Characterization of novel microsatellites from Drosophila transversa

We investigated a partial genomic library of Drosophila transversa for microsatellites and developed 12 markers for genetic analyses. This is the first time that microsatellite primers from the quinaria species group have been described. Four loci were cross-amplified in D. phalerata. Nine out of the 12 microsatellite markers developed are likely to be on the X chromosome.     

Development of polymorphic expressed sequence tag-derived microsatellites for the extension of the genetic linkage map of the black tiger shrimp (Penaeus monodon)

In this study, microsatellite markers were developed for the genetic linkage mapping and breeding program of the black tiger shrimp Penaeus monodon. A total of 997 unique microsatellite-containing expressed sequence tags (ESTs) were identified from 10 100 EST sequences in the P. monodon EST database. AT-rich microsatellite types were predominant in the EST sequences. Homology searching by the blastn and blastx programs revealed that these 997 ESTs represented 8.6% known gene products, 27.8% hypothetical proteins and 63.6% unknown gene products. Characterization of 50 markers on a panel of 35-48 unrelated shrimp indicated an average number of alleles of 12.6 and an average polymorphic information content of 0.723. These EST microsatellite markers along with 208 other markers (185 amplified fragment length polymorphisms, one exon-primed intron-crossing, six single strand conformation polymorphisms, one single nucleotide polymorphism, 13 non-EST-associated microsatellites and two EST-associated microsatellites) were analysed across the international P. monodon mapping family. A total of 144 new markers were added to the P. monodon maps, including 36 of the microsatellite-containing ESTs. The current P. monodon male and female linkage maps have 47 and 36 linkage groups respectively with coverage across half the P. monodon genome.     

Quantifying ascertainment bias and species-specific length differences in human and chimpanzee microsatellites using genome sequences

Surveys of variability of homologous microsatellite loci among species reveal an ascertainment bias for microsatellite length where microsatellite loci isolated in one species tend to be longer than homologous loci in related species. Here, we take advantage of the availability of aligned human and chimpanzee genome sequences to compare length difference of homologous microsatellites for loci identified in humans to length difference for loci identified in chimpanzees. We are able to quantify ascertainment bias for a range of motifs and microsatellite lengths. Because ascertainment bias should not exist if a microsatellite selected in one species is as likely to be longer as it is to be shorter than its homologue, we propose that the nature of ascertainment bias can provide evidence for understanding how microsatellites evolve. We show that bias is greater for longer microsatellites but also that many long microsatellites have short homologues. These results are consistent with the notion that growth of long microsatellites is constrained by an upper length boundary that, when reached, sometimes results in large deletions. By evaluating ascertainment bias separately for interrupted and uninterrupted repeats we also show that long microsatellites tend to become interrupted, thereby contributing a second component of ascertainment bias. Having accounted for ascertainment bias, in agreement with results published elsewhere, we find that microsatellites in humans are longer on average than those in chimpanzees. This length difference is similar among repeat motifs but surprisingly comprises two roughly equal components, one associated with the repeats themselves and one with the flanking sequences. The differences we find can only be explained if microsatellites are both evolving directionally under a biased mutation process and are doing so at different rates in different closely related species.     

Rapid isolation and characterization of microsatellites from the genome of pearl oyster (Pinctada martensi Dunker)

A new set of 34 microsatellite markers was isolated and characterized in pearl oyster Pincada martensi from a CA₁₆-enriched genomic library. Of 60 microsatellite markers screened in a wild population which contained 30 individuals, 34 markers occurred unambiguous bands, including seven monomorphic loci, 5 primer pairs (HHM844, HHM852, HHM888, HHM891, HHM899) were duplicated. These polymorphic markers showed high genetic variability represented mainly by the number of alleles, which ranged from 2 to 6, the expect heterozygosity which ranged from 0.1653 to 0.7622, and the polymorphic information content which ranged from 0.1516 to 0.7279. These markers could increase the available molecular resources that can be used to analyze genetic diversity and structure of pearl oyster.     

Development and characterization of polymorphic microsatellites from Prunus avium‘Napoleon’

Primers were developed for 21 microsatellite loci isolated by enrichment from Prunus avium‘Napoleon’. Twelve loci contained uninterrupted dinucleotide repeats and nine were more complex. Nineteen primer pairs (EMPA001–019) showed single locus polymorphisms in a cultivar survey of 14 sweet cherries, with two to seven alleles per locus. Three primer pairs in combination (EMPA014, 015 and 018) discriminated all cultivars. Two primer pairs for loci monomorphic in P. avium were included: EMPA020 revealed segregation in an interspecific progeny and EMPA021 revealed polymorphism in P. dulcis. Twelve primer pairs reliably amplified products in three peach cultivars of which seven revealed polymorphisms.     

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。     

Development and mapping of microsatellites from a microdissected BTA 11-specific DNA library

A chromosome-specific library was developed for Bos taurus autosome 11 by chromosome microdissection and microcloning using a bovine primary fibroblast culture, obtained from a t(X;23) heifer, that spontaneously developed a translocation chromosome involving bovine chromosome 11. The library was screened using (AC)12 oligos, positive clones selected, sequenced and primers developed to generate bovine chromosome 11-specific microsatellite markers. This study suggests that chromosome-specific libraries have great potential for development of microsatellite markers for the construction of marker-saturated linkage maps for each chromosome.     

蜜蜂全基因组中微卫星的丰度及其分布

查找出蜜蜂基因组中由1～6个碱基重复单元组成的简单序列重复,分析蜜蜂基因组中微卫星的分布频率,并比较其在各染色体中的分布频率。微卫星在蜜蜂基因组中的分布频率为1/0·804kb,其中二碱基重复序列占26·86%,是最丰富的重复单元,而六、一、三、四、五碱基重复单元序列分别占24·74%,22·19%,13·65%,10·98%,2·59%。同时发现富含A和T碱基的微卫星占主导地位,富含G和C碱基的微卫星数量较少。第4,1,3条染色体微卫星分布频率较高,而第11,14,12条染色体微卫星分布频率较低。     

Use of short microsatellites from database sequences to generate polymorphisms among Lycopersicon esculentum cultivars and accessions of other Lycopersicon species

A search of nearly 2000 sequences from Solanaceae species in the EMBL and Genbank databases yielded 220 microsatellites. Among these were 80 microsatellites from 675 Lycopersicon entries. Dinucleotide repeats, as well as (CAA)_n and (TAA)_n repeats, were over-represented in non-coding DNA. The other trinucleotide repeats were predominantly found in exonic DNA. PCR analysis of 44 of the microsatellite-containing Lycopersicon loci identified 36 primer pairs that yielded well-scorable fragments, or groups of fragments, in L. esculentum cultivars and accessions of Lycopersicon species. Twenty-nine of these amplified bands that were polymorphic among the four Lycopersicon species. Ten primer pairs generated polymorphic bands among seven tomato cultivars. Upon examining the number of microsatellites and the degree of polymorphisms in relation to the repeat type and motif, the type of DNA the microsatellite resided in, the length of the microsatellite, and the presence of imperfections in the microsatellite, only two significant correlations were found. (i) Imperfect repeats were less polymorphic among species than perfect repeats. (ii) The percentage of loci polymorphic among cultivars increased from 6% for the shortest loci (with eight or less repeat units) to 60% for the group with the longest repeats (12 repeat units or longer). Among the species, however, all length classes contained about 83% polymorphic loci. In general, 2–4 alleles were found for each locus among the samples of the test set. In a few cases, up to eight alleles were found. A combination of these microsatellite loci can therefore be useful in distinguishing cultivars of tomato, which are genetically very closely related to each other. Received: 9 August 1996 / Accepted: 23 August 1996     

Dinucleotide-repeat polymorphism in DNA of rainbow trout and its application in fisheries science

Dinucleotide–repeat DNA polymorphisms for rainbow trout, Oncorhynchus mykiss , are described. The potential applications of these markers in fisheries science are discussed.     

Motif mismatches in microsatellites: insights from genome-wide investigation among 20 insect species

We present a detailed genome-wide comparative study of motif mismatches of microsatellites among 20 insect species representing five taxonomic orders. The results show that varying proportions (∼15–46%) of microsatellites identified in these species are imperfect in motif structure, and that they also vary in chromosomal distribution within genomes. It was observed that the genomic abundance of imperfect repeats is significantly associated with the length and number of motif mismatches of microsatellites. Furthermore, microsatellites with a higher number of mismatches tend to have lower abundance in the genome, suggesting that sequence heterogeneity of repeat motifs is a key determinant of genomic abundance of microsatellites. This relationship seems to be a general feature of microsatellites even in unrelated species such as yeast, roundworm, mouse and human. We provide a mechanistic explanation of the evolutionary link between motif heterogeneity and genomic abundance of microsatellites by examining the patterns of motif mismatches and allele sequences of single-nucleotide polymorphisms identified within microsatellite loci. Using Drosophila Reference Genetic Panel data, we further show that pattern of allelic variation modulates motif heterogeneity of microsatellites, and provide estimates of allele age of specific imperfect microsatellites found within protein-coding genes.     

Developing microsatellite markers from cDNA: a tool for adding expressed sequence tags to the genetic linkage map of the chicken

A chicken embryonic cDNA library was screened with a (TG)₁₃ probe in order to develop polymorphic microsatellite markers. The redundancy of the embryonic cDNA library with a chicken brain cDNA library, which was used for microsatellite development in a previous study, was extremely high. Of the 300 (TG)₁₃ positive clones, only 80 were unique for the embryonic cDNA library. Still, nine expressed sequences derived from the embryonic cDNA library were mapped in the Wageningen (WAU) resource population. In addition seven microsatellite markers from the chicken brain cDNA library, which were monomorphic or unlinked in the two international reference families in the previous study, were also mapped in the WAU population. Three of the 16 mapped chicken expressed sequence tags (ESTs) showed relatively high percentages of sequence similarity to sequences found in other species. As two of these genes, RAB6 and ZFX/ZFY, have been mapped in humans, they contribute to the comparative map of the chicken.     

用商品群作为参考系构建猪的微卫星连锁图谱

由 19头杂种公猪 [皮特兰× (皮特兰×汉普夏 ) ]、5 2头杂种母猪 [Leicoma× (大约克×长白 ) ]及其 332头后代组成的商品群作为参考系 ,选择 172个微卫星标记和 3个Ⅰ类标记 (RYR1、PIT1、PRKAG3)对参考系的个体进行遗传标记分型 ,应用CRIMAP(2 4 )构建猪的整个基因组微卫星连锁图谱。采用多重PCR方法对微卫星标记进行扩增 ,用ABI 377测序仪进行电泳分离 ,应用Genescan 3 0和Genotyper 2 0软件进行基因型检测。 3个Ⅰ类标记用PCR RFLP技术进行分型。CRIMAP程序分析表明 :所构建的猪常染色体性平均连锁图谱的总长度为 2 36 8 7cM ,X染色体的长度为 14 3 10cM ,遗传标记的平均间距为 16 3cM ,亲本的微卫星标记座位的杂合度平均为 0 70。此连锁图谱的构建将为商品猪群的生长、胴体、肉质以及繁殖性状的QTL扫描打下基础。     

赤拟谷盗全基因组和EST中微卫星的丰度

微卫星是近年大力开发的一种分子标记,为了推进赤拟谷盗Tribolium castaneum(Herbst)遗传学相关研究,对赤拟谷盗全基因组和EST中由1~6个碱基重复单元组成的简单序列重复进行分析,进而对其微卫星的丰度和分布进行比较分析。微卫星在赤拟谷盗EST中的分布频率为1/0.87kb,其中单碱基重复序列占71.25%,是最丰富的重复单元,而六、三、四、二,五碱基重复单元序列分别占23.93%,2.94%,1.56%,0.17%,0.15%。全基因组中微卫星的分布频率为1/3.65kb,其中六碱基重复序列占61.96%,是最丰富的重复单元,而三,四,一,五,二碱基重复单元序列分别占14.35%,13.75%,4.68%,3.60%,1.69%。同时发现富含A和T碱基的微卫星占主导地位,富含G和C碱基的微卫星数量较少。进一步的分析显示,微卫星在每条染色体上的丰度存在很大的相似性。     

Toward developing a genome-wide microsatellite marker set for linkage analysis in the rhesus macaque (Macaca mulatta): identification of 76 polymorphic markers

Linkage analysis can be problematic in humans because of the lack of large, multigenerational pedigrees and the difficulties in obtaining phenotypic data on all family members. In contrast, large, captive colonies of rhesus macaque are a potentially valuable resource for linkage studies because detailed phenotypic and genealogical data are kept, inbreeding is avoided, and DNA samples can usually be obtained. Microsatellite marker sets for genome-wide screening are available in a number of species, but not for the rhesus macaque. We tested primers to 400 human microsatellite markers from a genome-wide mapping set using DNA from nine unrelated female rhesus macaques. We found that 76 (19%) of the primers amplified a polymorphic product using the standard protocols for human DNA. The average heterozygosity of the markers in humans was 0.80, compared to 0.65 in the rhesus macaques. This study provides preliminary data, which could be used toward the development of a linkage mapping set in this species. There would be a need, however, to confirm the Mendelian inheritance of the markers.     

Fifteen new synteny assignments of microsatellites to the bovine genome

A panel of 36 hamster-bovine hybrid cell lines was used to assign 15 bovine microsatellites. Locus identification, synteny group and/or chromosome assignment and registration number were as follows: ILSTS001 (U22, Chr07, D7S13), ILSTS002 (U09, Chr18, D18S7), ILSTS004 (U10, Chr01, D1S28), ILSTS005 (U05, Chr10, D10S25). ILSTS006 (U22, Chr07, D7S8), ILSTS008 (U24, Chr14, D14S15), ILSTS010 (U27, DU27S11), ILSTS011 (U24, Chr14, D14S16), ILSTS012 (U16, Chr11, D11S26), ILSTS015 (U07, Chr25, D25S3), ILSTS016 (U04, Chr21, D21S22), ILSTS017 (X, DXS11), ILSTS018 (U15, Chr06, D6S15), ILSTS019 (U07, Chr25, D25S7) and ILSTS020 (U05, Chr10, D10S27). These results contribute to the international effort to improve the bovine genetic map.     

Extending the chicken-human comparative map by placing 15 genes on the chicken linkage map

To increase the number of type I loci on the chicken linkage map, chicken genes containing microsatellite sequences (TAn, CAn, GAn, An) were selected from the nucleotide sequence database and primers were developed to amplify the repeats. Initially, 40 different microsatellites located within genes were tested on a panel of animals from diverse breeds, and identified 17 polymorphic microsatellites. These polymorphisms allowed us to add 15 new genes to the chicken linkage map. In addition, two genes were added to the chicken map by fluorescent in situ hybridization. As the map position of the human homologues of 13 of these genes is known, these markers extend the comparative map between chicken and man. Our results confirm and refine conserved regions between chicken and man on chicken chromosomes 2 and 7 and on linkage group E29C09W09. Furthermore, an additional conserved region is identified on chromosome 7.