牙鲆微卫星标记的开发及多态性分析

将牙鲆基因组DNA经限制性内切酶RsaⅠ和BstUI双酶切后采用FIASCO法构建酶切片段基因组文库。共挑取269个克隆,177个为阳性克隆,阳性克隆率为65.79%。经测序后获得191条微卫星序列,其中完美型占74.35%;非完美型占14.66%;混合型占10.99%。用引物设计软件Primer Premier 5.0设计引物153对,挑选其中的50对合成并在32个野生牙鲆个体中进行扩增,共31个位点具有多态性,统计结果后使用POPGENE软件进行分析,平均等位基因个数为3.939 4,平均有效等位基因数为3.052 2,平均观测杂合度为0.650 1,平均期望杂合度为0.586 6,各引物的Hardy-Weinberg平衡指数在0.012 587~0.984 917变动。这些筛选出的多态性微卫星标记可应用于进一步的牙鲆遗传多样性分析、家系分析及遗传图谱的构建等工作中。     

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 of Bgt. SSR markers of Bgt need to be further explored.     

黄鳝微卫星标记筛选及其在不同性别表型群体中的遗传多态性

采用FIASCO(Fast isolation by AFLP sequences containing repeats)法进行黄鳝微卫星标记筛选并将其应用于黄鳝性别差异群体的遗传变异分析.黄鳝基因组DNA经酶切、微卫星核心序列探针杂交、T载体连接并转化至DH5a中,构建黄鳝基因组微卫星富集文库.随机挑选75个阳性克隆测序,结果发现其中20个含有微卫星序列,利用软件成功设计了15对微卫星引物,经过黄鳝基因组DNA的PCR扩增及电泳检测,筛选8对多态性微卫星引物并对黄鳝3种不同性别表型群体的基因组DNA进行PCR扫描,结果显示:在8个微卫星座位中,共检测到51个等位基因,平均每个座位的等位基因数为6.375个,等位基因频率分布为0.001 2～0.603 3;黄鳝雌性表型群体、间性表型群体、雄性表型群体的平均遗传杂合度分别为0.637 1、0.696 9、0.734 5;平均多态信息含量分别为0.565 6、0.641 6、0.685 8,表明这些微卫星标记在黄鳝不同性别表型群体的遗传多态性发生了改变,它们可作为黄鳝遗传背景分析的分子标记.     

A FIASCO-Based Approach for Detection and Diagnosis of Puccinia graminis f. sp. tritici in China

Stem or black rust of wheat, caused by the fungus Puccinia graminis Pers. f. sp. tritici Eriks.＆E. Henn. （Pgt）, has historically caused severe losses to wheat （Triticum aestivum L.） production worldwide. In the Fujian and Guangdong provinces of China, six moderate-to-severe epidemics of wheat stem rust have occurred, which caused destructive losses of wheat between 1949 and 1966, although these were brought under control by integrated management. A rapid and reliable detection of the pathogen will contribute to the accurate forecast and seasonal control of this disease. The objective of this study was to develop a diagnostic molecular marker generated from simple sequence repeats （SSR） for the early rapid identiifcation of P. graminis. The genomic DNA of P. graminis, Puccinia striiformis, Puccinia triticina and seven other species was ampliifed by a pair of SSR primers generated by the FIASCO （fast isolation by AFLP sequences containing repeats） enrichment protocol. The primer set Pgtw （f）/Pgtw （r） generated a polymorphic pattern displaying a 330-bp DNA fragment speciifc for P. graminis whereas no DNA fragment was obtained from other non-target wheat fungal pathogens. The detection limit of the primer was 1 ng DNA in a 25-mL PCR reaction. The SSR markers of P. graminis can also be used to detect the presence of latent hyphae in Pgt-infected wheat leaves as early as 30 h post-inoculation. A rapid approach to distinguish P. graminis from similar pathogenic fungi would be anticipated in further study.     

Isolation of novel microsatellite loci in Orchesella villosa (Arthropoda, Collembola)

An experimental procedure using biotin-labelled probes and streptavidin-bound magnetic beads (FIASCO) was used to produce a microsatellite-enriched library for the collembolan Orchesella villosa. PCR primers were successfully constructed for seven loci containing, respectively, five pure, one interrupted, and one compound dinucleotide microsatellite repeats. As a preliminary test of their variability, we investigated 15 individuals from 5 locations inside a dismissed mining area in southern Tuscany. All microsatellite loci showed high levels of polymorphism. The mean number of different alleles at each locus across populations was 10.1 and observed heterozygosity per locus was 0.13–0.86. Only 2 out of the 7 loci appeared to be in Hardy–Weinberg equilibrium. The potential application of these loci to test the effects of environmental contamination on the genetic structure of exposed populations is discussed.     

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.     

小麦秆锈菌特异性SSR分子标记的开发

 【目的】研发简单、快速、准确的分子检测技术用于小麦秆锈菌的准确诊断和秆锈病的早期预警。【方法】采用FIASCO法构建秆锈菌基因组微卫星富集文库,分离微卫星DNA序列,设计合成小麦秆锈菌的特异性引物。【结果】根据小麦秆锈菌基因组微卫星富集文库,设计1对小麦秆锈菌特异性微卫星引物Pgtfssr1(f/r),可在来自中国不同麦区的20份小麦秆锈菌分离物基因组DNA中扩增出395 bp的特异性片段,而在小麦条锈菌、小麦叶锈菌和其它麦类病原真菌中未扩增出该特异性片段。病菌侵入寄主30 h后便可检测到特异性DNA片段的存在,灵敏度达到1 ng•μL-1模板DNA浓度水平。【结论】成功研发出小麦秆锈菌的特异性SSR分子标记,为小麦秆锈病早期诊断在生产上的应用奠定了基础。     

磁珠富集法制备莲藕基因组的微卫星分子标记

采用磁珠富集法FIASCO，设计生物素标记的寡核苷酸Biotin-（AG）13作探针，分离莲藕（Nelumbo nucifera Gaertn．）的微卫星分子标记，从所获得的阳性克隆中选取42个测序，结果36个克隆（85．0％）含有微卫星序列，其中23个（63．9％）重复数在10以上，19个（52．8％）为完美型。设计并且合成24对微卫星引物，经筛选其中16对为有效引物（66．7％），能扩增出多态性条带。     

Isolation of microsatellite loci and reliable genotyping using noninvasive samples of a critically endangered primate,Trachypithecus leucocephalus

Genetic information can be critical in identifying conservation priorities and developing conservation strategies. There is an urgent need for noninvasive genetic tools to study the wild populations of Asian colobine monkeys. The majority of these species are threatened with habitat destruction, population reduction and even extinction, but generally lack information on their genetic diversity and population structure. Genetic sampling and tissue collection have been scarce in these species owing to strict regulations on manipulation of endangered species, and the difficulties and risks associated with capturing these arboreal and fast‐moving monkeys in the challenging environments that they inhabit. These difficulties have hindered the development of molecular genetic markers, which are usually derived from tissues or blood. In this study, we present a method for de novo microsatellite isolation and genotyping using DNA from noninvasive origins of a critically endangered Asian colobine, the white‐headed langur (Trachypithecus leucocephalus). Genomic DNA isolated from hair was shown to be sufficient for microsatellite enrichment and isolation, with similar isolation efficiencies as from tissue DNA. We identified and characterized 20 polymorphic microsatellite loci, and evaluated their amplification success and genotyping reliability with 86 field‐collected fecal samples. These results show that this panel of loci can produce reliable genotypes from fecal samples, and represent a useful tool for noninvasive investigation of genetic structure, individual identification and kinship assessment in this highly endangered species. Our approach can be applied to conservation genetic studies of other wild species that lack sequence information and tissue samples.