鲮微卫星DNA分子标记的筛选与遗传多样性分析

采用磁珠富集法对已建立的生长较快的西江段野生浅色鲮群体的极大、极小群体各31尾基因组DNA进行了微卫星序列的筛选,128个阳性克隆成功测序93个,其中80个为的微卫星序列,微卫星序列完美型占85%,非完美型占6.25%,混合型占8.75%。利用微卫星序列合成了23对微卫星引物,扩增结果表明,等位基因数为1～10个,扩增片段大小为92～283bp,其中14对引物扩增条带清晰,为中度或高度多态位点,极大、极小群体的平均有效等位基因数和平均多态信息含量分别为3.0784、3.2079和0.5675、0.5974,反映出2个群体遗传多样性均较丰富;高度多态性引物4的位点b,片段大小为119bp,在极大群体中出现频率为61.3%,在极小群体占22.6%,出现频率极大群体高于极小群体近3倍,初步可作为候选差异性分子标记。     

中国龙虾微卫星标记的筛选及遗传多样性分析

文章以M13通用引物和重复序列(CT)₁₅、(AT)₁₅引物, 利用PCR法对中国龙虾(Panulirus stimpsoni Hoehuis)部分基因组DNA文库进行筛选。共获得78个微卫星序列, 分别分布于55个阳性重组克隆中, 其中完美型(perfect)共50个, 占64%; 非完美型(imperfect)3个, 占3.8%; 混合完美型(compound perfect)6个, 占7.7%; 混合非完美型(compound imperfect)19个, 占24.5%。根据微卫星序列, 设计并筛选出15对微卫星多态性引物, 对中国龙虾的群体进行了遗传多样性分析。获得3~12个等位基因, 等位基因大小在78~425 bp之间, 基本符合引物设计的理论长度。期望杂合度范围为0.48~0.87, 平均值为0.71, 表明中国龙虾基因组微卫星具有较高的杂合度与遗传多样性。15个微卫星位点的PIC值从0.44到0.84, 平均值为0.60, 说明这些微卫星位点在中国龙虾基因组中包含丰富的遗传信息, 合适用于中国龙虾的各种分子标记及遗传学分析和应用。     

利用双重PCR技术研究长爪沙鼠的微卫星结构

目的为了建立快速检测长爪沙鼠群体遗传多样性的方法及获得Z：ZCLA长爪沙鼠封闭群现用微卫星位点的结构。方法利用17个微卫星位点（9个来自长爪沙鼠,8个来自大小鼠）进行了PCR反应体系及反应条件的优化,组合了6组双重PCR及两个复合式点样,用上述8个组合对普通级Z：ZCLA长爪沙鼠封闭群43、444、5三个世代核心群各100只种鼠进行遗传检测。结果三个世代的300只种鼠的检测结果表明,9个长爪沙鼠位点均为微卫星,其中7个位点为完全型的微卫星,1个为复合型,1个为不完全型,多态性主要表现在核心序列的重复;来自大小鼠的8个微卫星位点,有7个在Z：ZCLA长爪沙鼠核心群中得到有效扩增,只有3个位点在三个世代中均有出现,对测序结果分析后发现,其核心序列均为小卫星。结论来自长爪沙鼠的位点,无论结构还是遗传方式均符合微卫星遗传标记的特点,可用作检测长爪沙鼠的群体遗传多样性。     

微卫星DNA与生化标记分析对长爪沙鼠群体遗传分析的比较

目的比较生化标记和微卫星DNA标记方法对长爪沙鼠群体遗传分析的可靠性。方法应用27个生化位点和13个微卫星DNA位点,采用已建立的生化标记和微卫星DNA标记分析方法对国内2个长爪沙鼠群体进行遗传分析,计算并比较两种方法测得的各群体遗传参数。结果生化基因位点中有13个位点在整体中呈现遗传多态性,多态率为48.1%;微卫星位点中有11个位点在整体中表现出多态性,多态率均为84.6%。两种方法测得的平均有效等位基因数趋于一致,微卫星DNA的多态位点百分率和平均杂合度均明显高于生化标记方法。但生化标记和微卫星DNA检测对两个长爪沙鼠群体的遗传多样性差异反映一致,所反映的群体平衡状况也基本一致。结论生化标记分析和微卫星DNA方法均可较好地反映长爪沙鼠群体遗传结构。     

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。     

云南松基因组微卫星富集文库的构建

采用磁珠富集法构建云南松微卫星富集文库。云南松基因组DNA经RsaⅠ酶切,与特定接头连接,再用接头特异引物进行PCR扩增。连接扩增产物与用生物素标记的(AG)12、(AT)12、(CG)12、(GT)12、(ACG)12、(ACT)12和(CCA)8探针杂交,通过链霉亲和素偶联的磁珠捕捉含接头和微卫星序列的片段并扩增,将获得的片段连接到pMD-19T载体上,转化至大肠杆菌JM109感受态细胞中,成功构建了云南松微卫星富集文库。通过PCR检测从文库中筛选阳性克隆,在383富集阳性菌落中获得阳性克隆257个,经测序分析,在获得的159条序列中,有143条含有SSR,其中完美型占65.73%,非完美型占23.78%,混合型占10.49%。结果表明,磁珠富集法构建云南松基因组微卫星文库高效、可行的,文库的构建为微卫星位点的分离、遗传多样性的分析等奠定基础。     

长爪沙鼠小鼠肝炎病毒（MHV）RT-PCR检测方法的建立及初步应用

目的建立长爪沙鼠小鼠肝炎病毒（MHV）RT-PCR检测方法,应用于长爪沙鼠、小鼠等实验动物MHV的检测。方法根据已发表的小鼠肝炎病毒（MHV）S基因序列,设计合成引物。提取MHV细胞毒RNA,以其为模板,进行PCR扩增。优化反应条件,进行特异性、敏感性、稳定性、重复性试验。并对65只长爪沙鼠及12只小鼠进行检测。结果建立的MHVRT-PCR检测方法特异、敏感、稳定。以MHVRNA逆转录产物为模板,所能检测RNA最小模板浓度为3．1pg／μL,可检测病毒最小滴度为10^-3／mL。65只沙鼠经RT-PCR检测,均为阴性,12只小鼠经RT．PCR检测,有3只MHV阳性,测序结果与Genbank中MHV核酸序列同源性均为97％。结论建立的长爪沙鼠小鼠肝炎病毒（MHV）RT-PCR检测方法可用于长爪沙鼠、小鼠等实验动物MHV的检测。     

火红熊蜂微卫星标记的筛选及种特异性分析

火红熊蜂Bombus pyrosoma是中国特有的熊蜂资源, 在华北地区分布十分丰富, 是众多野生植物和农作物的重要传粉者。为了探究火红熊蜂的遗传结构及其进化关系, 首次开展了该种熊蜂卫星标记的筛选及其种特异性研究。本研究采用生物素-磁珠吸附分离法从火红熊蜂基因组中筛选微卫星标记, 并选取黑熊蜂亚属Melanobombus其他7种熊蜂进行了这些标记的种特异性验证。结果表明： 不同探针与磁珠杂交产生的微卫星标记阳性克隆率存在很大差异, TC探针的微卫星阳性克隆率最高为82%; TG探针次之, 为28%; AT和GC探针分离未获得微卫星标记克隆。根据获得的阳性克隆的序列设计引物进行筛选验证, 共获得31对熊蜂微卫星序列特异性引物。微卫星标记分析发现, 完美型(perfect)18个, 占58.1%; 非完美型(imperfect)10个, 占32.3%; 混合完美型(compound perfect)1个, 占3.2%; 混合非完美型(compound imperfect)2个, 占6.4%。不同探针的微卫星核心序列重复数不同, TC探针和TG探针的微卫星核心序列最高重复数分别为28和15个, 最低重复数分别为7和11个。利用所获得的31对微卫星引物对黑熊蜂亚属其他7种熊蜂进行检测, 有26对引物可以扩增所测试的7种熊蜂, 其他5对引物只能扩增部分熊蜂种类, 其中BPM5是火红熊蜂种特异性引物。本研究从火红熊蜂基因组中筛选的微卫星标记, 不仅可用于火红熊蜂的遗传结构、 分子进化和资源保护等方面的研究, 而且可进一步用于黑熊蜂亚属其他种群的遗传特性分析。     

卤虫(AC)_n和(AG)_n微卫星文库构建

卤虫Artemia作为重要的基础实验材料和经济动物资源,其微卫星位点具有重要的研究和应用价值。本研究使用磁珠富集法,成功构建了卤虫的(AC)_n和(AG)_n微卫星富集文库。通过对文库进行PCR方法鉴定和测序确认,所构建的(AC)_n和(AG)_n文库阳性克隆率分别为55.8%和34.5%。依据得到的微卫星序列,设计并合成了63对微卫星引物,随机以2个地理单元(青海、西藏)共18个卤虫基因组DNA为扩增模板,筛选出6对具有多态性的卤虫微卫星引物。本研究筛选出的微卫星多态位点可用于卤虫种群的遗传多样性评估、遗传图谱的构建和数量性状定位等后续工作。     

勒氏笛鲷微卫星位点的筛选及特征分析

采用PCR法快速筛选勒氏笛鲷(Lutjanus russelli)基因组文库, 以获得(CA)_n微卫星位点。勒氏笛鲷基因组DNA经限制性内切酶HaeⅢ+ DraⅠ双酶切后, 连接T-载体克隆, 构建基因组文库。以通用引物M13+/-与重复序列引物(CA)15对基因组文库进行筛选, 二次筛选后得到121个可能含有微卫星位点的阳性克隆。进行序列测定, 共获得53个CA(n≥7)重复序列, 重复次数主要分布于7~15(80.77%)。在所得微卫星序列中, 重复单元除CA外, 还观察到单碱基、三碱基、四碱基、五碱基重复单元。根据侧翼序列设计48对引物, 通过优化PCR反应条件, 可获得清晰可重复的目的条带。研究旨在为勒氏笛鲷遗传多样性研究及遗传图谱的构建等奠定基础, 为勒氏笛鲷资源的合理开发利用提供参考。     

长爪沙鼠的遗传多样性分析

利用17个微卫星DNA标记对Z:ZCLA长爪沙鼠封闭群、野生群和近交系进行遗传多样性分析, 评估群体内的遗传变异和群体间的遗传分化。结果表明：在Z:ZCLA封闭群和野生群中共有9个微卫星DNA标记获得稳定的结果, 分别为AF200940、AF200941、AF200942、AF200945、AF200946、AF200947、D11Mit128、PKC和 SCN, 共检测到41个等位基因, 每个基因的等位基因数从1～7不等, 片段大小在120～283 bp之间, 所有位点的平均期望杂合度(He)和多态信息含量(PIC)值分别为0.5032和0.4656, Z:ZCLA封闭群和野生群9个微卫星位点平均有效等位基因数分别为2.78和2.89, 平均基因杂合度分别为0.3704和 0.3893, 平均多态信息含量分别为0.3256和0.3344, 两个群体都表现为中度多态, Z:ZCLA封闭群较野生群稍低; 在3个近交系中共有8个位点获得稳定的扩增结果, 分别为AF200941、AF200942、AF200945、AF200946、AF200947、D11Mit128、PKC和 SCN, 共检测到11个等位基因, 片段大小在140~241 bp之间, 其中5个位点在群体内表现为单态纯合, 3个位点在群体内表现为单态杂合, 所有位点在群体内和群体间均呈单态性, 表明这3个长爪沙鼠品系基本符合近交系的要求, 微卫星标记技术适用于近交系长爪沙鼠的遗传检测。     

种间转移扩增筛选仙湖苏铁微卫星位点及其遗传多样性研究

运用刺叶苏铁、葫芦苏铁、海南苏铁的SSR引物,在仙湖苏铁中进行种间转移扩增,筛选得到7对引物能扩增出清晰的特异带,其中3对引物的扩增产物具有多态性.为验证微卫星的真实性,扩增产物切胶回收后克隆测序.结果表明：重复单元的数目变化是扩增片段长度多态性的主要来源.运用筛选出的3对 SSR标记对4个仙湖苏铁野生种群进行遗传结构研究,等位基因数从2~5,杂合度从0．000~0．667,期望杂合度为0．000~0．610.种群两两遗传分化系数从0~0．382.总体上仙湖苏铁遗传多样性水平低,而种群间遗传分化显著.STRUCTURE分析结果表明,4个野生种群可被分配到3个假想的遗传簇.BOTTLENECK 分析结果表明种群近期没有遭遇瓶颈效应.     

An integrated SSR and RFLP linkage map of Sorghum bicolor (L.) Moench.

We report the development, testing, and use (for genetic mapping) of a large number of polymerase chain reaction (PCR) primer sets that amplify DNA simple sequence repeat (SSR) loci of Sorghum bicolor (L.) Moench. Most of the primer sets were developed from clones isolated from two sorghum bacterial artificial chromosome (BAC) libraries and three enriched sorghum genomic-DNA (gDNA) libraries. A few were developed from sorghum DNA sequences present in public databases. The libraries were probed with radiolabeled di- and trinucleotide oligomers, the BAC libraries with four and six oligomers, respectively, and the enriched gDNA libraries with four and three oligomers, respectively. Both types of libraries were markedly enriched for SSRs relative to a size-fractionated gDNA library studied earlier. However, only 2% of the sequenced clones obtained from the size-fractionated gDNA library lacked a SSR, whereas 13% and 17% of the sequenced clones obtained from the BAC and enriched gDNA libraries, respectively, lacked a SSR. Primer sets were produced for 313 SSR loci. Two-hundred sixty-six (85%) of the loci were amplified and 165 (53%) of the loci were found to be polymorphic in a population composed of 18 diverse sorghum lines. (AG/TC)n and (AC/TG)n repeats comprised 91% of the dinucleotide SSRs and 52% of all of the SSRs at the polymorphic loci, whereas four types of repeats comprised 66% of the trinucleotide SSRs at the loci. Primer sequences are reported for the 165 polymorphic loci and for eight monomorphic loci that have a high degree of homology to genes. Also reported are the genetic map locations of 113 novel SSR loci (including four SSR-containing gene loci) and a linkage map composed of 147 SSR loci and 323 RFLP (restriction fragment length polymorphism) loci. The number of SSR loci per linkage group ranges from 8 to 30. The SSR loci are distributed relatively evenly throughout approximately 75% of the 1406-cM linkage map, but segments of five linkage groups comprising about 25% of the map either lack or contain few SSR loci. Mapping of SSR loci isolated from BAC clones located to these segments is likely to be the most efficient method for placing SSR loci in the segments.     

A genetic map of 1,000 SSR and DArT markers in a wide barley cross

A high-density genetic map was developed from an F1-derived doubled haploid population generated from a cross between cultivated barley (Hordeum vulgare) and the subspecies H. vulgare ssp. spontaneum. The map comprises 1,000 loci, amplified using 536 SSR (558 loci) and 442 DArT markers. Of the SSRs, 149 markers (153 loci) were derived from barley ESTs, and 7 from wheat ESTs. A high level of polymorphism (∼70%) was observed, which facilitated the mapping of 197 SSRs for which genetic assignments had not been previously reported. Comparison with a published composite map showed a high level of co-linearity and telomeric coverage on all seven chromosomes. This map provides access to previously unmapped SSRs, improved genome coverage due to the integration of DArT and EST-SSRs and overcomes locus order issues of composite maps constructed from the alignment of several genetic maps. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

用19个生化基因位点研究普通级Z:ZCLA长爪沙鼠的遗传多样性

目的调查Z:ZCLA长爪沙鼠原种群普通级长爪沙鼠的遗传多样性。方法用本实验室自行建立的长爪沙鼠19个生化基因位点的乙酸纤维素膜电泳技术并结合基本群体遗传学指标研究了普通级Z:ZCLA长爪沙鼠100个家系的遗传多态性。结果Z:ZCLA长爪沙鼠在Es-1、Car-2、Hbb、Gpi-1、Cs-1、Ce-2I、dh-l、Mod-1呈单态,在Es-2、Es-3、Es-4、Es-6、Es-8、Es-9、Es-10、pd-1、gm-1、Trf、Akp-1个位点上呈现多态性,等位基因从2~4个不等,平均等位基因数3.0,平均杂合度0.512,平均多态信息量0.455。结论提示目前本群遗传多样性水平处于中度多态。     

AFLP to assess genetic variation in laboratory gerbils (Meriones unguiculatus)

The amplified fragment length polymorphism (AFLP) technique has been increasingly employed for characterizing inbred breeds of animals and detecting strain-specific polymorphisms. The majority of animals studies conducted in biomedical research are performed on rodent species, among which laboratory-reared Mongolian gerbils can be included. Despite the wide use of gerbils in scientific studies, their genetics has rarely been studied. Therefore we investigated the genetic differentiation of laboratory bred gerbils by means of AFLP markers. Six EcoRI/TaqI primer combinations were selected among 13 different combinations to assess the genetic polymorphisms in four stocks of animals: Charles River (CR), Harlan (Ha), Parma (Pr), and Crossbred (Cb). CR and Ha gerbils were purchased from commercial vendors, while Pr and Cb were derived from animals bred in our animal colony. A total of 228 fragments ranging between 70 and 650 bp were obtained. The mean percentage of polymorphic loci across primer combinations was 7.5%. Calculation of genetic distances through application of different algorithms (Nei's, BSI, and Jaccard's indexes) confirmed the poor genetic diversity between stocks. Nevertheless, a differentiation of the Pr and Cb stocks from the more homogeneous CR and Ha was revealed, in agreement with the different breeding derivation and management of the stocks.     

Simple sequence repeat (SSR) markers survey of the cassava (Manihot esculenta Crantz) genome: towards an SSR-based molecular genetic map of cassava

The development of PCR-based, easily automated molecular genetic markers, such as SSR markers, are required for realistic cost-effective marker-assisted selection schemes. This paper describes the development and characterization of 172 new SSR markers for the cassava genome. The placement of 36 of these markers on the existing RFLP framework map of cassava is also reported. Two similar enrichment methods were employed. The first method yielded 35 SSR loci, for which primers could be designed, out of 148 putative DNA clones. A total of 137 primer pairs could be designed from 544 putative clones sequenced for the second enrichment. Most of the SSRs (95%) were di-nucleotide repeats, and 21% were compound repeats. A major drawback of these methods of SSR discovery is the redundancy – 20% duplication; in addition, primers could not be designed for many SSR loci that were too close to the cloning site – 45% of the total. All 172 SSRs amplified the corresponding loci in the parents of the mapping progeny, with 66% of them revealing a unique allele in at least one of the parents, and 26% having unique alleles in both of the parents. Of the 36 SSRs that have been mapped, at least 1 was placed on 16 out of the 18 linkage groups of the framework map, indicating a broad coverage of the cassava genome. This preliminary mapping of the 36 markers has led to the joining of a few small groups and the creation of one new group. The abundance of allelic bridges as shown by these markers will lead to the development of a consensus map of the male- and female-derived linkage groups. In addition, the relatively higher number of these allelic bridges, 30% as against 10% for RFLPs in cassava, underscores SSR as the marker of choice for cassava. The 100% primer amplification obtained for this set of primers also confirms the appropriateness of SSR markers for use in cassava genome analysis and the transferability of the technology as a low-cost approach to increasing the efficiency of cassava breeding. Current efforts are geared towards the generation of more SSR markers to attain a goal of 200 SSR markers, or 1 SSR marker every 10 cM. Received: 15 November 1999 / Accepted: 14 April 2000     

Simple sequence repeat analyses of interspecific hybrids and MAALs of <Emphasis Type="Italic">Oryza </Emphasis><Emphasis Type="Italic">officinalis</Emphasis> and <Emphasis Type="Italic">Oryza sativa</Emphasis>

Wild rice is a valuable resource for the genetic improvement of cultivated rice (Oryza sativa L., AA genome). Molecular markers are important tools for monitoring gene introgression from wild rice into cultivated rice. In this study, Simple sequence repeat (SSR) markers were used to analyze interspecific hybrids of O. sativa-O. officinalis (CC genome), the backcrossing progenies and the parent plants. Results showed that most of the SSR primers (335 out of 396, 84.6%) developed in cultivated rice successfully amplified products from DNA samples of wild rice O. officinalis. The polymorphism ratio of SSR bands between O. sativa and O. officinalis was as high as 93.9%, indicating differences between the two species with respect to SSRs. When the SSR markers were applied in the interspecific hybrids, only a portion of SSR primers amplified O. officinalis-specific bands in the F(1) hybrid (52.5%), BC(1) (52.5%), and MAALs (37.0%); a number of the bands disappeared. Of the 124 SSR loci that detected officinalis-specific bands in MAAL plants, 96 (77.4%) showed synteny between the A and C-genomes, and 20 (16.1%) showed duplication in the C-genome. Sequencing analysis revealed that indels, substitution and duplication contribute to the diversity of SSR loci between the genomes of O. sativa and O. officinalis.     

Development of codominant simple sequence repeat, single nucleotide polymorphism and sequence characterized amplified region markers for the pea root rot pathogen, Aphanomyces euteiches

Three kinds of genetic markers including simple sequence repeats (SSRs), single nucleotide polymorphisms (SNPs) and sequence characterized amplified regions (SCARs) were developed from Aphanomyces euteiches. Of 69 loci tested, seven SSR, two SNP and two SCAR markers were codominantly polymorphic. These codominant markers and dominant markers described herein will facilitate population genetic and evolutionary studies of this important plant pathogen.