ISOLATION OF 105 MICROSATELLITE LOCI FROM AN OVINE GENOMIC LIBRARY ENRICHED FOR MICROSATELLITES

This paper reports on the development of a small-insert (～700 bp) total-genomic library for sheep specifically designed for enrichment for microsatellite (ms) loci. Four enriched libraries were prepared by amplification of the primary library with CA₁₅, CA₁₁, TG₁₅ and TG₁₁ oligonucleotide primers. A total of 11,020 clones was recovered, screened for dinucleotide repeats and over 500 positive clones sequenced. Sequence analysis indicated low clone redundancy and yielded 105 new ovine ms loci. Seventy-two percent of the new loci were found to be polymorphic in the sires of the AgResearch International Mapping Flock (IMF). The 105 new microsatellite loci increase the number of microsatellites available for sheep by >7%.     

High-resolution genetic map of bovine chromosome 29 through focused marker development

Chromosome-specific libraries aid in the development of genetic maps and focus marker development in areas of the genome with identified quantitative trait loci (QTL). A small-insert BTA29 library constructed by microdissection of a 1:29 Rb-fusion cell line, was screened for dinucleotide repeats (CA)(15) and/or (GA)(15) with the goal of generating new genetic markers for this, the smallest bovine autosome. A total of 90 primer pairs were designed and 82 of these successfully amplified bovine genomic DNA by PCR. In addition to these 82 loci, primer pairs were developed for nine putative genes identified from the sequenced clones by BLAST searches of GenBank. A somatic cell panel was used to test for synteny of the new loci with two previously mapped BTA29 markers located on the MARC bovine linkage map. A total of 75 of the 82 microsatellite (ms) loci were integrated into the MARC bovine linkage map. Linkage analysis placed 69 ms markers on BTA29, five on BTAX and one on BTA1. Combined results of the somatic cell and linkage analyses place 79 new markers (ms and gene-related) on BTA29, six loci on BTAX and two loci on BTA1. The results of this effort significantly increase the marker density on BTA29, expanding the ability to fine map QTL associated with this chromosome.     

Parentage testing and linkage analysis in the horse using a set of highly polymorphic microsatellites

Ten (TG)_n positive clones, isolated from an equine genomic library and sequenced, contained 12–19 uninterrupted TG repeats. Primers for polymerase chain reaction (PCR) were synthesized and nine of these (TG)_n loci (HTG7-15) were successfully amplified and utilized in this study together with five previously reported equine microsatellite loci (HTG2-6). The PCR products were analysed by polyacrylamide gel electrophoresis followed by automated laser fluorescence detection or autoradiography. All microsatellites showed polymorphism and stable Mendelian inheritance. Differences in microsatellite variability between horse breeds were detected. A linkage analysis comprising HTG2-15, one coat colour gene and 16 genetic blood markers enabled addition of HTG2 to linkage group U2 and a new linkage group (U6) was established comprising the loci HTG7 and HTG12. Close linkage was excluded within a set of eight microsatellites. The estimated probability of exclusion in four breeds for a parentage test based on these eight loci varied between 0.96 and 0.99.     

Structure and level of genetic diversity in various bean types evidenced with microsatellite markers isolated from a genomic enriched library

We have constructed a common bean genomic library enriched for microsatellite motifs (ATA), (CA), (CAC) and (GA). After screening, 60% of the clones selected from the library enriched for the (ATA) repeat contained microsatellites versus 21% of the clones from the library enriched for (GA) (CA) and (CAC) repeats. Fifteen primer pairs have been developed allowing for the amplification of SSR loci. We have evaluated the genetic diversity of these loci between 45 different bean lines belonging to nine various quality types. A total of 81 alleles were detected at the 15 microsatellite loci with an average of 5.3 alleles per locus. We have investigated the origin of allelic size polymorphism at the locus PvATA20 in which the number of repeats ranges from 24 to 85. We have related these large differences in repeat number to unequal crossing-over between repeated DNA regions. The diversity analysis revealed contrasted levels of variability according to the bean type. The lower level was evidenced for the very fine French bean, showing the effect of breeders intensive selection.     

Characterization of porcine polymorphic microsatellite loci

Twenty-seven (CA)_n and two (GA)_n microsatellite clones were isolated out of a size-selected genomic pig library. These were sequenced and the number of uninterrupted dinucleotides was found to range from 12 to 26. Flanking primers were chosen for 11 dinucleotide repeats and optimal conditions for polymerase chain reaction (PCR) amplifications were established. Different microsatellite loci were amplified simultaneously by combining primer sets. Related and unrelated pigs were screened for length polymorphisms of the different microsatellite loci. The polymorphic information content (PIC) of these loci ranged between 0.62 and 0.83. Segregation studies in pig reference families established Mendelian inheritance. Locus S0022 was found to be X-linked.     

Isolation and characterization of microsatellite loci from the rust pathogen,Melampsora lini

We developed and characterized primers for 11 variable microsatellite loci present in the genome of the flax rust, Melampsora lini. The microsatellite loci were identified by sequencing clones from a library of EcoRI DNA fragments enriched for four simple sequence repeat motifs (AAG, AAT, TC and TG). All 11 primer pairs successfully amplified DNA fragments from a sample of 102 M. lini isolates (98 isolated from Linum marginale and four from Linum usitatissimum), revealing a total of 32 alleles. Allelic diversity at the 11 loci ranged from 0.030 to 0.449.     

Isolation and characterization of 70 novel microsatellite markers from ostrich (Struthio camelus) genome.

Microsatellite markers are widely used in linkage mapping, parentage testing, population genetic studies, and molecular evolution studies in many agricultural species, while only a limited number of ostrich (Struthio camelus) microsatellites have been isolated. Thus, we constructed a random small-insert genomic library and a microsatellite-enriched library containing CA repeats. Fourteen clones containing CA repeats were isolated from 3462 clones in the non-enriched library by radioactive screening and 248 positive clones were isolated from 300 sequenced clones from the enriched library by PCR screening. After the enrichment procedures, the proportion of clones containing CA repeats was raised to 78.8%, compared with 0.4% in the non-enriched libraries, indicating that the enrichment value approaches 200 fold, which decreased the time and cost of cloning. The number of complete simple CA repeats in these positive clones ranged from 5 to 29. The primers for 94 of these microsatellites were developed and used to detect polymorphisms, of which 61 loci exhibited length polymorphisms in 17 unrelated ostrich individuals. The new polymorphic microsatellite markers we have identified and characterized will contribute to the ostrich genetic map, parentage testing, and comparative genomics between avian species.     

Development of 47 new microsatellite markers from a BTA6 library

Chromosome-specific libraries provide a means to isolate genetic markers from specific chromosomal regions. A small-insert BTA6 library, constructed by microdissection, was screened for dinucleotide repeats (CA)15 and (GA)15. A total of 47 new microsatellite loci were developed and tested for polymorphism and informativeness using the MARC bovine mapping family.     

Microsatellite DNA markers in the giant freshwater prawn, Macrobrachium rosenbergii: a tool for genetic analysis

Eight microsatellite loci were identified and characterized in the commercially important giant freshwater prawn, Macrobrachium rosenbergii. The microsatellite loci were detected by the random screening for dinucleotide repeat units within a partial genomic library developed for the species with biotinylated probes (CA)(15) , (AT)(15) and (GA)(15) . All the eight loci were found to be polymorphic. The number of alleles and observed heterozygosities per locus ranged between three to 16 and 0.22 to 0.71, respectively. These microsatellite markers will be useful for the conservation and management of wild and cultured stocks and population genetic studies of freshwater prawn.     

Characterization of microsatellite markers in peach [Prunus persica (L.) Batsch]

Microsatellites have emerged as an important system of molecular markers. We evaluated the potential of microsatellites for use in genetic studies of peach [Prunus persica (L.) Batsch]. Microsatellite loci in peach were identified by screening a pUC8 genomic library, a λZAPII leaf cDNA library, as well as through database searches. Primer sequences for the microsatellite loci were tested from the related Rosaceae species apple (Malus×domestica) and sour cherry (Prunus cerasus L.). The genomic library was screened for CT, CA and AGG repeats, while the cDNA library was screened for (CT)_n- and (CA)_n-containing clones. Estimates of microsatellite frequencies were determined from the genomic library screening, and indicate that CT repeats occur every 100 kb, CA repeats every 420 kb, and AGG repeats every 700 kb in the peach genome. Microsatellite- containing clones were sequenced, and specific PCR primers were designed to amplify the microsatellite- containing regions from genomic DNA. The level of microsatellite polymorphism was evaluated among 28 scion peach cultivars which displayed one to four alleles per primer pair. Five microsatellites were found to segregate in intraspecific peach-mapping crosses. In addition, these microsatellite markers were tested for their utility in cross-species amplification for use in comparative mapping both within the Rosaceae, and with the un- related species Arabidopsis thaliana L. Received: 18 June 1999 / Accepted: 6 December 1999     

文蛤微卫星DNA的筛选及其特性分析

采用磁珠富集分离法从文蛤(Meretrix meretrix)的基因组中筛选得到49条微卫星DNA序列,其中两碱基重复有3种类型36个序列,四碱基重复有14种类型26个序列.重复次数在5～30之间的序列占75.6%,30次以上重复的序列占24.4%,最高重复次数为100.根据重复单元的排列特点,完美型、非完美型及混合型序列所占比例分别为61.2%、14.5%和24.5%.本研究中构建的文蛤微卫星文库将在文蛤种质资源评价及分子遗传学研究中发挥作用.     

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 .     

中缅树鼩微卫星分子标记的筛选

目的 筛选中缅树鼩微卫星分子标记,逐步填补中缅树鼩特异性遗传标记的空白.方法 建立中缅树鼩基因小片段插入文库,利用5’端地高辛标记的(CA)15探针从约1500个菌落中选出36个阳性克隆.对这些克隆进行测序,发现其中15个含有重复序列,其中1个为重复克隆,1个因两端序列太短而不能设计引物.结果 用Primer3软件设计...     

Construction and characterization of a bovine BAC library with four genome-equivalent coverage

A bovine artificial chromosome (BAC) library of 105 984 clones has been constructed in the vector pBeloBAC11 and organized in 3-dimension pools and high density membranes for screening by PCR and hybridization. The average insert size, determined after analysis of 388 clones, was estimated at 120 kb corresponding to a four genome coverage. Given the fact that a male was used to construct the library, the probability of finding any given autosomal and X or Y locus is respectively 0.98 and 0.86. The library was screened for 164 microsatellite markers and an average of 3.9 superpools was positive for each PCR system. None of the 50 or so BAC clones analysed by FISH was chimeric. This BAC library increases the international genome coverage for cattle to around 28 genome equivalents and extends the coverage of the ruminant genomes available at the Inra resource center to 15 genome equivalents.     

Construction of a cytogenetically anchored microsatellite map in rabbit

Rabbit (Oryctolagus cuniculus) represents a valuable source of biomedical models and corresponds to a small but active economic sector in Europe for meat and fur. The rabbit genome has not been thoroughly studied until recently, and high-resolution maps necessary for identification of genes and quantitative trait loci (QTL) are not yet available. Our aim was to isolate over 300 new and regularly distributed (TG)n or (TC)n rabbit microsatellites. To achieve this purpose, 164 microsatellite sequences were isolated from gene-containing bacterial artificial chromosome (BAC) clones previously localized by fluorescence in situ hybridization (FISH) on all the rabbit chromosomes. In addition, 141 microsatellite sequences were subcloned from a plasmid genomic library, and for 41 of these sequences, BAC clones were identified and FISH-mapped. TC repeats were present in 62% of the microsatellites derived from gene-containing BAC clones and in 22% of those from the plasmid genomic library, with an average of 42.9% irrespective of the microsatellite origin. These results suggest a higher proportion of (TC)n repeats and a nonhomogeneous distribution of (TG)n and (TC)n repeats in the rabbit genome compared to those in man. Among the 305 isolated microsatellites, 177 were assigned to 139 different cytogenetic positions on all the chromosomes except rabbit Chromosome 21. Sequence similarity searches provided hit locations on the Human Build 35a and hypothetical assignments on rabbit chromosomes for ten additional microsatellites. Taken together, these results report a reservoir of 305 new rabbit microsatellites of which 60% have a cytogenetic position. This is the first step toward the construction of an integrated cytogenetic and genetic map based on microsatellites homogeneously anchored to the rabbit genome.     

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...     

Microsatellite loci in the phytoparasitic nematode Globodera.

A Globodera pallida genomic library, population Guiclan (Pa2/3), was screened for TG and TC microsatellite motifs. Screening of 50,000 clones revealed 48 positive matches. After sequencing, primers were designed to amplify 14 microsatellite loci. The specificity of the loci was tested with DNA templates of other populations of G. pallida, and also on other species of Globodera. Appearance of amplification products on several of these DNA templates showed that the microsatellite flanking regions are relatively conserved between G. pallida populations as well as between Globodera species. Evidence for allele polymorphism between individuals was demonstrated by using nine loci primers, in G. pallida population Guiclan and from a population of a closely related species G. "mexicana". Some alleles appeared to be species specific.     

Microsatellites in the silkworm, Bombyx mori: abundance, polymorphism, and strain characterization.

We have isolated and characterized microsatellites (simple sequence repeat (SSR) loci) from the silkworm genome. The screening of a partial genomic library by the conventional hybridization method led to the isolation of 28 microsatellites harbouring clones. The abundance of (CA)n repeats in the silkworm genome was akin to those reported in the other organisms such as honey bee, pig, and human, but the (CT)n repeat motif is less common compared to bumble bee and honey bee genomes. Detailed analysis of 13 diverse silkworm strains with a representative of 15 microsatellite loci revealed a number of alleles ranging from 3 to 17 with heterozygosity values of 0.66-0.90. Along with strain-specific microsatellite markers, diapause and non-diapause strain-specific alleles were also identified. The repeat length did not show any relationship with the degree of polymorphism in the present study. The co-dominant inheritance of microsatellite markers was demonstrated in F1 offspring. A list of primer sequences that tag each locus is provided. The availability of microsatellite markers can be expected to enhance the power and resolution of genome analysis in silkworm.     

Developing microsatellites when they are rare: trinucleotide repeat loci in the northern mockingbird Mimus polyglottos

The great value of microsatellite loci to population studies spurs their development. However, finding loci is difficult when microsatellites are uncommon in the genome. Because trinucleotide-repeat loci are rare in northern mockingbirds Mimus polyglottos, we sought a new method for developing a suite of loci. Here we show that a bacteriophage cloning vector, Lambda Zap Express (Stratagene, La Jolla, CA, USA) has several features which make it suitable for this purpose. Using this vector, we made a library of 150 000 size-selected clones and screened with an AAT₁₀ probe; 97 positives were identified. From these, 12 pairs of PCR primers were developed, nine of which amplify polymorphic loci. Certain combinations of these primer pairs enable simultaneous amplification of up to three loci.