牦牛基因组微卫星富集文库的构建与分析

根据生物素与链亲和素的强亲和性原理,用链亲和素磁珠亲和捕捉与生物素标记的微卫星寡核苷酸探针(CA)12、(CCG)8、(CAG)8、(TTTC)8退火结合的含有接头和牦牛微卫星序列的单链限制性酶切片段,获得单链目的片段,经PCR扩增形成双链,然后克隆到pMD18-T载体上,转化至DH5α中,首次成功构建牦牛基因组微卫星富集文库。测序结果发现,阳性克隆率为77％(37／48),说明构建的牦牛基因组微卫星富集文库是一个高质量的文库。牦牛富集微卫星文库的建立和牦牛微卫星的筛选将为下一步进行牦牛基因组结构的分析、牦牛遗传连锁图谱的构建、分子进化和系统发育研究、标记辅助选择以及经济性状的QTL定位提供大量的微卫星标记。     

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。     

藏酋猴微卫星富集文库的构建及微卫星分子标记的筛选

通过磁珠富集法构建了藏酋猴AC重复和AAAG重复的微卫星富集文库,分离微卫星序列并对其进行分析。将藏酋猴基因组DNA经Sau3AI酶切后纯化回收,连接特定接头。用生物素标记的探针与酶切片段杂交,捕获300～1000bp片段,随后将获得的片段连接到pMD-19T载体上,转化至JM109中,成功构建藏酋猴微卫星富集文库。(AC)n富集文库和(AAAG)n富集文库的阳性克隆率分别为50%和10%左右。根据测序得到的48个微卫星序列成功设计了24对引物,最终筛选出6个微卫星标记,这些标记将为藏酋猴的遗传多样性研究、圈养种群结构的分析和遗传图谱的构建等奠定一定的基础。     

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.     

岩原鲤微卫星富集文库的构建及微卫星分子标记的筛选

用磁珠富集法构建了岩原鲤Procypris rabaudi AC重复和GATA重复的微卫星富集文库.采用PCR方法分别以人工合成的oligoA和探针(AC)12或(GATA)6为引物筛选含有微卫星的阳性克隆.(AC)n 富集库和(GATA)n富集库的阳性克隆率分别为30%和7%左右.对40个AC重复的阳性克隆和30个GATA重复的阳性克隆测序,共获得61个微卫星序列,其中包含了一个三碱基重复(TGA)的微卫星序列.选择设计了19对AC重复和16对GATA重复的微卫星引物以及1对TGA重复的微卫星引物,通过PCR优化,共有20对引物能够产生稳定、清晰的目的产物带.为了检测获得的岩原鲤微卫星座位是否能用于近缘物种的研究,我们将20对岩原鲤微卫星引物用于中华倒刺鲃Spinibarbus sinensis基因组DNA PCR扩增,有60%的引物对能产生特异性的目的条带.本研究获得的20个岩原鲤微卫星座位可以用于岩原鲤及近缘物种的遗传多样性、种群遗传结构等的进一步研究.     

东方粘虫(Pseudaletia separata(Walker))微卫星富集文库的构建与分析

根据生物素与链亲和素的强亲和性原理,用包被链亲和素的磁珠亲和捕捉与生物素标记的微卫星寡核苷酸探针(CA)15、(GA)15、(GTT)12、(GAT)12、(TAGA)8 和 (GTGA)8退火结合的含接头和微卫星序列的单链粘虫基因组DNA限制性酶切片段,获得单链目的片段.经PCR扩增形成双链后连接到pGEM-T 载体上,再转化到DH5α热感受态细胞中,首次成功构建粘虫基因组微卫星富集文库.随机抽样(16次抽样,每库共12～114个克隆)测序发现,6个文库总的微卫星阳性克隆率30.07%,CA/GAT/GTT 等3个文库的阳性克隆率达到40%以上.单次抽样最高阳性克隆率达到56%.粘虫微卫星富集文库的建立和高多态性微卫星单拷贝位点的筛选将为粘虫的生态遗传学研究、连锁图谱构建、分子进化和系统发育研究等提供大量遗传标记,对昆虫迁飞的分子生态研究也有重大意义.     

乌苏里拟鲿微卫星文库的构建及分析

<正>乌苏里拟鲿(Pseudobagras ussuriensis)又名乌苏里逘(Leiocassis ussuriensis),隶属于鲶形目,鲿科,拟鲿属,主要分布于我国东北地区,但在我国其他各大水系也有分布[1]。乌苏里拟鲿肉质细嫩、味道鲜美、风味独特、适口性好、市场价值高,深受广大消费者和养殖户青睐。在20世纪80年代,乌苏里拟鲿的天然捕捞量曾仅次于同科的黄颡鱼(Pelteobagrus fulvidsco),然而由于捕捞强度的持续增长、环境污染及生境破坏等因素的影响,     

黑斑原(鱼兆)微卫星DNA富集文库构建与鉴定

采用磁珠富集法,利用生物素标记的(CA)12寡核苷酸探针从黑斑原(鱼兆)基因组DNA MboI酶切的400—1000 bp片段中筛选CA/GT微卫星位点,洗脱的杂交片段克隆到pMD18-T载体上构建富集微卫星基因组文库后,通过PCR筛选检测出720个阳性克隆,占所有克隆的89.2%,从阳性克隆中随机选取139个进行测序,序列分析发现,124个克隆含有7个以上的重复序列,其中完全的为80个(64.5%),不完全的为40个(32.3%),复合的为15个(3.2%),重复次数范围为7—165次,平均为52次。在124条序列中共59条可以设计引物。     

藏羚羊(AC)_n,(AG)_n微卫星文库构建

藏羚羊Pantholops hodgsonii是我国珍稀的动物资源,开发其微卫星标记具有重要的科研与应用价值。本研究采用FIASCO法(Fast Isolation by AFLP Sequences Containing repeats)构建了藏羚羊的(AC)n和(AG)n微卫星富集文库。各文库随机挑取150个白色菌落,分别筛选到76个(AC)n和61个(AG)n阳性克隆;经测序得到含AC重复的微卫星序列37条和含AG重复的序列29条。研究共设计、合成微卫星引物47对,以扩增、电泳等方法进行筛选,最终获得15个可稳定扩增且具有多态的微卫星标记。筛选出的引物可用于评估藏羚羊的核DNA遗传多样性、遗传结构、种群动态以及构建藏羚羊遗传图谱等。     

两种书虱微卫星富集文库的构建及比较

利用链霉亲和素与生物素之间的强亲和性原理,将链霉亲和素偶联的磁珠与微卫星探针(AC)12、(TC)12、(ATC)8、(ATG)8、(AAC)8、(ATAC)6及(GATA)6退火结合后,再亲和捕捉含接头和微卫星序列的单链书虱基因组DNA限制性酶切目的片段,经PCR扩增形成双链后进行克隆、建库。结果表明本研究成功构建了嗜卷书虱和嗜虫书虱共13个微卫星富集文库,包括6个嗜卷书虱文库,7个嗜虫书虱文库,其平均阳性克隆率为71.17%。经检测发现共得到两种书虱260个微卫星位点。这两种书虱微卫星富集文库的建立和高多态性微卫星位点的筛选将为嗜卷书虱和嗜虫书虱的种群遗传与进化、基因连锁图谱构建、分子系统发育研究等提供大量分子遗传标记,对其在实仓中的持续控制提供遗传学信息。     

Development of eighteen tetranucleotide microsatellite markers in Tibetan Macaque (Macaca thibetana) and genetic diversity analysis of captive population

In this study, eighteen tetranucleotide microsatellite loci were isolated from AAAG-enriched and GATA-enriched libraries of the Tibetan macaque, Macaca thibetana. These loci were tested on 53 individuals of M. thibetana, and most loci were proved to be highly polymorphic. A total of 109 alleles were detected with an average of 6.06 alleles per locus. The PIC values of these loci ranged from 0.192 to 0.879, with an average of 0.624. The observed and expected heterozygosities ranged from 0.170 to 0.800 and from 0.217 to 0.898, with an average of 0.583 and 0.675, respectively. 5 loci significantly deviated from Hardy–Weinberg equilibrium (HWE). Significant linkage disequilibrium (LD) was found between 9 pairs of loci. The newly identified polymorphic markers would facilitate the study of M. thibetana on the population structure and genetic diversity.     

High-throughput microsatellite isolation through 454 GS-FLX Titanium pyrosequencing of enriched DNA libraries

Microsatellites (or SSRs: simple sequence repeats) are among the most frequently used DNA markers in many areas of research. The use of microsatellite markers is limited by the difficulties involved in their de novo isolation from species for which no genomic resources are available. We describe here a high-throughput method for isolating microsatellite markers based on coupling multiplex microsatellite enrichment and next-generation sequencing on 454 GS-FLX Titanium platforms. The procedure was calibrated on a model species (Apis mellifera) and validated on 13 other species from various taxonomic groups (animals, plants and fungi), including taxa for which severe difficulties were previously encountered using traditional methods. We obtained from 11,497 to 34,483 sequences depending on the species and the number of detected microsatellite loci ranged from 199 to 5791. We thus demonstrated that this procedure can be readily and successfully applied to a large variety of taxonomic groups, at much lower cost than would have been possible with traditional protocols. This method is expected to speed up the acquisition of high-quality genetic markers for nonmodel organisms.     

基于粪便DNA 的藏狐微卫星位点筛选及个体识别

藏狐是我国青藏高原东部多房棘球绦虫和石渠棘球绦虫最主要的野生动物终末宿主。棘球绦虫会导致一类称为棘球绦虫病的致死性人兽共患疾病,青藏高原东部牧区是该病重要的流行区。因此作为终末宿主,评估藏狐种群的棘球绦虫感染率对于该病的流行病学研究意义明显。而要获取这方面信息,首先必须了解藏狐的种群数量。为此,我们基于非损伤取样的原则,使用藏狐新鲜粪便作为研究材料,从已发布的藏狐及近缘种的48个微卫星位点中筛选了11 个用于藏狐粪便DNA 多态性分析。对2011 -2012 年7 -8 月间收集的128 份有效藏狐粪便样品(2011 年68 份,2012 年60 份)进行特异性PCR 扩增,并用琼脂糖凝胶电泳和荧光引物标记法进行基因分型,根据各位点的等位基因频率计算出各位点的基因型数(N),期望杂合度(He )、观测杂合度(Ho)、多态信息含量(PIC)以及不同个体基因型相同概率值(PI)。结果发现,各位点N 介于4 - 7,H e为0.66 - 0. 80,H o为0.17 -0.68,PIC 为0.5496 - 0.7623。11 个位点的累积PI 值满足个体识别的需要(PIbiased = 1. 283 × 10 ^{- 11} ;PIsi bs =7.572 × 10 ^{- 5} )。但是,由于粪便DNA 质量差异较大,不同位点的扩增成功率差异较大(0.176 - 0. 926)。我们发现,按照扩增成功率由高到低排列,前6 个微卫星位点(P03,CXX172,CPH6,CPH8,P01i,P08)的扩增成功率均超过0.6,且累积PI 值小于0.004 (PIbiased =2.775 × 10 ^{- 7} ;PIsibs = 3. 606 ×10 ^{- 3} ),表明这6 个位点可以对藏狐进行个体识别。因此,针对本研究的数据,制定了如下的个体识别原则: (1)只有粪便DNA 至少成功扩增出前6 个微卫星位点的样品可以进入下一步分析; (2)所有位点的信息均相同的两个样品被认为是来自同一个体;(3)保险起见,如果仅有一对位点信息不相等,此两个样品依然被判定来自同一个体。在此基础上,我们从2011 年样品中识别出30 个藏狐个体,从2012 年样品中识别出21 个个体。     

Characterization of new Schistosoma mansoni microsatellite loci in sequences obtained from public DNA databases and microsatellite enriched genomic libraries

In the last decade microsatellites have become one of the most useful genetic markers used in a large number of organisms due to their abundance and high level of polymorphism. Microsatellites have been used for individual identification, paternity tests, forensic studies and population genetics. Data on microsatellite abundance comes preferentially from microsatellite enriched libraries and DNA sequence databases. We have conducted a search in GenBank of more than 16,000 Schistosoma mansoni ESTs and 42,000 BAC sequences. In addition, we obtained 300 sequences from CA and AT microsatellite enriched genomic libraries. The sequences were searched for simple repeats using the RepeatMasker software. Of 16,022 ESTs, we detected 481 (3%) sequences that contained 622 microsatellites (434 perfect, 164 imperfect and 24 compounds). Of the 481 ESTs, 194 were grouped in 63 clusters containing 2 to 15 ESTs per cluster. Polymorphisms were observed in 16 clusters. The 287 remaining ESTs were orphan sequences. Of the 42,017 BAC end sequences, 1,598 (3.8%) contained microsatellites (2,335 perfect, 287 imperfect and 79 compounds). The 1,598 BAC end sequences 80 were grouped into 17 clusters containing 3 to 17 BAC end sequences per cluster. Microsatellites were present in 67 out of 300 sequences from microsatellite enriched libraries (55 perfect, 38 imperfect and 15 compounds). From all of the observed loci 55 were selected for having the longest perfect repeats and flanking regions that allowed the design of primers for PCR amplification. Additionally we describe two new polymorphic microsatellite loci.     

微卫星标记在分子生态学中的应用及其位点的分离策略

微卫星DNA作为一种优良的遗传标记在分子生态学领域得到了广泛应用,本文综述了其在分子种群生物学、分子环境遗传学、分子适应等研究领域中的应用情况.微卫星位点的获得是开展各项研究的前提,传统的构建微卫星文库再杂交筛选的方法工作量大、效率低,因而在实践过程中又产生了富集文库法、PIMA法、FIASCO法等新的分离策略.本文对几种微卫星位点分离技术进行介绍并对其进行分析比较,为分子生态学研究过程中微卫星位点筛选方法的选择提供参考.     

Chicken microsatellite markers isolated from libraries enriched for simple tandem repeats

The total number of microsatellite loci is considered to be at least 10-fold lower in avian species than in mammalian species. Therefore, efficient large-scale cloning of chicken microsatellites, as required for the construction of a high-resolution linkage map, is facilitated by the construction of libraries using an enrichment strategy. In this study, a plasmid library enriched for tandem repeats was constructed from chicken genomic DNA by hybridization selection. Using this technique the proportion of recombinant clones that cross-hybridized to probes containing simple tandem repeats was raised to 16%, compared with < 0·1% in a non-enriched library. Primers were designed from 121 different sequences. Polymerase chain reaction (PCR) analysis of two chicken reference pedigrees enabled 72 loci to be localized within the collaborative chicken genetic map, and at least 30 of the remaining loci have been shown to be informative in these or other crosses.     

Difference in types of radiation-induced structural chromosome aberrations and their incidences between Chinese and Syrian hamster spermatozoa

The effects of ionizing radiations on sperm chromosomes were studied in the Chinese hamster (Crisetulus griseus) and the Syrian (golden) hamster (Mesocrisetus auratus). Testes of mature male Chinese hamsters (CH) were irradiated with X-rays (0.91, 1.82 and 3.63 Gy) and γ-rays (1.10, 2.15, 2.95 and 4.01 Gy) at a single acute dosage, whereas the irradiation was done with lower doses of X-rays (0.45, 0.91 and 1.82 Gy) and γ-rays (0.49, 0.99 and 1.98 Gy) in mature male Syrian hamsters (SH), taking the higher radiosensitivity of this species into consideration. They were mated with normal females within 6 days of exposure. Sperm-derived chromosomes were analyzed in 1125 and 1966 fertilized ova of the CH and the SH, respectively. In both species, there was no great difference in the induction of structural chromosome aberrations between X-irradiated and γ-irradiated spermatozoa. Chromosome-type aberrations were predominantly induced. The incidence of breakage-type aberrations increased linearly, and that of exchange-type aberrations linear-quadratically with increase of dosage. A species-specific difference in chromosomal radiosensitivity of spermatozoa was clear. In spite of the same radiation dosage, the incidence of chromosomally abnormal spermatozoa in the SH was about twice as high as that in the CH (e.g., 27.0% vs. 14.7% at 0.91 Gy of X-rays). The incidences of breakage-type aberrations (69–89%) were far higher than those of exchange-type aberrations (11–31%) in the SH, while the disparity of the two incidences was much smaller in the CH (46–65% vs. 35–54%). Exchange-type aberrations consisted of both chromosome-type and chromatid-type in the SH, while almost all of them were of the chromosome-type in the CH. These results suggest that the DNA-repairing capacity of oocytes is much higher in the CH than in the SH. Moreover, it seems likely that radiation-induced sperm DNA damage is repaired with both pre-replication repair (excision repair) and post-replication repair systems in SH oocytes, whereas the excision repair system operate most exclusively in CH oocytes.     

Bacterial artificial chromosome library for genome‐wide analysis of Chinese hamster ovary cells

Chinese hamster ovary (CHO) cell lines are widely used for scientific research and biotechnology. A CHO genomic bacterial artificial chromosome (BAC) library was constructed from a mouse dihydrofolate reductase (DHFR) gene‐amplified CHO DR1000L‐4N cell line for genome‐wide analysis of CHO cell lines. The CHO BAC library consisted of 122,281 clones and was expected to cover the entire CHO genome five times. A CHO chromosomal map was constructed by fluorescence in situ hybridization (FISH) imaging using BAC clones as hybridization probes (BAC‐FISH). Thirteen BAC‐FISH marker clones were necessary to identify all the 20 individual chromosomes in a DHFR‐deficient CHO DG44 cell line because of the aneuploidy of the cell line. To determine the genomic structure of the exogenous Dhfr amplicon, a 165‐kb DNA region containing exogenous Dhfr was cloned from the BAC library using high‐density replica (HDR) filters and Southern blot analysis. The nucleotide sequence analysis revealed a novel genomic structure in which the vector sequence containing Dhfr was sandwiched by long inverted sequences of the CHO genome. Biotechnol. Bioeng. 2009; 104: 986–994. © 2009 Wiley Periodicals, Inc.     

Gametes and fertilization in the Chinese hamster

Freshly ovulated eggs are each surrounded by a compact cumulus oophorus. The overall diameter of the normal egg (including the zona pellucida) is about 100 μm. Cumulus cells, particularly those near the egg, are arranged redially in a viscous noncellular matrix. The spermatozoon is about 250 μm in length. The head a large acrosome, changes in which can be readily examined with the light (phase- contrast) microsope. When exposed to physiological salt solutions, testicular spermatozoa either were motionless or flexed the posterior half of their tails slowly. Spermatozoa from the caput epididymis were highly motile, flexing the entire tail. A few of them moved progressively. Mature spermatozoa from the vas deferens were highly motile and moved either straightforward or in a circle. They vibrated their tails stiffly without flexing them. In normally mated females, fertilization began sometime between 2 and 3 h after ovulation and was completed within the next 4 to 5 h. Spermatozoa swimming in the ampullary fluid or within the cumulus oophorus about the time of fertilization flexed the anterior half (which roughly corresponds to the midpieac region) of their tails. This peculiar movement may be homologous to the so-called “hyperactivation” of spermatozoa as reported in several other mammalian species. Actively motile spermatozoa within the cumulus or no the zona pellucida had either modified (“collapsed”) or no acrosomal caps. The sperm head usually passed verticually or nearly through the zona, but the path was oblique in some instances. In 54% of the recently fertilized eggs examined, the entire length of the sperm tail was within the perivitelline space; in the other 46% of the eggs varying lenghts of the tail remined the perivitelline space, the tails were extruded from the vitellus of many eggs even before the eggs began their first cleavage. When unfertilized eggs in the cumulus oophorus were inseminated with vas deferens spermatozoa in a modified Tyrode's solution (m-TALP), about 80% of them were ferrtilized by 4–6 h after insemination. The vast majority were monospermic. When eggs were freed from the cumulus prior to insemination, none were fertilized, suggesting that the cumulus cells or their matrix assisted capacitation and/or the acrosome reaction of the spermatozoa under the in vitro conditions employed. No eggs were fertilized by the testicular or caput epididymal spermatozoa regardless of the presence or absence of cumulus oophorus around the eggs at the time of insemination.