豚鼠基因组26个多态性微卫星标记的筛选

目的筛选豚鼠基因组的多态性微卫星标记,为豚鼠遗传质量控制及基因定位等工作奠定基础。方法采用磁珠富集法和豚鼠基因组数据库筛选法获取微卫星位点序列,通过分析和初步筛选,挑选部分候选位点,根据其序列设计引物,对5种不同来源的豚鼠基因组DNA标本进行PCR扩增,以期获得多态性分子标记。结果本实验采用磁珠富集法共获得微卫星序列304个,设计引物125对,最终获得多态性位点1个,暂未发现多态性的特异性位点17个;用数据库筛选法共获得微卫星序列292个,设计并合成相应引物178对,最终发现多态性位点25个,暂未发现多态性的特异性位点28个。结论本实验获得26个多态性微卫星标记,45个潜在的候选标记,为微卫星标记在豚鼠遗传质量监测及突变基因定位等工作的应用奠定了基础。     

磁珠富集法分离刀鲚微卫星标记

利用磁珠富集法分离微卫星序列,以开发长江刀鲚微卫星分子标记。将长江刀鲚基因组DNA经限制内切酶Mse I酶切,回收400-1 000 bp片段,安装接头,构建长江刀鲚全基因组PCR文库。用生物素标记的微卫星探针(CA)12与其杂交,磁珠富集含有微卫星序列的DNA片段。将洗脱所得片段进行PCR扩增,然后进行克隆。经过菌落PCR检验后挑选出118个阳性克隆进行测序,其中97条含有微卫星序列。用设计合成59对微卫星引物对30尾养殖长江刀鲚进行引物的多态性筛选,得到9对多态性引物。     

伊犁鲈微卫星位点的筛选及近缘物种通用性

为开发伊犁鲈(Perca schrenkii)分子标记用于鲈属鱼类种质资源保护,以伊犁鲈为材料,应用磁珠富集法进行了微卫星标记的筛选.从伊犁鲈尾鳍提取总DNA,进行酶切、接头连接、PCR扩增,再采用生物素标记(CA)15探针及生物素标记(TG)15探针对扩增产物进行杂交富集,经再次PCR扩增及T-A克隆,成功构建了伊犁鲈基因组微卫星富集文库.采用重复序列引物筛选获得阳性克隆,随机选取48个阳性克隆进行测序,测得序列46个,其中38个克隆含有微卫星序列,41个位点的微卫星重复数在8次以上.根据测得序列设计17对微卫星引物,均能在伊犁鲈群体中扩增获得目的条带.采用该17对引物对河鲈(P.fluviatilis)及黄金鲈(P.flavescens)群体样本进行扩增,10对引物具有通用性,其中6对在河鲈中具有高度多态性(PIC>0.5),5对在黄金鲈中具有高度多态性.     

背角无齿蚌基因组(GT)n微卫星DNA特征

利用磁珠富集法筛选背角无齿蚌(Anodonta woodiana)的微卫星分子标记,采用Sau3A1酶对完整DNA进行酶切,以生物素标记的(GT)15寡核苷酸探针从酶切片段中筛选微卫星序列.洗脱的杂交片段克隆到PGEM-T载体上构建富集微卫星基因组文库后,通过菌液PCR筛选检测出阳性克隆进行测序.结果表明:在筛选的18...     

巴氏蘑菇微卫星序列的分离及其对Co60辐射诱变菌株的鉴别

根据链霉素磁珠和生物素特异结合的特性,用生物素标记的二聚核苷酸重复序列探针从巴氏蘑菇的基因组中分离微卫星序列。将结合于链霉素磁珠上的标记探针同两端连接已知序列人工接头的巴氏蘑菇DNA酶切片段杂交。洗脱未杂交DNA片段后,用磁珠富集的片段建立微卫星文库。挑取522个菌落用对应重复序列为引物进行PCR筛选,得到48个阳性克隆,经测序有32个菌落含微卫星序列。微卫星富集效率为阳性克隆数的67%,总克隆数的6%。除去重复或无效的微卫星序列,在设计出的12对用于鉴别85个巴氏蘑菇的Co60辐射变异株微卫星引物中,有4对引物总共扩增出明显的变异菌株17个。证明有些微卫星位点可用于巴氏蘑菇辐射变异品种的指纹筛选与鉴别。     

藏鸡微卫星文库的构建与微卫星标记筛选

通过磁珠富集法构建了藏鸡基因组微卫星富集文库,分离微卫星序列并对其进行分析.将藏鸡基因组DNA经Sau3AI酶切后纯化回收,连接特定接头.用生物素标记的(CA)12探针与藏鸡基因组酶切回收片段杂交,捕获200～900 bp片段,随后将获得的片段连接到pMD 18-T载体上,转化至JM109中,成功构建藏鸡微卫星富集文库.从1200个转化子中获得了353个阳性克隆,随机挑选53个测序,根据测序结果成功设计了18对藏鸡微卫星引物,最终筛选出6个具有多态性的微卫星标记,其PIC值均大于0.5,同时可以用于研究藏鸡遗传多样性.实验结果表明磁珠富集法能够有效提高分离微卫星标记的效率.     

Dynal磁珠富集大熊猫微卫星标记

应用Dynal磁珠-生物素标记的微卫星探针与大熊猫基因组酶切片段杂交,捕获400—600bp含有微卫星序列的DNA片段,连接到pGEM-T载体中,构建富集微卫星序列的小片段插入文库。应用γ^32P标记的探针筛选文库,从2880个转化子中获得了260个阳性克隆。对54个序列进行了测序,并成功地设计了大熊猫微卫星引物37对。该方法能有效提高筛选微卫星标记的效率。     

青鱼微卫星标记的开发与特性分析

青鱼(Mylopharyngodon piceus)是中国最为重要的淡水养殖鱼类。开发青鱼的微卫星标记能为青鱼的遗传多样性分析提供更多工具。本研究使用磁珠富集法,利用生物素探针(CA)10和(GACA)6,富集得到青鱼基因组微卫星片段,进一步通过设计微卫星引物检验其在青鱼原种群体中的有效性和多态性水平。结果显示,所构建文库中849个克隆含有微卫星序列,通过利用PCR技术在吴江原种青鱼36个个体中进行多态性筛选,获得了25个多态性微卫星位点。其平均等位基因数(Na)和有效等位基因数(Ne)分别为7.08和3.526,平均观测杂合度(Ho)和期望杂合度(He)分别为0.602和0.619,平均多态信息含量(PIC)为0.568。其中,Mp23、Mp27和Mp35这3个位点极显著偏离哈迪-温伯格平衡(P 0.01)。本研究开发的微卫星标记能为青鱼种质资源的评价和保护等研究提供工具。     

曼氏无针乌贼GT微卫星位点的筛选

采用链霉亲和素包被的磁珠富集法筛选曼氏无针乌贼(Sepiella maindroni)微卫星位点。试验样品来自舟山六横岛,提取4个样品的DNA混合成DNA pool,用限制性内切酶Sau 3A I酶切。接上接头后构建基因组PCR文库,用生物素标记的(GT)15探针筛选。将筛选获得目的片段进行PCR扩增,连接pMD18-T载体,转入DH5α感受态大肠杆菌里,扩大培养后PCR筛选阳性克隆。总共选取278个克隆,对120个经过检测含有插入片段的克隆进行测序,发现102个克隆含有微卫星序列,阳性克隆比率为85%。除去重复测序和侧翼链不足的序列,可以设计引物的微卫星序列有64条。     

磁珠富集法筛选实验豚鼠微卫星分子标记

目的直接从实验豚鼠基因组DNA中筛选获得微卫星分子标记。方法应用磁珠和生物素标记的微卫星探针与豚鼠基因组酶切片段杂交,捕获200～1000 bp含有微卫星序列的DNA片段,连接到pMD-18V载体中,转化到感受态细胞E.coli DH5α中构建富集微卫星序列的小片段插入文库。然后用PCR法进行筛选。结果从约2000个转化子中获得240个阳性克隆。对其中98个进行了测序,并成功设计豚鼠微卫星引物17对。结论经过优化的磁珠富集法能够稳定、高效地获得豚鼠微卫星标记。本研究获得的微卫星位点将成为豚鼠遗传学研究的有力工具。     

Development and characterization of eight new microsatellite markers for the haremic sandperch,Parapercis cylindrica (family Pinguipedidae)

Eight di‐, tri‐ and tetranucleotide microsatellite markers were developed for the haremic sandperch Parapercis cylindrica using a linker‐ligated, magnetic bead enrichment protocol. Screening of at least 17 individuals showed these markers to be polymorphic with observed heterozygosity ranging from 0.381 to 1.000 (mean = 0.742) and the numbers of alleles ranging from three to 18. The average polymorphic information content for these eight loci was 0.723. These markers may be used for parentage studies aimed at exploring the complex mating strategies employed by this haremic coral reef fish and will be valuable for population genetic studies.     

Dinucleotide microsatellite loci isolated from flowering dogwood (Cornus florida L.)

We report eight variable dinucleotide microsatellite loci cloned from flowering dogwood (Cornus florida L.) using a biotin enrichment protocol. Degenerate oligonucleotide primer‐polymerase chain reaction (DOP‐PCR) was used to generate a population of DNA fragments, from which adenine‐cytosine dinucleotide (AC) and adenine‐guanine dinucleotide (AG) repeats were captured using biotinylated probes and streptavidin coated magnetic particles. The captured fragments were cloned into plasmids, and the plasmid library was screened for microsatellites using a simple PCR technique. Selected plasmids were sequenced, and PCR primers were designed and optimized using a thermal‐gradient thermocycler. The loci reported are highly variable with an average of 9.25 allele per locus and an average heterozygosity of 0.84.     

Development and use of simple sequence repeat SSR markers in Rubus species

The isolation of polymorphic codominant microsatellite markers in Rubus and in particular red raspberry will provide a tool to investigate gene flow between cultivated and wild raspberries. Microsatellite loci were isolated by screening a PstI size selected genomic library with AC₍₁₃₎ and AG₍₁₃₎. Positive clones were sequenced and primer pairs designed to the sequences flanking identified SSRs. One primer of each pair was fluorescently labelled to facilitate polymerase chain reaction (PCR) product identification on an automated DNA sequencer. We describe 10 polymorphic microsatellite loci developed and demonstrate their usefulness in different Rubus species.     

Isolation and characterization of 16 tetranucleotide microsatellite loci in the red panda (Ailurus fulgens)

A total of 15 polymorphic tetranucleotide microsatellite loci in the red panda, Ailurus fulgens, were characterized in this study. Based on evaluations of 33 red pandas, the number of observed alleles for each locus ranged from seven to 17 and the expected and observed heterozygosities were 0.412–0.897 and 0.121–0.909, respectively. The mean polymorphic information content was 0.721. These markers would greatly strengthen the utilization of microsatellite tools in genetic variation studies in red panda populations.     

Design and implementation of degenerate microsatellite primers for the mammalian clade

Microsatellites are popular genetic markers in molecular ecology, genetic mapping and forensics. Unfortunately, despite recent advances, the isolation of de novo polymorphic microsatellite loci often requires expensive and intensive groundwork. Primers developed for a focal species are commonly tested in a related, non-focal species of interest for the amplification of orthologous polymorphic loci; when successful, this approach significantly reduces cost and time of microsatellite development. However, transferability of polymorphic microsatellite loci decreases rapidly with increasing evolutionary distance, and this approach has shown its limits. Whole genome sequences represent an under-exploited resource to develop cross-species primers for microsatellites. Here we describe a three-step method that combines a novel in silico pipeline that we use to (1) identify conserved microsatellite loci from a multiple genome alignments, (2) design degenerate primer pairs, with (3) a simple PCR protocol used to implement these primers across species. Using this approach we developed a set of primers for the mammalian clade. We found 126,306 human microsatellites conserved in mammalian aligned sequences, and isolated 5,596 loci using criteria based on wide conservation. From a random subset of ~1000 dinucleotide repeats, we designed degenerate primer pairs for 19 loci, of which five produced polymorphic fragments in up to 18 mammalian species, including the distinctly related marsupials and monotremes, groups that diverged from other mammals 120-160 million years ago. Using our method, many more cross-clade microsatellite loci can be harvested from the currently available genomic data, and this ability is set to improve exponentially as further genomes are sequenced.     

Isolation and characterization of microsatellite loci in the great bustard,Otis tarda

With the aim to study population genetics of the endangered great bustard, Otis tarda, dinucleotide microsatellite loci were isolated using an adapted hybrid‐capture enrichment protocol. This work reports the characterization of a set of six polymorphic microsatellite markers within the great bustard (n = 52). Results from cross‐species amplifications in several other members of the family Otididae demonstrate that five primer pairs also successfully amplified homologous loci outside the species Otis tarda.     

Microsatellite loci from the house wren (Troglodytes aedon)

We cloned seven microsatellite loci from house wrens (Troglodytes aedon) using a biotin enrichment protocol. Starting with fragments generated using DOP–PCR, fragments containing microsatellite motifs AC and AAC were captured using biotinylated probes and streptavidin coated magnetic particles. Captured fragments were cloned into plasmids; prior to sequencing, the plasmids were screened for microsatellites using a simple PCR approach. Five of the loci showed variation in a sample of nine individuals.     

Isolation and characterization of microsatellite loci in Ononis repens,Leguminosae

Ten polymorphic microsatellites were isolated from Ononis repens, using a microsatellite enrichment protocol and selective hybridization with a mixed synthetic dinucleotide repeat probe. Primers for DNA amplifications using polymerase chain reaction (PCR) were designed and synthesized. The loci amplified in both O. repens and O. spinosa. All 10 loci were polymorphic in each of four populations studied. These microsatellite markers will be useful for population genetic analysis and biodiversity studies of O. repens and O. spinosa.     

Polymerase chain reaction primers for polymorphic microsatellite loci in the African armyworm,Spodoptera exempta (Lepidoptera: Noctuidae)

Eight pairs of polymerase chain reaction (PCR) primers that amplify polymorphic microsatellite loci were developed for the African armyworm, Spodoptera exempta (Walker) to be used in the study of its population dynamics in sub‐Sahara Africa where the species is a major pest of cereals and rangeland. A magnetic beads based enrichment protocol was used; it appears that previously reported scarcity of microsatellites in Lepidoptera species does not apply to the African armyworm. All the loci showed significant heterozygote deficit; possibly because the samples were laboratory reared from limited stock. Four primer pairs successfully amplified single fragments of beet and fall armyworm DNA of comparable size to the African armyworm alleles.