偃麦草E染色体组特异RAPD和SCAR标记的建立

用100条10碱基随机引物，以普通小麦中国春，中间偃麦草为材料进行RAPD分析，筛选到一个偃麦草染色体组特异引物OPF03，并从中间偃麦草中克隆了该引物的特异DNA片段OPF031291。将该片断与比萨偃麦草中的OPF031296（GenBank序号U43516）比较，同源性为88％。根据OPF031291的序列，设计了2对SCAR引物，利用OPF03和引物P3，P4对普通小麦，普通小麦－中间偃麦草的部分双二倍体，长穗偃麦草，中间偃麦草，小麦－二倍体长穗偃麦草代换系，附加系共6类材料进行了RAPD和SCAR分析，发现RAP标记OPR031291没有出现在含E^e染色体组的材料中，而标记SCAR982则出现于所有含E染色体组的材料中。说明，根据E^b染色体组特异RAPD标记转化的SCAR标记，可以同时检测小麦背景下的E^e染色体。     

对辣椒抗性基因Me3表现毒性的南方根结线虫 群体的SCAR分子标记

【目的】研究南方根结线虫（Meloidogyne incognita）Me3毒性变异群体的分子标记,用快速有效的分子方法来鉴定毒性变异的发生。【方法】以南方根结线虫的无毒群体以及抗性基因Me3毒性群体为材料,用根据南方根结线虫基因组设计的100对引物和19对来自文献的引物进行扩增,筛选出在3个种群中扩增的差异条带,设计SCAR引物,并建立多重PCR反应体系。【结果】得到了7对能在3个群体稳定扩增出差异条带的多态性引物,把其中的2个位点开发为SCAR标记,能够区分开3个线虫群体。多重PCR反应体系在无毒群体和Me3毒性群体中分别扩增出1条999 bp和1条629 bp的条带,在混合群体扩增出999和629 bp的2条带。【结论】成功开发了对Me3表现毒性的南方根结线虫变异群体的分子标记,可用多重PCR体系一次性鉴定Me3毒性变异的发生。     

SCAR分子标记鉴别烟粉虱和温室白粉虱

在简单重复序列PCR的基础上建立了烟粉虱和白粉虱两个物种DNA特异性片段的SCAR标记。验证表明,新建立的烟粉虱SCAR标记能特异性扩增不同地理种群的烟粉虱而不能扩增温室白粉虱;相反新建立的温室白粉虱SCAR标记则能特异性地扩增不同地理种群的温室白粉虱而不能扩增烟粉虱种群。两种SCAR标记能够快速、准确地鉴定烟粉虱和温室白粉虱。     

一个新的与稻瘟病菌无毒基因AVR-Pikm紧密连锁的SCAR标记

 无毒基因是病原物中决定寄主抗病性表达的功能基因,其功能的丧失导致毒性小种的产生。本研究利用随机引物扩增DNA多态性技术,从稻瘟病菌菌株S1522中新筛选到1个与无毒基因AVR-Pik^m紧密连锁的DNA标记OPE12₁₄₀₀。根据OPE12₁₄₀₀的核苷酸序列,设计了1对含有17个核苷酸的特异性SCAR引物,并利用该引物对无毒表型亲本S1522和毒性表型亲本S159及其有性杂交后代的108个菌株进行了PCR扩增。结果表明:所有无毒表型的菌株均能特异性扩增出1条与OPE12₁₄₀₀大小相近的DNA片段,而毒性表型的菌株除2个重组体外,均不能扩增出此片段。根据计算,这一SCAR标记与目标无毒基因AVR-Pik^m之间的遗传距离为1.89 cM,与本研究小组先前报道的另一个标记OPO12₁₀₀₀位于目标基因的同一侧,但与OPO12₁₀₀₀相比,距目标基因近了2.86 cM。本标记的获得将有助于确定AVR-Pik^m在染色体上的位置,有助于确定用于进一步筛选位于相反一侧的连锁标记的重叠群区域。     

月季种质鉴定和多样性分析

 利用RAPD 技术对28 个月季品种和2 个蔷薇品种进行了遗传多样性分析, 从145 个引物中筛选出12 个引物, 可以扩增出清晰、稳定和多态性高的产物。其中3 个引物从30 个材料中扩增出3 个品种特异的RAPD 分子标记, 并将其中的一个成功地转换成了SCAR 标记。利用筛选出的12 个引物扩增出的65 条多态性片段作了聚类分析, 对这些月季品种的亲缘关系和进化作了初步探讨。     

Variation in Pathogenicity Associated with the Genetic Diversity of Fusarium graminearum

We screened 188 isolates of Fusarium graminearum, which originated from northwest Europe, the USA and Nepal, for genetic diversity using a sequence-characterised amplified region polymorphism (SCAR). On the basis of this analysis, 42 of the 118 isolates were selected for random amplified polymorphic DNA (RAPD) analysis. Three groups were identified, two of which, A and B, contained the isolates from Nepal, and a third, group C, contained the isolates from Europe and the USA. In pathogenicity tests on wheat and maize seedlings, group C isolates were more pathogenic than the group A and B isolates. The isolates were assigned chemotypes based on their ability to produce the trichothecene mycotoxins nivalenol (NIV) and deoxynivalenol (DON). Isolates from group A were equally likely to produce NIV or DON while group B isolates produced predominantly NIV, and group C isolates produced predominantly DON. Within group A, isolates of the two chemotypes were equally pathogenic to wheat but isolates with the NIV chemotype were significantly more pathogenic to maize. The results confirm that distinct genetic groups exist within F. graminearum and demonstrate that these groups have different biological properties, especially with respect to their pathogenicity to two of the most economically important hosts of this pathogen.     

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。     

Development of molecular markers linked to the <Emphasis Type="Italic">Fom-1</Emphasis> locus for resistance to Fusarium race 2 in melon

Fusarium wilt, caused by Fusarium oxysporum f. sp. melonis (F.o.m), is a worldwide soil-borne disease of melon (Cucumis melo L.). The most effective control measure available is the use of resistant varieties. Resistance to races 0 and 2 of this fungal pathogen is conditioned by the dominant gene Fom-1. An F₂ population derived from the ‘Charentais-Fom1’ × ‘TRG-1551’ cross was used in combination with bulked segregant analysis utilizing the random amplified polymorphic DNA (RAPD) markers, in order to develop molecular markers linked to the locus Fom-1. Four hundred decamer primers were screened to identify three RAPD markers (B17₆₄₉, V01₅₇₈, and V06₁₀₉₂) linked to Fom-1 locus. Fragments amplified by primers B17₆₄₉ and V01₅₇₈ were linked in coupling phase to Fom1, at 3.5 and 4 cM respectively, whereas V06₁₀₉₂ marker was linked in repulsion to the same dominant resistant allele at 15.1 cM from the Fom-1 locus. These RAPDs were cloned and sequenced in order to design primers that would amplify only the target fragment. The derived sequence characterized amplified region (SCAR) markers SB17₆₄₅ and SV01₅₇₄ (645 and 574 bp, respectively) were present only in the resistant parent. The SV06₁₀₉₂ marker amplified a band of 1092 bp only in the susceptible parent. These markers are more universal than the CAPS markers developed by Brotman et al. (Theor Appl Genet 10:337–345, 2005). The analysis of 24 melon accessions, representing several melon types, with these markers revealed that different melon types behaved differently with the developed markers supporting the theory of multiple, independent origins of resistance to races 0 and 2 of F.o.m.     

3个奥利亚罗非鱼群体遗传多样性的RAPD分析和SCAR标记的转化

应用RAPD技术对3个奥利亚罗非鱼(Oreochromis aureus)群体(奥利亚罗非鱼83系、新引进的红色奥利亚罗非鱼与奥利亚罗非鱼02系)进行遗传多样性分析.根据扩增结果统计获得群体内和群体间的遗传距离:奥利亚罗非鱼83系、红色奥利亚罗非鱼、奥利亚罗非鱼02系群体内的遗传距离分别为0.021 0,0.090 3和0.014 9;群体间的遗传距离以奥利亚罗非鱼83系与红色奥利亚罗非鱼群体间的最大,为0.135 1,奥利亚罗非鱼83系与奥利亚罗非鱼02系的遗传距离最小,为0.061 0,红色奥利亚罗非鱼与奥利亚罗非鱼02系群体间的遗传距离为0.121 8.利用MEGA3.1软件进行聚类分析,奥利亚罗非鱼83系与奥利亚罗非鱼02系首先聚为一支,然后再与红色奥利亚罗非鱼聚为一支.3个奥利亚罗非鱼群体总的Shannon多样性指数为:0.234 0,总的Nei基因多样性指数为0.152 7,3个群体的遗传分化指数(Gst)为0.507 1.上述结果说明这3个群体的群体内遗传变异较小,但群体间存在一定程度的遗传变异和遗传分化.对RAPD分析中所获得的6条特异带进行SCAR转化.S97800和S4691400两个特异片段成功转化为SCAR标记.这些标记可作为鉴别3个奥利亚罗非鱼群体的遗传标记,并将为今后的选择育种提供分子标记.     

黄瓜抗炭疽病相关基因AFLP标记的SCAR转换

将黄瓜抗炭疽病相关基因的一个共显性AFLP标记成功地转换成了简单实用的SCAR标记。对AFLP标记片段进行序列测定,根据序列特点设计了特异的SCAR引物,引物长18～21 bp。PCR结果表明,引物对（1）可以扩增出131 bp和125 bp两条带,引物对（2）可以扩增出178 bp和172 bp两条带,分别为抗炭疽病的特征带和感炭疽病的特征带。将该两个标记命名为SCEM131/125和SCEM178/172,两对引物在F2单株和抗感病材料验证中符合率高达97.27%,可以用于黄瓜炭疽病的分子鉴定。