High similarity between flanking regions of different microsatellites detected within each of two species of Lepidoptera: Parnassius apollo and Euphydryas aurinia

Microsatellite flanking regions have been compared in two butterfly species. Several microsatellite flanking regions showed high similarity to one another among different microsatellites within a same species, but very few similarities were found between species. This can be the consequence of either duplication/multiplication events involving large regions containing microsatellites or of microsatellites imbedded in minisatellite regions. The multiplication of microsatellites might also be linked to mobile elements. Furthermore, crossing over between nonhomologous microsatellites can lead to the exchange of the flanking regions between microsatellites. The same phenomenon was observed in both studied butterfly species but not in Aphis fabae (Hemiptera), which was screened at the same time using the same protocol. These findings might explain, at least partially, why microsatellite isolation in Lepidoptera has been relatively unsuccessful so far.     

Assignment of five polymorphic ovine microsatellites to bovine syntenic groups

A panel of bovine somatic cell hybrids was used to map ovine microsatellites. Five of seven microsatellites were assigned to five bovine syntenic groups. These microsatellites were designated D5S10 (MAF23), D1S4 (MAF46), D13S1 (MAF18), D4S3 (MAF50), and DXS2 (MAF45), mapped to syntenic groups U3 (chromosome 5), U10 (chromosome 1), U11, U13, and the X chromosome, respectively. Two remaining sheep microsatellites amplified rodent DNA in the hybrid somatic cell panel, and were not assigned to bovine syntenic groups. Assignment of ovine-derived microsatellites to bovine syntenic groups provides additional evidence of the usefulness of microsatellites for mapping closely related species. The use of ovine and bovine microsatellites will aid in development of comparative genomic maps for these two species.     

Chloroplast microsatellites in Prunus,Rosaceae

Ten chloroplast microsatellite markers were developed from Japanese plum (Prunus salicina) based on nucleotide sequences of c. 4300 bp from six chloroplast regions. Out of 10 microsatellites, seven markers contained mononucleotide repeats. Almost all microsatellites displayed discrete amplified fragments for 17 species in Prunus. The microsatellites generated 46 different fragment types and differentiated all used species. Polymorphism was also observed within species for all microsatellites.     

The conservation of dinucleotide microsatellites among mammalian genomes allows the use of heterologous PCR primer pairs in closely related species

The high degree of polymorphism displayed by DNA microsatellites makes them useful as DNA markers in linkage studies. A search of the DNA sequence databases revealed that the locations of dinucleotide microsatellites are often conserved among mammalian species, enabling the prediction of the presence of DNA microsatellites using comparative genetic data. In closely related species such as cattle and sheep, this conservation was close enough to allow PCR primers designed for use in one species to be used to analyze microsatellite length polymorphism in the other. A total of 48 sets of primer pairs, flanking bovine microsatellites and giving polymorphic PCR products in that species, were tested with template DNA from sheep, horses, and humans. Specific products were obtained in 27 cases (56%) with ovine DNA, 20 of which (42%) showed polymorphisms. With equine DNA, 3 (6.2%) gave specific but monomorphic products, while no specific products were obtained using human DNA. The ability to use heterologous PCR primers, coupled with comparative mapping information will facilitate the use of DNA microsatellites in gene mapping studies in closely related species such as cattle and sheep, rat and mouse, or primates.     

Microsatellite markers for diverse Salix species

Forty‐six microsatellites were isolated from an enriched library of Salix burjatica and tested on 20 individuals (of nine species/hybrids) from the National Willows Collection (IACR‐Long Ashton Research Station, UK). Twenty‐nine were monomorphic, gave multilocus or unscorable patterns, or were duplicates. The remaining 17 microsatellites gave 2–22 alleles/locus. Three microsatellites successfully cross‐amplified in 31 additional Salix species. A further six were tested on panels comprising 6–25 individuals from the 31 species. Cross‐amplification was successful in all cases. These results suggest that the microsatellites isolated here should prove useful for population studies in a wide range of Salix species.     

Development of microsatellite markers in Fragaria, their use in genetic diversity analysis, and their potential for genetic linkage mapping.

We have developed 21 new microsatellites in the model diploid perennial species Fragaria vesca from an enriched genomic library developed using F. vesca 'Ruegen'. The transferability of the primer pairs to other Fragaria species was high; all 31 primer pairs produced amplicons in 3 accessions of the octoploid strawberry Fragaria x ananassa, whereas 24 (77%) amplified a product in 7 other diploid Fragaria species. We analysed the allelic variation among 15 F. vesca accessions using the 21 microsatellites reported here and 10 F. vesca microsatellites described previously. The level of polymorphism detected at these microsatellite loci was high; five loci were monomorphic. Only two microsatellites were required to unambiguously discriminate among the 15 F. vesca accessions. A preliminary survey of segregation in an F2 progeny indicates that 20 of the 26 polymorphic loci (77%) could be mapped.     

Morphometric and genetic characterization of sympatric populations of Clarias gariepinus and C. anguillaris from Senegal

A sample of African Clarias catfishes from the Senegal River was studied using morphometry, allozyme variation, microsatellites and RFLPs of mitochondrial DNA. They all confirmed the presence of two species , C. gariepinus and C. anguillaris . The two species were closely related genetically and no diagnostic loci were found in allozymes and microsatellites studies. Two of the 11 haplotypes of mtDNA observed were shared by both species. Three of the four assays (morphometry, allozymes and microsatellites) allowed a precise characterization of both. One specimen occupied an intermediate position in the analysis of the data; it was considered an F_l hybrid whose possible origin is discussed.     

Triplet-repeat microsatellites shared among hard and soft pines

Vascular plant species have shown a low level of microsatellite conservation compared to many animal species. Finding trans-specific microsatellites for plants may be improved by using a priori knowledge of genome organization. Fifteen triplet-repeat microsatellites from hard pine (Pinus taeda L.) were tested for trans-specific amplification across seven hard pines (P. palustris Mill., P. echinata Mill., P. radiata D. Don., P. patula Schiede et Deppe, P. halepensis Mill., P. kesiya Royle), a soft pine (P. strobus L.), and Picea rubens Sargent. Seven of 15 microsatellites had trans-specific amplification in both hard and soft pine subgenera. Two P. taeda microsatellites had conserved flanking regions and repeat motifs in all seven hard pines, soft pine P. strobus, and P. rubens. Perfect triplet-repeat P. taeda microsatellites appear to be better candidates for trans-specific polymorphism than compound microsatellites. Not all perfect triplet-repeat microsatellites were conserved, but all conserved microsatellites had perfect repeat motifs. Persistent microsatellites PtTX2123 and PtTX3020 had highly conserved flanking regions and a conserved repeat motif composition with variable repeat unit numbers. Using trinucleotide microsatellites improved trans-specific microsatellite recovery among hard and soft pine species.     

Discovery and cross-amplification of microsatellite polymorphisms in asterinid sea stars

Variation in tandem repeats of two- to six-base nucleotide motifs (microsatellites) can be used to obtain inexpensive and highly informative multi-locus data on population genetics.We developed and tested a large set of cross-amplifiable sea star (Asterinidae) microsatellite markers from a mixed pool of genomic DNA from eight species. We describe cloned sequences, primers, and PCR conditions, and characterize population-level variation for some species and markers. A few clones containing microsatellites showed considerable similarity to sequences (including genes of known function) in other sea stars and in sea urchins (from the Strongylocentrotus purpuratus complete genome). The pooled genomic DNA method was an efficient way to sample microsatellites from many species: we cloned 2-10 microsatellites from each of eight species, and most could be cross-amplified in 1-7 other species. At 12 loci in two species, we found 1-10 alleles per microsatellite, with a broad range of inbreeding coefficients. Measures of polymorphism were negatively correlated with the extent of cross-amplification.     

Evolution of microsatellites in Arabis petraea and Arabis lyrata, outcrossing relatives of Arabidopsis thaliana

We examined microsatellite variation in two diploid, outcrossing relatives of Arabidopsis thaliana, Arabis petraea and Arabis lyrata. The primer sequences were derived from A. thaliana. About 50% (14 loci) of the A. thaliana primers could successfully amplify microsatellites in the related species. Analysis of microsatellite structure in the related species showed that there had been large changes in the microsatellites: there were large differences in repeat numbers and many of the A. thaliana simple repeats were shorter in the related species. For the loci we compared, the related species had a much lower level of variability at the microsatellites than Japanese wild populations of A. thaliana. This is presumably related to the different microsatellite structures, because allozyme data showed that the outcrossing relatives were highly polymorphic compared to other outcrossing herbaceous species. Use of microsatellites in assessing variability or phylogenetic relationships between different species requires caution, because changes in microsatellite structure may alter evolutionary rates.      

Novel microsatellites from the green swordtail (Xiphophorus hellerii) also display polymorphism in guppy (Poecilia reticulata)

The green swordtail (Xiphophorus hellerii; Poeciliidae) is a popular ornamental freshwater fish species. Fourteen microsatellites were isolated from a green swordtail genomic library enriched for CA‐repeats. Thirteen microsatellites were polymorphic in green swordtail; interestingly four of them were tetranucleotide‐type. Cross‐species amplification showed that 10 of the 14 microsatellites were polymorphic in guppy (Poecilia reticulata; Poeciliidae) as well. No significant directional difference of allele length was seen between the two species at any of the loci tested.     

Characterization and isolation of novel microsatellites from the Drosophila dunni subgroup

We have isolated and characterized 77 novel microsatellites from two species, Drosophila dunni and Drosophila nigrodunni, which are closely related Caribbean-island endemics from the Drosophila cardini species group. These species are very distantly related to all other Drosophila from which microsatellites have previously been characterized. We find that the average length of microsatellites isolated in these species is quite small, with an overall mean length of 9.8 repeat units for dinucleotide microsatellites in the two study species. The nucleotide composition of dinucleotides differs between the two species: D. nigrodunni has a predominance of (AC/GT)n repeats, whereas D. dunni has equal numbers of (AC/GT)n and (AG/CT)n repeats. Tri- and tetranucleotide repeats are not abundant in either species. We assayed the variability of eight microsatellites in a closely related third species, Drosophila arawakana, using wild-caught individuals from the island of Guadeloupe. We found the microsatellites to be extremely variable in this population, with observed heterozygosities ranging from 0.541 to 0.889. DNA amplification trials suggest that these eight microsatellites are widely conserved across the D. cardini group, with five of the eight producing amplification products in every species tested. However, the loci are very poorly conserved over greater phylogenetic distances. DNA amplification of the microsatellite loci was unreliable in members of the closely related Drosophila quinaria, Drosophila calloptera, Drosophila guarani and Drosophila tripunctata species groups. Furthermore, these microsatellites could not be detected in the genome of Drosophila melanogaster, despite the conservation of microsatellite flanking regions at some loci. These data indicate that Drosophila microsatellite loci are quite short lived over evolutionary timescales relative to many other taxa.     

Nectomys squamipes microsatellites and homologous loci in sigmodontine rodents

Three monomorphic and four highly polymorphic microsatellites of Nectomys squamipes were isolated and characterised in a sample of 141 specimens from eight different Brazilian localities. These seven microsatellites and four others previously described in this species were tested in seven other nonfocus sigmodontine species. At least three loci were successfully amplified in every species, but none was amplified in all species. All sequenced products in nonfocus species showed (GT)(n) motifs as in N. squamipes. Several loci were amplified in Nectomys rattus and Oligoryzomys nigripes, while absence of PCR products was observed more frequently in Oxymycterus dasythricus and Akodon cursor. Two of three monomorphic loci in N. squamipes were polymorphic in other species.     

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.     

Chloroplast microsatellite primers for cacao (Theobroma cacao) and other Malvaceae

? Premise of the study: Chloroplast microsatellites were developed in Theobroma cacao to examine the genetic diversity of cacao cultivars in Trinidad and Tobago. ? Methods and Results: Nine polymorphic microsatellites were designed from the chloroplast genomes of two T. cacao accessions. These microsatellites were tested in 95 hybrid accessions from Trinidad and Tobago. An average of 2.9 alleles per locus was found. ? Conclusions: These chloroplast microsatellites, particularly the highly polymorphic pentameric repeat, were useful in assessing genetic variation in T. cacao. In addition, these markers should also prove to be useful for population genetic studies in other species of Malvaceae.     

Isolation and characterization of polymorphic microsatellite loci for the dace complex: Leuciscus leuciscus (Teleostei: Cyprinidae)

Ten novel polymorphic microsatellites were isolated from the dace complex (Leuciscus leuciscus), which is a European cyprinid species. Four multiplex polymerase chain reaction sets were optimized in order to genotype 26 polymorphic loci in all, including 16 previously published cyprinid-specific loci. The level of genetic diversity was assessed in 142 dace individuals. We also successfully applied 26 of the microsatellites to 10 related species. These primers thus will be useful to assess population structure of the dace and other cyprinid species, with application for conservation issues and phylogeographical approaches.     

微卫星标记及其在蚜虫种群生物学研究中的应用

微卫星（SSR）是以1～6个碱基为重复单位组成的简单串联重复序列,具有丰度高、多态性高、共显性遗传、选择中性和可重复性好等优点。微卫星标记技术可分为三种类型：基于杂交的SSR指纹,基于PCR的SSR指纹和单位点SSR的PCR扩增。前两者不需要知道基因组序列信息,属于多位点标记;后者则需要前期工作,属于单位点标记,即通常所指的微卫星标记。到目前为止,共在14种蚜中筛选到141个微卫星位点,并发表了相关的引物序列,这给今后的相关研究提供了丰富的共享资源。业已证明微卫星引物在邻近种之间有一定的通用性。微卫星标记严格遵守孟德尔遗传规律,已经被用来推断某些蚜虫有性生殖的情况。同种蚜虫中可能同时存在有性系和无性系,微卫星研究证明有性系比无性系有更高的遗传多样性,无性系通常表现出杂合过剩和连锁不平衡。蚜虫的迁飞规律适合用微卫星标记加以研究,已有的研究显示在具有高度迁飞特性的种类中,地理种群间的遗传分化程度低,基因频率的相似性高。存在广泛分布的相同的多位点基因型是迁飞性蚜虫的另一个重要特征。在我国,微卫星应用于蚜虫生物学研究的工作还较少,鉴于该种标记的优良特性和巨大的潜力,本文建议今后相关的研究应该首先考虑微卫星标记。     

Characterization and cross-species amplification of microsatellites from the endangered Hawksbill turtle (Eretmochelys imbricate)

Twelve novel polymorphic microsatellites were isolated from the endangered Hawksbill turtle (Eretmochelys imbricate). Eight of 12 markers were used to study genetic diversity of two sea turtle species: E. imbricate and green sea turtle (C. mydas). In E. imbricate, the average allele number of the eight microsatellites was 6.25/locus with a range of 3–13. The average expected and observed heterozygosity was 0.66 and 0.63 respectively. In C. mydas, the average allele number of the eight markers was 11.63/locus. The observed heterozyosity (0.68) was lower than the expected heterozyosity (0.79). Most of 12 microsatellites amplified specific and polymorphic PCR products in other six turtle species. Hence, the developed microsatellites would facilitate studies on genetic diversity and population structure of E. imbricate and other marine turtle species.     

Characterization of microsatellites located within the genes of goldfish (Carassius auratus auratus)

The goldfish (Carassius auratus auratus; Cyprinidae) is not only an important ornamental fish species, but also a useful model for biological studies. The sequence of goldfish genes present in the public database was searched for short tandem repeats, and 11 polymorphic microsatellites were detected within eight important genes. Two microsatellites were located in coding regions of the c‐myc and GAP‐43 genes, respectively, whereas the others were mainly located in 5′ and 3′ untranscribed regions of other genes, such as gonadotropin and activin. The average allele number of these microsatellites was 5.5 per locus with a range between 3 and 9. These microsatellites will be useful for ecological and population genetic studies of this species, as well as for the ornamental fish industry.     

Evolutionary history of microsatellites in the obscura group of Drosophila

The evolutionary origins of microsatellites are not well understood. Some investigators have suggested that point mutations that expand repeat arrays beyond a threshold size trigger microsatellites to become variable. However, little empirical data has been brought forth on this and related issues. In this study, we examine the evolutionary history of microsatellites in six species within the obscura group of Drosophila, tracing changes in microsatellite alleles using both PCR product size and sequence data. We found little evidence supporting a general role of point mutations triggering initial microsatellite expansion, and no consistent threshold size for expansion was observed. Flanking region length variation was extensive when alleles were sequenced in distantly related species, and some species possessed altogether different repeat arrays between the same primer binding sites. Our results suggest extreme caution in using microsatellite allele sizes for phylogenetic analyses or to infer divergences between populations.