微卫星分子标记及其在动物遗传育种中的研究进展

微卫星分子标记在基因组中具有含量丰富、多态性、共显性和易于检测等优点,是动物遗传育种应用中一种重要的分子标记技术。随着科学技术的飞速发展,微卫星标记的研究方法也日新月异:生物信息学技术的兴起使得微卫星位点的获得越来越方便;高通量测序技术的成熟使得开发新微卫星标记更加简单高效;自动化的序列分析仪器使得微卫星DNA的检测越来越快速准确;同时微卫星标记的应用范围也越来越广泛,其功能的开发正逐渐从刚开始的个别位点研究转变到全基因组微卫星分析。本研究简单介绍了微卫星分子标记的特点,重点综述了目前微卫星分子标记的获得、开发和筛选方法,及其在动物遗传育种中的应用,将为微卫星分子标记的研究提供理论支持。     

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.     

宽口光唇鱼微卫星位点的筛选与特征分析

通过FIASCO法(Fast Isolation by AFLP Sequences Containing Repeats)筛选宽口光唇鱼Acrossocheilus monticola(Günter)基因组微卫星位点,利用生物素标记的寡核苷酸探针(AC)8、(CT)8、(GGT)8、(GATA)8和(GATT)7首次成功构建宽口光唇鱼基因组微卫星富集文库。从文库中共筛选495个克隆测序,成功设计163对微卫星引物,经PCR扩增检测,获得了18个多态性微卫星标记。利用这18对多态性引物,分析了赤水河赤水市宽口光唇鱼种群的遗传多样性,数据显示:18对多态性微卫星引物的等位基因数为8～31个,平均等位基因数为19.6个,平均期望杂合度为0.8701,平均观测杂合度为0.8312,其中引物Am07、Am35、Am47、Am69、Am78和Am127显著偏离哈迪温伯格平衡(P<0.05),以上数据表明赤水河赤水市宽口光唇鱼种群的遗传多样性水平较高。筛选的微卫星标记对于宽口光唇鱼的遗传背景分析和生物种质资源的保护有重要作用。     

微卫星位点筛选方法综述

微卫星标记因其丰富的多态性和共显性等特点,已得到了广泛的应用.应用微卫星标记首先需要获得微卫星位点的序列信息,用来设计引物.获得微卫星位点的方法有多种,本文综述了获得和富集微卫星位点的常用方法.最简便、最省时的方法是从公共数据库(如EMBL、Genbank、EST数据库等)或已发表的文献中查找到微卫星位点,但只限于已经有序列数据发布的物种.第二种方法是种间转移扩增,即从相近物种的数据库中查找微卫星位点,或使用已有数据发表的遗传距离相近物种的微卫星标记.第三种方法是从基因组DNA中筛选微卫星位点,其中用于富集微卫星的方法有引物法、磁珠杂交法、尼龙膜杂交法以及RAPD技术法.     

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。     

花生微卫星标记的研究进展

近年来花生微卫星标记的开发取得了一定的进展,初步揭示了花生在DNA水平上的遗传多样性。花生微卫星标记的开发途径主要包括通过构建小片段基因组文库开发基因组SSR标记,根据花生EST序列开发EST-SSR标记,根据豆科植物序列信息和SSR标记开发花生SSR标记,将SSR标记与其它分子标记结合开发新的DNA标记,以及基于SSR核心序列开发ISSR标记。花生微卫星标记主要应用于遗传多样性研究、遗传图谱与品种指纹图谱构建以及分子标记辅助育种等领域。本文综述了花生SSR标记开发研究的进展及应用。     

花生微卫星标记的研究进展

近年来花生微卫星标记的开发取得了一定的进展, 初步揭示了花生在DNA水平上的遗传多样性。花生微卫星标记的开发途径主要包括通过构建小片段基因组文库开发基因组SSR标记, 根据花生EST序列开发EST-SSR标记, 根据豆科植物序 列信息和SSR标记开发花生SSR标记, 将SSR标记与其它分子标记结合开发新的DNA标记, 以及基于SSR核心序列开发ISSR标记。花生微卫星标记主要应用于遗传多样性研究、遗传图谱与品种指纹图谱构建以及分子标记辅助育种等领域。本文综述了花生SSR标记开发研究的进展及应用。     

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。     

应用微卫星标记对不同基因组组合生物型遗传多态性的研究(英文)

微卫星ＤＮＡ在真核基因组中分布频率为每２０～３０ｋｂ一个,具有片段长度短、序列 高度重复、种类多以及高度多态的特点,极适于遗传变异研究。本文报道：采用微卫星 标记对不同基因组组合的鱼类进行了基因组指纹图谱构建。受试材料为红鲤（ＲＣ）、红鲫 （ＲＡ）、镜鲤（ＭＣ）、鲤鲫杂种二倍体（ＣＡ）鲫鲤杂种三倍体（ＣＡＡ）,人工复合三倍体（ＣＣＡ） 等六种生物型。微卫星ＤＮＡ座位（探针）ＭＦＷ２、ＭＦＷ８和ＭＦＷ１６各自存在１２、１６和 １０个等位基因（Ｆｉｇ．１,２,＆３）。通过对微卫星标记图谱的量化分析,利用ＵＰＧＭＡ构建了 不同生物型的遗传关系树系图（Ｔａｂｌｅ　１,Ｆｉｇ．４）。本研究发现,徽卫星和ＲＡＰＤ分析两种 手段反映六种生物型之间的聚类模式完全一致。然而由微卫星标记获得的生物型内和生 物型之间的遗传距离均大于ＲＡＰＤ的,此结果表明微卫星标记在揭示群体内个体间差 异上有独到之处。     

DNA分子标记技术及其在水产动物遗传上的应用研究

随着DNA分子标记技术的发展,其在动物遗传上发挥了重大作用,使用DNA分子标记可以观察到整个基因组的遗传多样性。目前,在水产养殖种类中使用的遗传标记主要包括线粒体DNA、RFLP、RAPD、AFLP、微卫星、SNP和EST标记。DNA分子标记的应用使得人们对水产养殖动物的遗传多样性、近亲繁殖、种类和品系鉴定以及遗传连锁图谱建立的研究都取得了很大进展,也加快了数量性状位点(QTL)基因的鉴定作为分子标记辅助选择(MAS)的研究。将这些标记技术在水产动物上的应用进行了论述,以及如何从人类基因组工程和斑马鱼这种模式鱼的研究中得到启发,更好的应用于水产动物基因组学和遗传学研究做一讨论。     

Development,characterization and variability analysis of microsatellites in lychee (<Emphasis Type="Italic">Litchi chinensis</Emphasis> Sonn., Sapindaceae)

We report 12 microsatellites enriched in CT repeats obtained from a genomic library of the lychee (Litchi chinensis Sonn.) cultivar Mauritius. The polymorphisms revealed by these microsatellites were evaluated in a collection of 21 lychee cultivars. A total of 59 fragments were detected with these 12 SSRs, with an average of 4.9 bands/SSR. Three primer pairs seem to amplify more than a single locus. The mean expected and observed heterozygosities over the 9 single-locus SSRs averaged 0.571 (range: 0.137–0.864) and 0.558 (range: 0.169–0.779) respectively. The total value for the probability of identity was 7.53×10^-5. In addition, the selected SSRs were used to amplify DNA from four longan cultivars. Eleven of the 12 SSRs produced amplification fragments in longan, and eight of these fragments were polymorphic. All except two of the products amplified from longan were the same size as those amplified from lychee, suggesting a close genetic proximity between the two species. The SSRs studied produced 22 different patterns, allowing the unambiguous identification of 16 lychee and the 4 longan cultivars studied. Discrimination was possible with just four selected microsatellites. Two groups with two and three undistinguishable cultivars were obtained, reflecting probable synonymies. Unweighted pair-group method of artimetic averages (UPGMA) cluster analysis divided the lychee cultivars studied into two main groups, one consisting of ancient cultivars and the other with more diverse recent cultivars. This is the first report of microsatellite development in the Sapindaceae, and the results demonstrate the usefulness of microsatellites for identification, similarity studies and germplasm conservation in lychee and related species.Communicated by H.F. Linskens     

Characterization of microsatellites and development of chromosome specific STMS markers in bread wheat

Microsatellites, or simple sequence repeats (SSRs), have become the markers of choice for genetic studies with many crop species including wheat. Currently an international effort is underway to enrich the repertoire of available sequence tagged microsatellite site (STMS) markers in wheat. As a part of this effort, we have sequenced 43 clones obtained from a microsatellite-enriched wheat genomic library; 34 clones contained 41 different microsatellites. These microsatellites (mono-, di-, tri- nucleotide repeats) were classified as 19 simple perfect, 18 simple imperfect and 4 compound imperfect types. Dinucleotide repeats were the most abundant (70%). Primer pairs for only 16 microsatellites could be designed, since the flanking sequences of the others were either too short or were otherwise not suitable for designing the microsatellite specific primers. Microsatellite loci of the expected size and polymorphism were successfully amplified from 15 of these 16 primer pairs using three wheat varieties. 14 loci detected by 12 out of the 15 functional primer pairs were assigned to 11 specific chromosomes. An erratum to this article is available at .     

Level of genetic differentiation affects relative performances of expressed sequence tag and genomic SSRs

Microsatellites, also called simple sequence repeats (SSRs), are markers of choice to estimate relevant parameters for conservation genetics, such as migration rates, effective population size and kinship. Cross‐amplification of SSRs is the simplest way to obtain sets of markers, and highly conserved SSRs have recently been developed from expressed sequence tags (EST) to improve SSR cross‐species utility. As EST‐SSRs are located in coding regions, the higher stability of their flanking regions reduces the frequency of null alleles and improves cross‐species amplification. However, EST‐SSRs have generally less allelic variability than genomic SSRs, potentially leading to differences in estimates of population genetic parameters such as genetic differentiation. To assess the potential of EST‐SSRs in studies of within‐species genetic diversity, we compared the relative performance of EST‐ and genomic SSRs following a multispecies approach on passerine birds. We tested whether patterns and levels of genetic diversity within and between populations assessed from EST‐ and from genomic SSRs are congruent, and we investigated how the relative efficiency of EST‐ and genomic SSRs is influenced by levels of differentiation. EST‐ and genomic SSRs ensured comparable inferences of population genetic structure in cases of strong genetic differentiation, and genomic SSRs performed slightly better than EST‐SSRs when differentiation is moderate. However and interestingly, EST‐SSRs had a higher power to detect weak genetic structure compared to genomic SSRs. Our study attests that EST‐SSRs may be valuable molecular markers for conservation genetic studies in taxa such as birds, where the development of genomic SSRs is impeded by their low frequency.     

PERMANENT GENETIC RESOURCES: Development of 52 new polymorphic SSR markers from cherimoya (Annona cherimola Mill.): transferability to related taxa and selection of a reduced set for DNA fingerprinting and diversity studies

Fifty-two single locus polymorphic microsatellites were developed using two genomic libraries digested with HaeIII and RsaI of cherimoya cv. Fino de Jete enriched in CT/AG repeats. A total of 222 alleles were detected with the selected simple sequence repeats (SSRs). The observed and expected heterozygosities ranged from 0.08 to 0.73 and from 0.20 to 0.84, respectively. Most of the SSRs were transferable to other species in the Annonaceae. A set of 20 microsatellites was selected to facilitate the exchange of data among laboratories.     

Genome-wide characterization and analysis of microsatellite sequences in camelid species

Microsatellites or simple sequence repeats (SSRs) are among the genetic markers most widely utilized in research. This includes applications in numerous fields such as genetic conservation, paternity testing, and molecular breeding. Though ordered draft genome assemblies of camels have been announced, including for the Arabian camel, systemic analysis of camel SSRs is still limited. The identification and development of informative and robust molecular SSR markers are essential for marker assisted breeding programs and paternity testing. Here we searched and compared perfect SSRs with 1–6 bp nucleotide motifs to characterize microsatellites for draft genome sequences of the Camelidae. We analyzed and compared the occurrence, relative abundance, relative density, and guanine-cytosine (GC) content in four taxonomically different camelid species: Camelus dromedarius, C. bactrianus, C. ferus, and Vicugna pacos. A total of 546762, 544494, 547974, and 437815 SSRs were mined, respectively. Mononucleotide SSRs were the most frequent in the four genomes, followed in descending order by di-, tetra-, tri-, penta-, and hexanucleotide SSRs. GC content was highest in dinucleotide SSRs and lowest in mononucleotide SSRs. Our results provide further evidence that SSRs are more abundant in noncoding regions than in coding regions. Similar distributions of microsatellites were found in all four species, which indicates that the pattern of microsatellites is conserved in family Camelidae.     

Development of SSR markers for the phylogenetic analysis of almond trees from China and the Mediterranean region.

Expressed sequence tag (EST) derived simple sequence repeats (SSRs, microsatellites) were screened and identified from 3863 almond and 10 185 peach EST sequences, and the spectra of SSRs in the non-redundant EST sequences were investigated after sequence assembly. One hundred seventy-eight (12.07%) almond SSRs and 497 (9.97%) peach SSRs were detected. The EST-SSR occurs every 4.97 kb in almond ESTs and 6.57 kb in peach, and SSRs with di- and trinucleotide repeat motifs are the most abundant in both almond and peach ESTs. Twenty one EST-SSRs were thereafter, developed and used together with 7 genomic SSRs, to study the genetic relationship among 36 almond (P. communis Fritsch.) cultivars from China and the Mediterranean area, as well as 8 accessions of other related species from the genus Prunus. Both EST-derived and genomic SSR markers showed high cross-species transferability in the genus. Out of the 112 polymorphic alleles detected in the 36 cultivated almonds, 28 are specific to Chinese cultivars and 25 to the others. The 44 accessions were clustered into 4 groups in the phylogenetic tree and the 36 almond cultivars formed two distinct subgroups, one containing only Chinese cultivars and one of unknown origin and the other only those originating from the Mediterranean area, indicating that Chinese almond cultivars have a distinct evolutionary history from the Mediterranean almond. Our preliminary results indicated that common almond was more closely related to peach (P. persica (L.) Batsch.) than to the four wild species of almond, (P. mongolica Maxim., P. ledebouriana Schleche, P. tangutica Batal., and P. triloba Lindl.). The implications of these SSR markers for evolutionary analysis and molecular mapping of Prunus species are discussed.     

核盘菌和灰葡萄孢基因组中的简单重复序列分析

利用公共的真菌基因组数据库资源, 对核盘菌（Sclerotinia sclerotiorum）和灰葡萄孢（Botrytis cinerea）基因组中SSRs的结构类型、分布、丰度及最长序列等进行了系统分析, 并与已经研究过的禾谷镰孢菌（Fusarium graminearum）, 稻瘟病菌（Magnaporthe grisea）和黑粉菌（Ustilago maydis）等几种植物病原真菌基因组中的SSRs进行了比较。结果表明: 核盘菌和灰葡萄孢基因组中的SSRs非常丰富, 分别为6 539和8 627个, 并且在结构类型和分布规律上具有一定的相似性; 与其他几种病原真菌相比, 核盘菌和灰葡萄孢基因组中长重复的四、五、六核苷酸基序更为丰富, 从而使得这两种真菌具有更高的变异性。同时, 我们发现真菌基因组中SSRs的丰度与基因组的大小及GC含量没有必然的关系。文章对核盘菌和灰葡萄孢基因组中SSRs的丰度、出现频率及最长基序的分析为快速、便捷地设计多态性丰富的SSRs引物提供了有益的信息。     

Development of a set of highly polymorphic genomic microsatellites (gSSRs) in Sitka spruce (Picea sitchensis (Bong.) Carr.)

Robust, polymorphic microsatellite DNA markers (simple sequence repeats—SSRs) are valuable tools for a range of tree conservation and breeding applications. SSRs are routinely used in the study of population genetic structure and diversity, pedigree reconstruction and genetic linkage mapping. Their abundance in the genome, co-dominant inheritance and potential for cross-species amplification make microsatellites highly prized markers. This paper characterises 22 novel genomic polymorphic microsatellite loci for Sitka spruce (Picea sitchensis (Bong.) Carr.). Amplification of DNA from Sitka spruce material was carried out both with a set of unrelated trees to obtain diversity statistics for each locus, and with the progeny of a full-sib family to test simple Mendelian inheritance. Observed heterozygosity ranged from 0.38 to 0.91 and allele number per locus ranged from 6 to 21, with a mean of 12.2. In addition, the primer pairs were tested with DNA from Norway spruce (P. abies) and white spruce (P. glauca) to investigate their potential for cross-species amplification and ten loci amplified in all three species. The results from these genomic microsatellites are compared to data generated from microsatellites derived from Picea EST libraries. In summary, this novel, highly polymorphic markers represent a significant addition to the rapidly expanding Picea genomics tool-box. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Eucalyptus microsatellites mined in silico: survey and evaluation

Eucalyptus is an important short rotation pulpy woody plant, grown widely in the tropics. Recently, many genomic programmes are underway leading to the accumulation of voluminous genomic and expressed sequence tag sequences in public databases. These sequences can be utilized for analysis of simple sequence repeats (SSRs) and single nucleotide polymorphism (SNPs) available in the transcribed genes. In this study, in silico analysis of 15,285 sequences representing partial and full-length mRNA from Eucalyptus species for their use in developing SSRs or microsatellites were carried out. A total of 875 EST-SSRs were identified from 772 SSR containing ESTs. Motif size of 6 for dinucleotide and 5 for trinucleotide, tetranucleotide, and pentanucleotides were considered in locating the microsatellites. The average frequency of identified SSRs was 12.9%. The dinucleotide repeats were the most abundant among the dinucleotide, trinucleotide and tetranucleotide motifs and accounted for 50.9% of the Eucalyptus genome. Primer designing analysis showed that 571 sequences with SSRs had sufficient flanking regions for polymerase chain reaction (PCR) primer synthesis. Evaluation of the usefulness of the SSRs showed that EST-derived SSRs can generate polymorphic markers as all the primers showed allelic diversity among the 16 provenances of E. tereticornis.