Genetic diversity in Spanish donkey breeds using microsatellite DNA markers

Genetic diversity at 13 equine microsatellite loci was compared in five endangered Spanish donkey breeds: Andaluza, Catalana, Mallorquina, Encartaciones and Zamorano-Leonesa. All of the equine microsatellites used in this study were amplified and were polymorphic in the domestic donkey breeds with the exception of HMS1, which was monomorphic, and ASB2, which failed to amplify. Allele number, frequency distributions and mean heterozygosities were very similar among the Spanish donkey breeds. The unbiased expected heterozygosity (H_E) over all the populations varied between 0.637 and 0.684 in this study. The low G_ST value showed that only 3.6% of the diversity was between breeds (P < 0.01). Significant deviations from Hardy-Weinberg equilibrium were shown for a number of locus-population combinations, except HMS5 that showed agreement in all analysed populations. The cumulative exclusion probability (PE) was 0.999 in each breed, suggesting that the loci would be suitable for donkey parentage testing. The constructed dendrogram from the D_A distance matrix showed little differentiation between Spanish breeds, but great differentiation between them and the Moroccan ass and also with the horse, used as an outgroup. These results confirm the potential use of equine microsatellite loci as a tool for genetic studies in domestic donkey populations, which could also be useful for conservation plans.     

Linkage of the gene for equine combined immunodeficiency disease to microsatellite markers HTG8 and HTG4; synteny and FISH mapping to ECA9

Equine combined immunodeficiency disease (CID) is caused by homozygosity for an autosomal recessive gene. To identify linked markers for the disease, we studied a family segregating for the equine CID gene. A stallion and 19 of his CID-affected offspring were tested for marker segregation at 23 microsatellite DNA loci. His CID-affected offspring inherited only one of his two alleles at the HTG8 and HTG4 loci, namely HTG8–186 and HTG4–124 , respectively. Lod scores for linkage to the CID gene using a Θ of 0·01were 5·34 for HTG8 and 2·37 for HTG4. The apparent genotypes also suggested linkage disequilibrium between the HTG8–186 allele and the gene for CID. The gene for the DNA protein kinase catalytic subunit ( DNA-PK ) was recently suggested as a candidate gene for equine CID. A defect of this gene causes a disease in mice that is similar to equine CID. Therefore, we investigated whether this gene might be associated with the microsatellite markers. Analysis of a somatic cell hybrid panel demonstrated synteny of DNA-PK with HTG4 and HTG8 (Kentucky Synteny Group 3). Fluorescence in situ hybridization (FISH) studies demonstrated that DNA-PK is located on horse chromosome ECA9p12. This work supports the hypothesis of DNA-PK as the probable cause of equine CID.     

Microsatellite DNA markers for rice chromosomes

We found 369 complete microsatellites, of which (CGG/GCC)_n was the most frequent, in 11 798 rice sequences in the database. Of these microsatellites, 35 out of 45 could be successfully converted into microsatellite DNA markers using sequence information in their flanking regions. Thus, the time and labor used to develop new microsatellite DNA markers could be saved by using these published sequences. Twenty eight polymorphic markers between Asominori (japonica) and IR24 (indica) have been correctly mapped on the rice genome and microsatellites appear to be randomly distributed in the rice chromosomes. Integration of these markers with the published microsatellite DNA markers showed that about 35% of the rice chromosomes were covered by the 56 microsatellite DNA markers. These microsatellites were hypervariable and were easily to assay by PCR; they were distributed to all chromosomes and therefore, one can easily select plants carrying desired chromosome regions using these microsatellite DNA markers. Thus, microsatellite maps should aid the development of new breeds of rice saving time, labor, and money.     

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     

Isolation of thirty‐one new porcine microsatellites from a microsatellite enriched microdissected chromosome 8 library

Abstract

Chromosome 8 (SSC8) is an important one in the swine genome because it has been shown to harbor several economically important quantitative trait loci (QTL). The entire porcine chromosome 8 was microdissected and amplified by degenerate oligonucleotide primer (DOP) PCR. The PCR product was then enriched for (CA)_n microsatellites by hybridization to a biotinylated CA repeat oligonucleotide and captured by streptavidin‐coated magnetic beads. The captured DNA was cloned into a TA cloning vector. Screening with an isotopically labeled CA oligonucleotide probe resulted in the isolation of 31 informative and polymorphic microsatellite clones. Seventeen of those were mapped to chromosome 8, 12 to chromosome 15, 1 to chromosome 10 and 1 to chromosome X. The markers were all placed on the USDA‐MARC porcine genetic linkage map.     

Cloning of highly polymorphic microsatellites in the horse

We have isolated equine microsatellites by screening a genomic library with (TG)n and (TC)n probes. TG microsatellites were found to be more abundant than TC repeats, with an estimated frequency of one per 100,000bp. Sequence analysis of eight TG-positive clones revealed varying structures of the repeat regions; perfect stretches of TG repeats, imperfect stretches of TG repeats and compound regions of TG and TC repeats. Five loci were analysed by PCR and showed extensive polymorphism; three to seven alleles and heterozygosities of 0.40-0.76 were observed when screening 20-30 unrelated individuals. The high degree of polymorphism, their abundance and the possibility of automating the typing procedure make these loci ideal for standardized paternity testing in the horse. Furthermore, we demonstrate that single hairs can be used as starting material for the PCR analysis.     

Genetic variations in horse using microsatellite markers

Genetic variations has been analyzed using five microsatellite markers (AHT4, HTG10, ABS2, ABS23 and CA245) in three horse breeds in Egypt (Arabian, Thoroughbred and Egyptian Native). All the microsatellites typed in this study can be considered informative they produced a number of alleles ranged from eight alleles for the microsatellites ABS23, CA245 to 13 alleles for the microsatellite HTG10. The most polymorphic microsatellite was HTG10. The values of He for the five microsatellite studied were: 0.754, 0.829 and 0.807 for the breeds Arabian, Thoroughbred and Egyptian Native, respectively. The highest He value for all markers was detected in Thoroughbred breed, then The Egyptian Native and lastly in The Arabian breed. The mean values of PIC which obtained from the present study ranged from 0.686 to 0.764. Fst value may indicate the presence of gene flow between horse breeds. The values of genetic distances and phylogeny tree proved that Arabian and Native horses are coming from one ancestor while the Thoroughbred is coming from another ancestor. The values obtained for allele diversity, heterozygosity, inbreeding measurements and gene diversity showed that horse breeds understudy, moreover the present study results points to the usefulness of evaluations of diversity using molecular markers for the choice of breeds worthy of conservation.     

Characterization of equine microsatellites and microsatellite-linked repetitive elements (eMLREs) by efficient cloning and genotyping methods.

We performed efficient cloning and genotyping methods for isolation of a large number of polymorphic microsatellites. The methods contain the time-efficient cloning method of constructing microsatellite-enriched libraries and the economic genotyping method of fluorescent labeling of PCR products. Eighty novel equine microsatellites cloned were efficiently isolated from the enrichment library and analyzed for genotype polymorphism. Of these, 72 microsatellites were analyzed with a good resolution. The average heterozygosity of all loci was 0.52, and the number of alleles ranged from one to 9 with an average of 4.5 alleles. The other eight loci showed multiple bands of PCR products, suggesting the occurrence of microsatellites in a repetitive element, in which the number of microsatellite repeats varies among different members of the repetitive element. We found five homologous groups at flanking regions in comparison with the flanking regions of microsatellites from DNA databases. One of them showed homology to equine repetitive element-2. In the other four homologous groups, the two groups were named equine microsatellite-linked repetitive element-1 (eMLRE-1) and equine microsatellite-linked repetitive element-2 (eMLRE-2) as novel equine repetitive elements identified from equine genome. These data should help the analysis of equine DNA sequences and the design of equine genome markers.     

Instability of (GATA)<Subscript> n</Subscript> microsatellite loci in the parthenogenetic Caucasian rock lizard<Emphasis Type="Italic"> Darevskia unisexualis</Emphasis> (Lacertidae)

Mini- and microsatellites, comprising tandemly repeated short nucleotide sequences, are abundant dispersed repetitive elements that are ubiquitous in eukaryotic genomes. In humans and other bisexual species hypervariable mini- and microsatellite loci provide highly informative systems for monitoring of germline and somatic instability. However, little is known about the mechanisms by which these loci mutate in species that lack effective genetic recombination. Here, multilocus DNA fingerprinting was used to study M13 minisatellite and (GATA) _n microsatellite instability in the parthenogenetic Caucasian rock lizard Darevskia unisexualis (Lacertidae). DNA fingerprinting of 25 parthenogenetic families, from six isolated populations in Armenia (comprising a total of 84 siblings), using the oligonucleotide (GATA)₄ as a hybridization probe, revealed mutant fingerprinting phenotypes in 13 siblings that differed from their mothers in several restriction DNA fragments. In three families (8 siblings), the mutations were present in the germline. Moreover, the mutant fingerprint phenotypes detected in siblings were also present in population DNA samples. No intrafamily variations in DNA fingerprint patterns were observed with the M13 minisatellite probe. Estimates of the mutation rate for (GATA) _n microsatellite loci in D. unisexualis showed that it was as high as that seen in some bisexual species, reaching 15% per sibling or 0.95% per microsatellite band. Furthermore, in one case, a somatic (GATA) _n microsatellite mutation was observed in an adult lizard. These findings directly demonstrate that mutations in (GATA) _n microsatellite loci comprise an important source of genetic variation in parthenogenetic populations of D. unisexualis.Communicated by G. P. Georgiev     

Strong association between polymorphisms in an intronic microsatellite and in the coding sequence of the BoLA-DKB3 gene: implications for micro-satellite stability and PCR-based DRB3 typing

A highly polymorphic microsatellite in the bovine DRB3 gene was characterized by polymerase chain reaction (PCR) analysis and DNA sequencing. A very strong association between expressed DRB3 polymorphism and microsatellite alleles was revealed by PCR analysis of genomic DNA from 116 animals representing three breeds of cattle. The results indicated a low frequency of microsatellite length mutations as the association was consistent over breeds. The DRB3 microsatellite may be utilized in a PCR-based typing method of bovine class II alleles. The microsatellite polymorphism did not distinguish all known DRB3 alleles, but it was shown that this method may be complemented by the use of allele-specific PCR based on the extensive polymorphism in the DRB3 exon 2. The DNA sequences of seven microsatellite alleles, associated with different class II hap-lotypes, were determined. The DRB3 microsatellite is composed of three repeat motifs, a stretch of at least 10 uninterrupted (TG)_n dinucleotides, a long but interrupted stretch of (GA)_n dinucleotides, and a few (CAGA)_n tetranucleotides. There were pronounced sequence differences beween alleles and the results indicated that the evolution of this microsatellite has involved length mutations of the dinucleotide repeats as well as point mutations causing interruptions in the dinucleotide repeats.     

Isolation and characterization of polymorphic microsatellite loci from bluefin leatherjacket (Navodon septentrionalis Gunther, 1877)

The first set of 18 polymorphic microsatellite markers were developed from bluefin leatherjacket (Navodon septentrionalis Gunther, 1877). From a (GT)_n-enriched genomic library, we got 121 microsatellites, of which 60 were randomly selected for designing microsatellite primers. Eighteen of these loci were polymorphic in a test population of 32 individuals with alleles ranging from 2 to 9, and expected and observed heterozygosities from 0.1463 to 0.8517 and from 0.1562 to 1.0000, respectively. No significant linkage disequilibrium between pairs of loci was found, but three loci significantly deviated from Hardy–Weinberg equilibrium after Bonferroni correction. These polymorphic microsatellite loci should provide sufficient level of genetic diversity to investigate population structure in bluefin leatherjacket.     

Phylogenetic distribution and genetic mapping of a (GGC)n microsatellite from rice (Oryza sativa L.)

DNA microsatellites are ubiquitously present in eukaryotic genomes [30] and represent a vast source of highly informative markers [30, 33, 34, 2]. We describe in this article a (GGC)_n microsatellite which is widely distributed in eukaryotic genomes. Using polymerase chain reaction (PCR) techniques and DNA sequencing, we demonstrated for the first time in plant species that a (GGC)_n microsatellite locus is moderately polymorphic. Six alleles are present at this locus in rice and length polymorphisms are caused by variation in the number of tandem GGC repeats. By scoring a backcross mapping population, we were able to demonstrate that this locus is stably inherited and does not link to any known RFLP markers on the rice RFLP map. Our results suggest that DNA microsatellites should be useful in plants for construction of genetic linkage maps, extension of the existing genetic linkage maps, linkage analysis of disease and pest resistance genes, and the study of population genetics.     

Characterization of 24 porcine (dA-dC)n-(dT-dG)n microsatellites: genotyping of unrelated animals from four breeds and linkage studies

Twenty-four PCR primer pairs were designed for the detection of porcine microsatellites. Polymorphism was investigated in 76 unrelated animals from four different breeds: Duroc, Landrace, Hampshire, and Yorkshire. Compared with human microsatellites, a general lower heterozygosity was detected; however, for each microsatellite a significant variation between breeds in number of alleles and heterozygosity was seen. Mean heterozygosity was found to be significantly higher (P<0.01%) in the Yorkshire breed than in the other three breeds. Linkage analyses with the CEPH linkage packet were performed in a backcross family comprising 45 animals, of which 43 had informative meioses. Ten of the microsatellites could be assigned to six different linkage groups, demonstrating that linkage mapping with microsatellites can be carried out with great efficiency in a relatively small number of animals. Four of the linkage groups represent Chromosomes (Chrs) 4, 6, 7, and 8 respectively, while two linkage groups are unassigned.The nucleotide sequence data reported in this paper have been submitted to GenBank and have been assigned the accession numbers listed in Table 1.     

Characterization and analysis of microsatellite loci in Elymus caninus (Triticeae: Poaceae)

 Microsatellites are highly variable DNA sequences that can be used as markers for the genetic analysis of plants. The potential of microsatellite markers for use in a genetic diversity study in Elymus species was evaluated. Genomic libraries of Elymus caninus were constructed. The libraries were screened with two dinucleotide, (GA)n and (GT)n, and two trinucleotide repeats, (TCT)n and (CAC)n. A total of 19 positive clones were found for the two dinucleotide repeats; no positive clone was found for the trinucleotide repeats. Positive clones were sequenced to confirm the presence of microsatellites and to generate polymerase chain reaction (PCR) primers based on the sequences flanking the microsatellite. All sequenced (GA)n clones have repeats of n>10; over half of the (GT)n microsatellites have n<10 repeats. Primer pairs were designed and evaluated for 8 selected microsatellites. PCR products were amplified from 15 Elymus caninus accessions. The number of alleles found for the eight loci varied from 1 for ECGA89 and ECGT35 to 13 for ECGA22, as determined by non-denaturing polyacrylamide electrophoresis. Six microsatellite loci were found to be polymorphic in E. caninus. The eight primer pairs were tested on three other species; seven were successful in amplifying DNA from Elymus alaskanus and E. mutabilis, and four amplified DNA from E. caucasicus. Based on these results, microsatellites appear to be useful markers in detecting variation in E. caninus. Received: 8 September 1997/Accepted: 6 October 1997     

Polymerase chain reaction primers for polymorphic microsatellite loci from the túngara frog Physalaemus pustulosus

We developed eight PCR?primer pairs of polymorphic microsatellite loci for the túngara frog Physalaemus pustulosus. Genomic libraries were enriched for one of four microsatellite repeat sequences (CA_n, GA_n, ATG_n and TAGA_n). Following characterization of microsatellite loci by sequencing, primers were designed and PCR conditions optimized. Microsatellite PCR‐amplification was tested in 37 frogs from 8 populations in Costa Rica and Panama. Primer sequences, PCR conditions, allelelic diversities and observed as well as expected heterozygosities in the screened populations are described.     

Isolation of microsatellite loci in green abalone (Haliotis fulgens) and cross‐species amplification in two other North American red (Haliotis rufescens) and pink (Haliotis corrugata) abalones

Microsatellite loci were isolated in Haliotis fulgens using a (CT)_n enriched‐genomic library. From 33 sequenced clones, 21 microsatellites regions were identified, 15 with the expected (CT)_n. Eight microsatellite loci were amplified, six of which were polymorphic with a range of three to 20 alleles, and five cross‐amplified in two other species (Haliotis rufescens and Haliotis corrugata). These microsatellites will be useful as population genetic markers in the three species.     

Polymorphic microsatellite loci isolated from the marine sponge Scopalina lophyropoda (Demospongiae: Halichondrida)

A total of seven microsatellites out of 88 isolated from a genomic library enriched for (CA)_n and (GA)_n repeats were characterized in the Mediterranean marine sponge Scopalina lophyropoda. The microsatellite motifs were large (34.81 ± 13.9 bp) and imperfect. The seven microsatellite loci were screened in 30 individuals collected from Blanes, northwestern Mediterranean. All of them were polymorphic (allele numbers and observed heterozygosities ranged from 3 to 6 and from 0.16 to 0.76, respectively). No significant linkage disequilibrium between pairs of loci and no departure from Hardy–Weinberg equilibrium were found. These markers are therefore promising for studies of the population structure of the species.     

Abundance, variability and chromosomal location of microsatellites in wheat

The potential of microsatellite sequences as genetic markers in hexaploid wheat (Triticum aestivum) was investigated with respect to their abundance, variability, chromosomal location and usefulness in related species. By screening a lambda phage library, the total number of (GA)_n blocks was estimated to be 3.6 x 10⁴ and the number of (GT)_n blocks to be 2.3 x 10⁴ per haploid wheat genome. This results in an average distance of approximately 270 kb between these two microsatellite types combined. Based on sequence analysis data from 70 isolated microsatellites, it was found that wheat microsatellites are relatively long containing up to 40 dinucleotide repeats. Of the tested primer pairs, 36% resulted in fragments with a size corresponding to the expected length of the sequenced microsatellite clone. The variability of 15 microsatellite markers was investigated on 18 wheat accessions. Significantly, more variation was detected with the microsatellite markers than with RFLP markers with, on average, 4.6 different alleles per microsatellite. The 15 PCR-amplified microsatellites were further localized on chromosome arms using cytogenetic stocks of Chinese Spring. Finally, the primers for the 15 wheat microsatellites were used for PCR amplification with rye (Secale cereale) and barley accessions (Hordeum vulgare, H. spontaneum). Amplified fragments were observed for ten primer pairs with barley DNA and for nine primer pairs with rye DNA as template. A microsatellite was found by dot blot analysis in the PCR products of barley and rye DNA for only one primer pair.     

Isolation and cross-amplification of microsatellites in pink abalone (Haliotis corrugata)

Ten novel microsatellite loci were isolated in pink abalone, Haliotis corrugata, using (GT)₁₅ and (CT)₁₅ enriched genomic libraries. Two previously reported Haliotis kamtschatkana microsatellites cross‐amplified in H. corrugata. A set of 12 polymorphic microsatellites were evaluated in a wild population sample (N = 49). The number of alleles ranged from two to 55, and the observed and expected heterozygosities ranged from 0.104 to 0.939 and from 0.213 to 0.982, respectively. Significant deviations from Hardy–Weinberg equilibrium at three loci and no linkage disequilibrium were observed. Haliotis corrugata microsatellites cross‐amplified in other abalone species, two in H. fulgens, and seven in H. rufescens.     

Development of microsatellite markers and analysis of intraspecific genetic variability in chickpea (Cicer arietinum L.)

Paucity of polymorphic molecular markers in chickpea (Cicer arietinum L.) has been a major limitation in the improvement of this important legume. Hence, in an attempt to develop sequence-tagged microsatellite sites (STMS) markers from chickpea, a microsatellite enriched library from the C. arietinum cv. Pusa362 nuclear genome was constructed for the identification of (CA/GT) _n and (CT/GA) _n microsatellite motifs. A total of 92 new microsatellites were identified, of which 74 functional STMS primer pairs were developed. These markers were validated using 9 chickpea and one C. reticulatum accession. Of the STMS markers developed, 25 polymorphic markers were used to analyze the intraspecific genetic diversity within 36 geographically diverse chickpea accessions. The 25 primer pairs amplified single loci producing a minimum of 2 and maximum of 11 alleles. A total of 159 alleles were detected with an average of 6.4 alleles per locus. The observed and expected heterozygosity values averaged 0.32 (0.08–0.91) and 0.74 (0.23–0.89) respectively. The UPGMA based dendrogram was able to distinguish all the accessions except two accessions from Afghanistan establishing that microsatellites could successfully detect intraspecific genetic diversity in chickpea. Further, cloning and sequencing of size variant alleles at two microsatellite loci revealed that the variable numbers of AG repeats in different alleles were the major source of polymorphism. Point mutations were found to occur both within and immediately upstream of the long tracts of perfect repeats, thereby bringing about a conversion of perfect motifs into imperfect or compound motifs. Such events possibly occurred in order to limit the expansion of microsatellites and also lead to the birth of new microsatellites. The microsatellite markers developed in this study will be useful for genetic diversity analysis, linkage map construction as well as for depicting intraspecific microsatellite evolution.