Heterodera filipjevi许昌群体SCAR标记的建立

菲利普孢囊线虫Heterodera filipjevi是禾谷类作物上重要的病原线虫之一,严重影响禾谷类作物的产量和品质。本课题组前期研究发现菲利普孢囊线虫许昌群体是一个新的致病型,其在小麦上的致病力强于其他多个群体,危害更为严重。本研究旨在建立菲利普孢囊线虫许昌群体的快速、准确的分子检测体系,为Heterodera filipjevi许昌群体的监测和防控及抗病品种的选育利用奠定基础。本研究采用随机扩增多态性DNA(RAPD)和序列特征扩增区域(SCAR)的方法,对黄淮麦区5个重要小麦孢囊线虫致病型共9个线虫群体进行RAPD分析和SCAR标记转化,并通过增加除黄淮麦区外的线虫群体验证所获得的致病型相关分子标记的特异性和有效性。本研究共筛选了331条RAPD引物,筛选出2个菲利普孢囊线虫许昌群体相关的RAPD标记,引物S86可以扩增出1条约550 bp的多态性片段,引物S178可以扩增出1条约1 200 bp的多态性片段,并将这2个RAPD标记成功转化为SCAR标记。SCAR标记的特异性检测结果表明这两个SCAR标记只在许昌群体上有特异性扩增,可以用于许昌群体的分子检测。     

小麦条锈菌新菌系V26的SCAR检测标记

 建立小麦条锈菌生理小种的快速分子检测技术体系对小麦条锈菌的监测和防治策略的制定具有重要价值。条锈菌V26是近年来出现的,对我国目前小麦抗病育种中普遍应用的抗条锈病基因Yr26具有毒性的新菌系。该菌系的出现,对我国当前小麦生产、抗病育种都造成了严重威胁。本研究选用189条随机引物对CYR29、CYR31、CYR32、CYR33、T4、Su11-4和V26等7个条锈菌生理小种(菌系)进行了扩增,筛选V26的特异性RAPD片段,并对其进行克隆和测序。根据测序结果,设计并合成SCAR特异性引物, 将V26的RAPD标记转化为稳定的SCAR标记。使得对该菌系的快速检测成为可能,同时也将会为条锈菌新小种的监测提供更为准确的科学依据。     

小麦叶锈菌生理小种MFR的分子鉴定研究

 用AFLP方法对来自中国和墨西哥的23个小麦叶锈菌生理小种进行分析,共筛选了64对引物,获得一对引物(M₀₅/E₀₃)可在MFR小种中扩增出一条特异性DNA片段,进行回收、克隆、测序,结果表明该片段具有325个碱基。根据特异性片段序列设计出SCAR标记引物,对60个叶锈菌生理小种分离物进行回检结果表明,研制的SCAR标记能够准确区分MFR生理小种。本实验结果为小麦锈菌生理小种分子检测体系的建立奠定了基础     

小麦条锈菌生理小种条中32号及水源14致病类型在甘肃的流行与发展趋势

小麦条锈菌新毒性小种的产生与发展是造成条锈病大流行及品种抗锈性丧失的主导因素。1999～2004年采用条锈菌生理小种变异监测、致病性及寄生适合度测定、毒谱分析、哺育品种调查等方法,对条中32号及水源14致病类型的流行预测进行了系统研究。结果表明,条中32号及水源14致病类型已经成为甘肃省第一、二位优势小种,两小种毒性谱宽、致病力强、寄生适合度高、哺育品种面积大,其流行趋势已超过条中31号,将继31号后成为危害甘肃省小麦生产的流行小种。小麦条锈菌毒性群体结构已进入以条中32号和水源14致病类型为代表的Hybrid46和水源11致病类群占优势的新时期,这也对全国小麦条锈菌群体组成产生重要影响。     

马铃薯晚疫病菌抗甲霜灵的SCAR标记

建立马铃薯晚疫病菌抗甲霜灵SCAR(sequenced characterized amplified region,序列特征扩增区域)标记,以马铃薯晚疫病菌对甲霜灵高抗菌株HD01-3和对甲霜灵高感菌株DK98-1为亲本,通过无性单游动孢子分离和有性杂交获得菌株HD01-3无性后代群体、菌株DK98-1无性后代群体以及F1代分离群体,以此为试验材料,利用BSA法(bulked segregant analysis,分离群体分组分析法)构建抗感基因池对后代菌系的甲霜灵抗性进行RAPD(random amplified polymorphic DNA,随机扩增多态性DNA)分析。从178条RAPD随机引物中找到一条特异性引物S2054,其可以扩增出一个与晚疫病菌对甲霜灵抗性相关的遗传标记,将该特异条带回收、克隆、测序,发现此标记大小为457bp,根据测序结果设计特异PCR引物,用于扩增抗感基因池,成功地将特异RAPD标记转化为SCAR标记。初步建立了马铃薯晚疫病菌抗甲霜灵SCAR标记,辅助监测晚疫病菌对甲霜灵的抗性。     

2006-2007年甘肃省小麦条锈菌生理小种监测结果

报道了2006-2007年对甘肃省684份小麦条锈菌标样的监测结果。监测出31个生理小种及致病类型。小种消长变化总趋势与以往基本相同,仍以Hybrid46及水源11致病类群为主,所不同的是流行小种的出现频率不断上升。水源11 14类型仍为第1位流行小种,出现频率为26.2%~33.5%。条中32号为第2位流行小种,出现频率为16.1%~23.6%。次要小种水源11 4、水源11 7、水源11 5出现频率年度间略有不同。而条中31号已降为稀有小种。Yr9、Yr3b+4b、Yrsu为甘肃的主要致病基因。抗锈育种应以条中32号、水源11 14、水源11 4、水源11 7为主,兼顾Hybrid46和水源11致病类群中其他致病类型。     

小麦品种与条锈菌小种间的相互作用机制

为了在小麦多品种、条锈菌多小种体系中研究小种间的相互关系,1995年9月至1997年5月在田间设置方块圃,以全感、水平抗性和垂直抗性三种不同抗病类型的6个品种及条中29、水源11致病类型I与混合小种(条中29:水源11致病类型I=1:1)三类小种组合为试材,对各品种-小种组合的流行速率进行方差分析。结果表明:在多数品种上,条中29与水源11生理小种间存在互作关系,表现为协同作用,且混小种的流行速率明显高于单小种的流行速率。     

用于土壤中生防芽孢杆菌B006和BH1检测的SCAR标记及其特异性

生防芽孢杆菌Bacillus spp.菌株B006和BH1对引发多种根腐病的真菌具有拮抗作用,研发特异性标记是研究该菌株在土壤中生态行为的关键.本研究采用16个RAPD随机引物对B006和BH1以及包括芽孢杆菌、鞘氨醇杆菌Sphingosinc spp.和假单胞菌Pseudomonas spp.在内的25株参试菌株的基囚组DNA多态性进行了分析,其中利用单引物B19和C01可分别扩增到仅出现于B006和BH1基因图谱中的特异性条带(大小分别为730bp和658bp).为提高扩增的特异性和稳定性,根据B006和BH1的RAPD标记中核苷酸序列分别设计了6对和12对引物并用于菌株特异性条带的筛选,从中挑选出2对特异性最好的引物转化为SCAR引物,命名为SCAR-B006和SCAR-BH1,扩增产物大小分别为523bp和658bp.在自然土中进一步验证SCAR标记的特异性,结果表明仅在加有菌株B006或BH1的土壤样品中可扩增到相应的SCAR条带,说明了这种以PCR为基础的方法可用于土壤中菌株B006和BH1的快速鉴别.     

2002~2003年甘肃省小麦条锈菌生理小种监测结果

2002～2003年对甘肃省421份小麦条锈菌标样进行了监测,结果表明有28个生理小种及致病类型小种消长变化总趋势与以往基本相同,仍以Hybrid46及水源11致病类群为主;不同的是流行小种类型发生变化。水源14类型已成为第1位流行小种,出现频率为23.1%～27.9%。条中32号为第2位流行小种,出现频率为12.4%～17.4%。水4、水7、水5出现频率也逐年上升,而条中31号已降为次要小种;Yr9、Yr3b+4b、Yrsu为甘肃的主要致病基因。抗锈育种应以条中32号、水14、水4为主,兼顾Hybrid46和水源11致病类群中其它致病类型。     

基于KASP技术的小麦条锈菌SNP分子标记开发与评价

为开发用于小麦条锈菌Puccinia striiformis f. sp. tritici群体遗传研究的竞争性等位基因特异性PCR-单核苷酸多态性(kompetitive allele specific PCR-single nucleotide polymorphism,KASPSNP)标记,以中国小麦条锈菌流行小种CYR32的基因组为参考,与美国小麦条锈菌流行小种PST78和印度小麦条锈菌流行小种38S102的基因组进行比对,根据比对到的SNP位点设计KASP-SNP引物,用64个中国小麦条锈菌标样对其进行筛选,同时用13对多态性引物组成的简单重复序列(sim‐ple sequence repeat,SSR)分子标记分析这64个标样,并利用Powermarker 3.25和Structure 2.3软件通过多态性指数和群体遗传结构分析来评价KASP-SNP和SSR两种分子标记。结果显示,共比对到29 929个SNP位点,设计出462对KASP-SNP引物,经64个中国小麦条锈菌标样筛选到43对多态性较好的引物,所开发的这43对KASP-SNP引物多态性信息含量指数平均为0.346,基因多样性指数平均为0.420,而SSR引物的2种指数分别为0.237和0.265,前者较后者分别高出46.0%和58.5%。2种标记结果的群体遗传结构分析可得到类似结果,最佳聚类数K值都为4,云南菌系是遗传结构相对最简单的菌系,湖北菌系是遗传结构相对最复杂的菌系,但个别菌株的遗传划分存在较大差异。表明本研究开发的KASP-SNP分子标记多态性较SSR分子标记更加丰富,具有较好的应用前景。     

感染“中四”小麦条锈菌T4菌系SCAR检测标记的开发

正条锈病是由小麦条锈菌(Puccinia striiformis f.sp.tritici)引起的世界范围内小麦上最重要的病害之一。利用抗病品种是防治该病最经济、有效的措施。但是由于小麦条锈菌的高度变异性,品种抗病性很容易被条锈菌新小种所克服。因此,持续监测条锈菌生理小种的动态变化,及时发现新小种,对     

RAPD技术和聚类分析在小麦条锈病菌生理小种研究中的应用

 试用45个随机引物对我国小麦条锈病菌的6个生理小种(CYR17,SU11,CYR23,CYR28,CYR29和CYR30)进行了RAPD分析,其中10个适宜引物具有鉴别作用,共扩增出89条RAPD图带,多态性为73%。自CYR23、CYR28和CYR29三个生理小种中扩增出了特征性RAPD图带。根据RAPD分析结果,将这一结果与6个小种对不同小麦品种苗期和成株期致病性的聚类分析作了比较。可将供试小种分为两组,第一组包括CYR17、SU11和CYR23,其中前两者的相似性最大;CYR28和CYR29也较相近,属于第二组;新小种CYR30与其它小种的相关性尚待进一步研究。     

Use of the DNA sequence of the intergenic spacer region between the 16S and 23S rRNA genes for the identification of Clavibacter michiganensis subsp. insidiosus at the molecular level

A study has been performed to identify Clavibacter michiganensis subsp. insidiosus at the molecular level, using the polymerase chain reaction (PCR) technique with oligonucleotide primers based on specific sequence recognition of the intergenic spacer region between the 16S and 23S rRNA genes. The pair of primers was designed on the basis of available DNA sequence data for that region in C. m. insidiosus and other bacteria. Using this pair of primers, a large amount of an amplified DNA fragment of 218 bp in length was obtained from C. m. insidiosus. The specificity of this amplification was proved by PCR analysis, using the above-mentioned pair of primers and templates from different bacteria, some related to C. m. insidiosus. The PCR products were analysed using agarose gel electrophoresis.     

马铃薯青枯病的PCR检测

以我国11个省(市、区)和6种不同寄主植物的43个青枯菌Ralstonia solanacearum代表性菌株和4个国外青枯菌菌株为试材,采用15条随机引物进行了RAPD分析,筛选到了1条青枯菌所特有的DNA片段,根据其碱基序列,设计了特异PCR引物,对不同青枯菌DNA进行扩增,并以马铃薯的其它病原细菌为对照,只有青枯菌可获得773bp的DNA扩增产物.经过对PCR反应模板制备程序的简化和优化,利用本研究设计的特异引物,直接对马铃薯病块茎的DNA粗提液进行扩增,获得了773bp片段.此技术可望用于马铃薯种薯青枯病菌的检测,大大缩短检测时间,提高检测效率.     

源于华山新麦草抗条锈病基因 YrH122遗传分析和SSR标记

 H122是1个通过杂交和回交选育的普通小麦-华山新麦草易位系。为明确其抗条锈病基因及遗传特点,建立抗病基因SSR标记,利用我国小麦条锈菌流行小种CYR29、CYR31、CYR32、CYR33和致病类型Su11-4、Su11-11对H122进行苗期抗性鉴定,根据鉴定结果选用CYR32、CYR33和Su11-4对其与铭贤169杂交F1、F2及BC1代进行了遗传分析,同时应用258对SSR引物对将H122/铭贤169 F2代接种Su11-4的185个单株构建的作图群体进行了PCR扩增和电泳分析。结果表明,H122对供试小种均表现免疫或近免疫,对CYR32的抗病性由1对显性基因控制,对CYR33的抗病性由1对隐性基因控制,对Su11-4的抗病性亦由1对显性基因控制,将其暂命名为YrH122。筛选到3个与YrH122连锁的SSR标记Xbarc229、Xwmc339和Xwmc93,遗传距离分别为7.7、4.3和11.0 cM,并将该基因定位于小麦染色体1DL上。SSR标记回检显示,YrH122来源于华山新麦草。通过基因来源、分子检测及染色体位点比较,YrH122可能是1个不同于目前已知抗条锈病基因的新基因。     

Development of a PCR-based diagnostic test for Spongospora subterranea f.sp. nasturtii, the causal agent of crook root of watercress (Rorippa nasturtium-aquaticum)

The polymerase chain reaction (PCR) technique was utilized to obtain internal transcribed spacer ribosomal DNA (ITS rDNA) and small-subunit (18S) rDNA sequences from UK isolates of Spongospora subterranea f.sp. nasturtii , a plasmodiophorid pathogen of watercress ( Rorippa nasturtium-aquaticum ). ITS sequence data obtained from S. subterranea isolated from a range of UK sites were found to be identical. PCR primers were designed using these sequences and were shown to be capable of specific amplification of S. subterranea f.sp. nasturtii DNA from plant tissue and from water samples containing zoospores of the pathogen. As little as 5 ng total genomic DNA from infected plant material, or 1000 zoospores, was required for consistently successful amplification of DNA. A filtration-based method for obtaining pathogen DNA for PCR from watercress-bed water was developed.     

In planta-polymerase-chain-reaction detection of the wilt-inducing pathotype ofFusarium oxysporumf.sp.cicerisin chickpea (Cicer arietinumL.)

A 1.6 kb fragment of random amplified polymorphic DNA (RAPD-PCR, polymerase chain reaction), which was specific for race 5, a wilt-inducing isolate ofFusarium oxysporumf.sp.ciceris(Foc), was cloned and sequenced. This fragment was not detected in RAPD-PCR reactions with DNA from yellowing-inducing pathotypes ofFoc, or from other fungi tested. Specific PCR primers were designed from the sequence data and used to detect the presence of the fungus in genomic DNA isolated from symptomless chickpea plants, 16 days after inoculation. A single, 1.5 kb PCR product was only observed in PCR reactions with DNA from plants infected with a wilt-inducing isolate. No products were observed in reactions with DNA from plants infected with yellowing-inducing pathotypes, or from DNA isolated from uninfected chickpea cultivar controls. Southern hybridization demonstrated homology between the second PCR product and the original specific wilt-associated RAPD fragment. PCR products were detected with DNA extracted from roots and stem tissue, but no fungal DNA was detected in leaf tissue of the same infected plants. In a blind trial, the specific primers correctly identified the fungal pathotype in four different, wilt-infected chickpea cultivars.     

Morphological and molecular characterization of Colletotrichum spp. from citrus orchards affected by postbloom fruit drop in Brazil

Brazilian isolates of Colletotrichum spp. from citrus orchards affected by postbloom fruit drop were examined for colony colour, mycelial growth, benomyl-resistance, pathogenicity, and genetic variability by random amplified polymorphic DNA (RAPD) analysis. All isolates were obtained from flowers and persistent calyxes from different citrus hosts from Sao Paulo, Brazil. DNA polymorphisms detected after amplification with random 10-mer primers were used to classify the isolates into two groups. Group I isolates grew rapidly on potato-dextrose agar (PDA) and were sensitive to benomyl, and group II isolates grew slowly on PDA and were benomyl-resistant. Colletotrichum acutatum was analyzed by RAPD and had high genetic similarity with group II isolates of Colletotrichum from citrus. Probably, the group I is C. gloeosporioides and group II is C. acutatum.     

Development and evaluation of a one‐hour DNA extraction and loop‐mediated isothermal amplification assay for rapid detection of phytoplasmas

A rapid DNA extraction and loop‐mediated isothermal amplification (LAMP) procedure was developed and evaluated for the detection of two specific groups of phytoplasmas from infected plant material. Primers based upon the 16–23S intergenic spacer (IGS) region were evaluated in LAMP assays for amplification of group 16SrI (aster yellows group) and group 16SrXXII (Cape St Paul wilt group) phytoplasma strains. DNA could be extracted from leaf material (16SrI phytoplasmas) or coconut trunk borings (16SrXXII phytoplasmas) onto the membranes of lateral flow devices, and small sections of these membranes were then added directly into the LAMP reaction mixture and incubated for 45 min at 65°C. Positive reactions were detected through the hydroxyl napthol blue colorimetric assay within 1 h of the start of DNA extraction, and were confirmed by subsequent agarose gel electrophoresis of the LAMP products. The level of detection was comparable to that obtained by nested PCR using conventional 16S rDNA phytoplasma‐specific primers. Furthermore, the assays were specific for the phytoplasmas they were designed to detect – the 16SrI assay only detected 16SrI phytoplasmas and not those from any other phylogenetic groups, whilst the 16SrXXII assay only detected 16SrXXII phytoplasmas. The DNA extractions and LAMP assay are easy to perform, requiring minimal equipment, and may therefore form the basis of a rapid and reliable field‐detection system for phytoplasmas.