位,确定节粮快大型肉鸡配套选育目标,以江丰矮小黄鸡H系为材料,与三个正常型黄鸡(A系、B系、C系)进行正反交试验,比较分析6个组合(HA、HB、HC、AH、BH、CH)因dw基因作用在肉鸡生产性能、胫长、屠宰性能等方面表现的差异。结果显示:正交、反交的后代公鸡63日龄体重及胫长差异不显著(P>0.05);100日龄后代母鸡体重、胫长均差异极显著(P<0.01),其中正交后代母鸡(含dw基因)体重比反交母鸡体重轻29.72%,胫长短25.76%,符合伴性遗传规律;屠宰率则各组合差异不显著(P>0.05);反交不含dw基因的母鸡(AH、BH、CH)腿肌率比正交后代含dw基因的母鸡(HA、HB、HC)高。试验结果表明,江丰矮小黄鸡品系的矮脚基因遗传模式属于伴性遗传;100日龄后代母鸡因dw基因的作用使体重轻29.72%、胫长短25.76%,与国内外多数学者的研究结果相符合。

Transient Expression of BYDV-MP in Nicotiana benthamiana

[Objective]The aim of this study was to identify transient expression of movement protein （MP） gene in Nicotinana benthaminana rapidly and further investigate the function of this exogenous gene. [Method]The movement protein gene of barley yellow dwarf virus （BYDV） was cloned into potato virus X （PVX） viral vector of pGR107,and PVX-recombinant vector was obtained. After electroporation of Agrobacterium tumefaciens,PVX was inoculated into the lower leaves of tobacco by Agrobacterium infiltration assay to observe the infection of virus on tobacco. [Result]After infection for 7 days,upper non-inoculated leaves of tobacco infected by the PVX-recombinant vector showed the virus infection symptoms,while the control group had no viral infection phenomenon. Daily follow-up observations for two groups revealed that tobacco infected by PVX-recombinant vector had severe symptoms of virus infection and curling leaves,or even led to necrosis both in infiltrated and systemic leaves in late period. However,tobacco infected by PVX vector had only slight symptoms of virus infection and could recover from infection. RT-PCR of the infected tobacco indicated that exogenous gene BYDV-MP had a normal transcription and expression in tobacco. [Conclusion]As a determinant factor for viral disease,BYDV-MP promotes the systemic infection rate of PVX and its symptom. In addition,it is feasible to express exogenous MP gene in Nicotiana benthaminan via PVX expression vector.

微量元素和维生素互作对47~70日龄优矮母鸡生长性能的影响

本试验旨在研究不同水平梯度微量元素和维生素互作对47-70日龄优矮母鸡生长性能的影响。选择体重相近、健康的47日龄优矮母鸡2520只,随机分为6组,每组7个重复,每个重复60只。试验采用3微量元素水平×2维生素水平因子设计,微量元素水平分为(1000、500mg·kg^(-1)和0 mg·kg^(-1))和维生素水平分为(200 mg·kg^(-1)和0 mg·kg^(-1))。试验阶段为47~70日龄,试验周期24 d。结果表明,主效应(微量元素):各组优矮母鸡的初始均重、末均重、日增重、日采食和料肉比均无显著差异,但其料肉比随着微量元素含量降低而降低,但差异不显著;主效应(维生素):各组优矮母鸡的初始均重、末均重和日采食量均无显著差异,200 mg·kg^(-1)维生素组优矮母鸡的料肉比显著低于0 mg·kg^(-1)维生素组。微量元素与维生素之间无交互作用。综上,从47~70日龄优矮母鸡日粮同时除去微量元素和维生素显著提高料肉比,其提高料肉比的效果为维生素大于微量元素,而单独剔除微量元素对其生长性能无影响。