Isolation and mapping of polymorphic microsatellites in cattle

Summary
A partial plasmid library with bovine genomic inserts of about 500 basepairs was screened with a (dC-dA)_n-(dG-dT)_n oligonucleotide probe for the repeated nucleotide motif (CA)_n. Eleven positive clones (0.3% of all colonies screened) were discovered and were subsequently isolated and sequenced. Eight microsatellite loci were analysed, one with eight alleles, one with seven alleles, three with six alleles, one with three alleles and two with two alleles. Six of these microsatellites were mapped by PCR-analysis of a panel of somatic hybrid lines.     

Pig microsatellites isolated from cosmids revealing polymorphism and localized on chromosomes

One of the most widely studied simple sequences in the mammalian genome is the (TG)_n dinucleotide sequence. Because these microsatellites are highly polymorphic, we chose to study microsatellites from cosmids to provide genetic markers for the porcine genome. After screening a porcine cosmid library with a (CA)₁₀ probe, 20 cosmids containing microsatellites were subcloned and 17 microsatellites identified by sequencing. Oligonucleotide primers flanking the repeat were designed for seven (TG)_n microsatellites with n > 14. These seven microsatellites revealed polymorphism and were regionally assigned to chromosomes by fluorescent in situ hybridization of initial cosmids. These seven loci will be useful for both the construction of the genetic map and as landmark loci on the physical map of the porcine genome.     

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.     

Characterization and linkage mapping often sheep microsatellite markers derived from a sheep x hamster cell hybrid

A cosmid library has been constructed from a sheep x hamster cell hybrid containing sheep chromosome t₁, rob (6;24). Clones containing sheep DNA were identified by hybridizing to a total sheep genomic DNA probe. Small fragments (<500 bp) containing (AC)_n microsatellites were subcloned and sequenced. Ten microsatellite markers were characterized and six were mapped back to chromosomes 6 and 24. The remaining microsatellites mapped to chromosome 26, which was shown to be present in a small proportion of cells of the cell line.     

Development and mapping of ten porcine microsatellite markers

Thirty (TG)_n microsatellite clones were isolated from a pig genomic library, sequenced, and tested for their suitability to detect polymorphism on a panel of animals by means of the polymerase chain reaction. Ten of these clones were developed into suitable markers and subsequently segregation of these markers was determined in the five PiGMaP reference pedigrees. A linkage analysis was performed on these 10 microsatellites together with 365 other loci that have been typed on these reference families. Eight of the microsatellites have been mapped to eight different linkage groups that have been previously assigned to different chromosomes (chromosomes 1, 6, 7, 9, 14, 15, 17 and 18). Of the remaining two markers, one is X-linked and the other shows no linkage. The number of alleles detected by these microsatellites, in the reference pedigrees, varied from six to sixteen and the heterozygosity varied from 42 to 85% in the 26 unrelated founder animals of these reference pedigrees.     

A small-insert bovine genomic library highly enriched for microsatellite repeat sequences

A bovine genomic phagemid library was constructed with randomly sheared DNA. Enrichment of this single-stranded DNA library with CA or GT primers resulted in 45% positive clones. The 14% of positive clones with (CA · GT)>12, and not containing flanking repetitive elements, were sequenced, and the efficiency of marker production was compared with random M13 bacteriophage libraries. Primer sequences and genotyping information are presented for 390 informative bovine microsatellite markers. The genomic frequency for 11 tri- and tetranucleotide repeats was estimated by hybridization to a lambda genomic library. Only GCT, GGT, and GGAT were estimated to have a frequency of >100 per genome. Enrichment of the phagemid library for these repeats failed to provide a viable source of microsatellite markers in the bovine. Comparison of map interval lengths between 100 markers from the enriched library prepared from randomly sheared DNA and M13 bacteriophage libraries prepared from Mbo1 restriction digests suggested no bias in skeletal genomic coverage based on source of small insert DNA. In conclusion, enrichment of the bovine phagemid library provides a sufficient source of microsatellites so that small repeat lengths and flanking repetitive sequences common in the bovine can be eliminated, resulting in a high percentage of informative markers.The nucleotide sequence data reported in this paper have been submitted to GenBank and have been assigned the accession numbers U25689 and U25690.     

Characterization of 42 highly polymorphic bovine microsatellite markers

We have isolated 42 highly polymorphic microsatellite (GT/CA)_n markers from Japanese black cattle Wagyu ( Bos taurus ). Forty-one of the markers were assigned to bovine autosomes with lod scores > 6, through linkage analyses performed on the International Bovine Reference Family Panel (IBRP). The remaining marker showed X-linked inheritance. These markers exhibited an average heterozygosity value of 0.67 with between four and 17 alleles on the IBRP.     

Turkey microsatellite DNA loci amplified by chicken-specific primers

Forty-eight primer-pairs complementary to unique DNA sequences flanking chicken (genus Gallus ) genomic (TG)_n microsatellite repeats were previously designed. These primer-pairs were used in the polymerase chain reaction to amplify turkey (genus Meleagris ) genomic DNA loci. Results indicated that the majority (92%) of these primer-pairs generated amplification products in turkey genomic DNA. Hybridization using end-labelled (TG)₈ as a probe showed that, out of 41 primer-pairs tested, only 14 generated an amplification product that also contained a detectable (TG)_n microsatellite repeat when turkey DNA was the template. Among 18 primerpairs tested for polymorphism, using three commercial turkey lines, five were found to exhibit length polymorphism, three of which did not contain a detectable TG repeat. Therefore, a significant portion of chicken microsatellite markers can be useful for genomic mapping and linkage analysis in the turkey, reducing the costs involved in producing turkey-specific microsatellite markers.     

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     

Isolation and Enrichment of Abundant Microsatellites from a Channel Catfish (Ictalurus punctatus) Brain cDNA Library

Efforts to construct a genetic linkage map of channel catfish have involved identification of random genomic microsatellite markers, as well as anchored Type I loci (expressed genes) from channel catfish. To identify Type I markers we constructed a directional cDNA library from brain tissue to obtain expressed catfish sequences that could be used for single nucleotide polymorphism (SNP) marker development. These cDNA sequences surprisingly contained a high proportion of microsatellites (about 14%) in noncoding regions of expressed sequence tags (ESTs), many of which were not associated with known sequences. To further identify cDNAs with microsatellites and reduce the number of sequencing reactions needed for marker development, we enriched this library for repeat sequences and sequenced clones from both directions. A total of 1644 clones from seven repeat-enriched captures (CA, GT, CT, GA, MTT, TAG, and TAC) were sequenced from both ends, and 795 nonredundant clones were assembled. Thirty-seven percent of the clones contained microsatellites in the trimmed sequence. After assembly in the TIGR Catfish Gene Index (CfGI), 154 contigs matched known vertebrate genes and 92 contigs contained microsatellites. When BLAST-matched orthologues were available for similarity alignments, 28% of these contigs contained repeats in the 5'-UTR, 72% contained repeats in the 3'-UTR, and 8% contained repeats at both ends. Using biotinylated repeat oligonucleotides coupled with streptavidin-coated magnetic beads, and rapid, single-pass hybridization, we were able to enrich our plasmid library greater than two-fold for repeat sequences and increase the ability to link these ESTs with known sequences greater than six-fold.     

Isolation and enrichment of abundant microsatellites from a channel catfish (Ictalurus punctatus) brain cDNA library

Efforts to construct a genetic linkage map of channel catfish have involved identification of random genomic microsatellite markers, as well as anchored Type I loci (expressed genes) from channel catfish. To identify Type I markers we constructed a directional cDNA library from brain tissue to obtain expressed catfish sequences that could be used for single nucleotide polymorphism (SNP) marker development. These cDNA sequences surprisingly contained a high proportion of microsatellites (about 14%) in noncoding regions of expressed sequence tags (ESTs), many of which were not associated with known sequences. To further identify cDNAs with microsatellites and reduce the number of sequencing reactions needed for marker development, we enriched this library for repeat sequences and sequenced clones from both directions. A total of 1644 clones from seven repeat-enriched captures (CA, GT, CT, GA, MTT, TAG, and TAC) were sequenced from both ends, and 795 nonredundant clones were assembled. Thirty-seven percent of the clones contained microsatellites in the trimmed sequence. After assembly in the TIGR Catfish Gene Index (CfGI), 154 contigs matched known vertebrate genes and 92 contigs contained microsatellites. When BLAST-matched orthologues were available for similarity alignments, 28% of these contigs contained repeats in the 5'-UTR, 72% contained repeats in the 3'-UTR, and 8% contained repeats at both ends. Using biotinylated repeat oligonucleotides coupled with streptavidin-coated magnetic beads, and rapid; single-pass hybridization, we were able to enrich our plasmid library greater than two-fold for repeat sequences and increase the ability to link these ESTs with known sequences greater than six-fold.     

Isolation of 105 microsatellite loci from an ovine genomic library enriched for microsatellites

This paper reports on the development of a small-insert (approximately 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%.     

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.     

Transfer of bovine microsatellites to the cervine (Cervus elaphus)

Bovine microsatellites were used to amplify DNA of red deer ( Cervus elaphus ). Fourteen of 27 bovine systems (52%) displayed polymorphism, while no (CA)_n-repeat was detected in seven systems and six systems gave no amplificates in red deer. The allele number ranged from 2 to 7, the polymorphism information content between 0.24 and 0.76. The results demonstrate that transfer of microsatellite systems between families of the same order (artiodactyla) is possible. Molecular genetic research will help to clarify the differentiation and ecology of wild animals and will contribute to define criteria needed for the preservation of endangered species.     

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.     

Rapid isolation of CA microsatellites from the tilapia genome

We have developed (CA)n microsatellite markers for the cichlid fish, Oreochromis niloticus using a variation of the hybrid capture method. The resulting genomic library was highly enriched in repetitive DNA with 96% of clones containing CA repeats. The number of repeats ranged from four to 45 with an average of 19. Two-thirds of the sequenced clones had 12 or more repeats and sufficient flanking sequence to design primers. The resulting markers were tested in an F2 cross of O. niloticus x O. aureus. Nearly 90% of the markers amplified in this cross and 74% of these were informative. This work demonstrates the importance of minimizing the number of polymerase chain reaction (PCR) amplification cycles before and after the enrichment steps to reduce PCR recombination and the generation of chimaeric clones.     

Polymorphic CA/GT and GA/CT microsatellite loci for Ostrinia nubilalis (Lepidoptera: Crambidae)

Ten polymorphic dinucleotide (CA/GT and GA/CT) microsatellite loci suitable for population genetic screening were characterized from enriched partial Ostrinia nubilalis genomic libraries. Sequence from 126 enriched small insert genomic library clones identified 25 CA/GT and 58 GA/CT loci that were unique. Perfect repeats tended to be short (n = 10–12). Ten microsatellites, PCR amplified from a Crawfordsville Iowa population showed a mean of 10 alleles per locus (range six to 20), and six of 10 loci showed heterozygote deficiency. Amplification of eight loci was observed in the sister species O. furnicalis.     

Microsatellite repeats are not randomly distributed within Norway spruce (Picea abies K.) expressed sequences.

A Norway spruce (Picea abies K.) cDNA library obtained from vegetative bud tissue was screened for the presence of (AG)n and (AC)n microsatellite repeats. Ten (AG)n and six (AC)n microsatellites were found, with an average length of 25.5 repeat units. Most of the microsatellites are simple perfect repeats. The microsatellite distribution within the clones is clearly non-random, with different classes of repeats lying in different positions relative to the coding region and in a highly conserved orientation. An estimate of the frequency of dinucleotide microsatellites in expressed regions was obtained, showing that SSRs (simple sequence repeats) are found in genes about 20 times less frequently than in random genomic clones, with (AG)n repeats more frequent than (AC)n repeats. Potential applications of these sequences as expressed region-based molecular markers are shown by developing six SSR markers for the detection of natural variation in Norway spruce populations and testing two of them for the identification of illegitimate progenies from a mapping population.     

Fifty microsatellite markers for Japanese quail

A Japanese quail genomic library enriched for (CA/GT)n simple sequence repeats was screened and positive clones were sequenced. Fifty original microsatellite sequences were isolated that consisted mainly of perfect repeats of the dinucleotide (CA/GT)n motif and a corresponding number of polymerase chain reaction (PCR) primer pairs complementary to unique DNA sequences flanking the microsatellite repeats were designed to detect the repeats. Forty-six percent (23 of 50) of the markers revealed polymorphism in two unrelated quail individuals (one male and one female) randomly sampled from a population of wild quail origin. All 50 primer pairs were tested in the PCR for their ability to amplify chicken genomic DNA. Amplification products were obtained for 14 (28.0%) of the markers at the annealing temperature optimized for quail. These results provide an opportunity to begin characterizing the quail genome for the development of a genetic map for this economically valuable species and the eventual construction of a comparative genetic map in Phasianidae, which comprises a number of agriculturally important species of poultry.     

Microsatellite markers isolated from the wild medicinal plant Centella asiatica (Apiaceae) from an enriched genomic library

? Premise of the study: Microsatellite markers for Centella asiatica, an important medicinal herb, were developed and characterized to promote genetic and molecular studies. ? Methods and Results: A GA/GT-enriched genomic library was constructed from an accession from Madagascar. Roughly 75% of the 768 clones of the enriched library contained microsatellites. Eighty sequences containing microsatellites were obtained from 96 positive clones. Specific primers were designed for 20 loci, and 17 of them displayed polymorphism when screened across 17 C. asiatica accessions, with an average of 4.3 alleles per locus. The observed and expected heterozygosity values averaged 0.114 and 0.379, respectively. ? Conclusions: This is the first report constructing an enriched genomic library and identifying microsatellite markers from C. asiatica. These 17 polymorphic microsatellite markers are a useful resource for this plant, applicable for diversity studies, pedigree analyses, and genetic mapping.