Amplification Of Microsatellite Loci Research Articles

Isolation and characterization of microsatellite loci in the highland papaya Vasconcellea × heilbornii V. Badillo (Caricaceae)

AbstractNine polymorphic microsatellite loci were identified in the tropical plant Vasconcellea ×heilbornii and used to estimate allelic diversity in two populations of southern Ecuador. Allelic richness ranged from two to five alleles, observed heterozygosity ranged from 0.150 to 0.947 and expected heterozygosity ranged from 0.186 to 0.701. Most of these markers also amplified microsatellite loci in two other Vasconcellea species (Vasconcellea stipulata and Vasconcellea cundinamarcensis). Hence, these markers will be useful for population genetic analysis and the evaluation of genetic diversity and gene flow in these species.

Characterization of microsatellite markers for the mangrove tree Avicennia germinans L. (Avicenniaceae)

AbstractWe describe 10 primers for amplification of microsatellite loci for the mangrove, Avicennia germinans. Eight loci were isolated from a DNA sample from the Pacific coast of Baja California, Mexico and two loci were isolated from a DNA sample from the Atlantic coast of Bermuda. Polymorphism was investigated in a population from the Mexican Pacific coast (n = 15) and in four samples scattered throughout the range of the species. Total number of alleles for the species ranged from two to 10. Observed heterozygosity in the Mexican Pacific coast population ranged from 0.27 to 0.60, with two loci having fixed alleles.

Development and characterization of microsatellite loci in the endangered oyster mussel Epioblasma capsaeformis (Bivalvia: Unionidae)

AbstractPrimers for 10 polymorphic microsatellite loci were developed and characterized for the endangered oyster mussel Epioblasma capsaeformis from the Clinch River, Tennessee. Microsatellite loci also were tested in four other populations or species. Amplification was successful for most loci in these closely related endangered species or populations; therefore, a high level of flanking sequence similarity was inferred for this group of species and populations. Allelic diversity ranged from nine to 20 alleles/locus, and averaged 13.6/locus. This study demonstrated the feasibility of using polymerase chain reaction (PCR) primers to amplify microsatellite loci across freshwater mussel species to conduct population genetics studies.

Characterization and cross‐species amplification of microsatellite loci in the plant pathogenic fungus Leptosphaeria maculans

AbstractSeven polymorphic microsatellite markers suitable for population genetic studies and genetic mapping were developed for Leptosphaeria maculans, a fungal pathogen of canola (Brassica napus). Polymorphism was evaluated using 14 isolates from diverse geographical locations. Each locus had either two or three alleles. Cross‐species amplification was observed for almost all loci in L. biglobosa ‘brassicae’ and L. maculans ‘lepidii’.

Identification of microsatellite DNA markers for population structure analysis in Indian major carp, Cirrhinus mrigala (Hamilton-Buchanan, 1882)

Summary Forty-four primer sequences available for four cyprinid fishes were tested to amplify microsatellite loci in Indian major carp, Cirrhinus mrigala. A total of 12 loci were successfully amplified with clear scorable patterns and five thereof were polymorphic. Suitability of the identified polymorphic loci in population structure analysis of C. mrigala was assessed. Genetic variation was examined in 76 specimens collected from five different rivers. The mean observed heterozygosity ranged from 0.247 to 0.333. Significant heterogeneity in allele frequencies was detected, indicating that the samples analysed did not belong to homogenous populations. The identified microsatellite markers are promising for the analysis of intraspecific divergence in C. mrigala across its distribution range.

Cross‐species amplification of microsatellite loci in aphids: assessment and application

AbstractDespite the relative ease of isolating microsatellites, their development still requires substantial inputs of time, money and expertise. For this reason there is considerable interest in using existing microsatellites on species from which markers were not cloned. We tested cross‐species amplification of 48 existing aphid loci in species of the following genera: Aphidinae: Aphidini: Aphis and Rhopalosiphum; Aphidinae: Macrosiphini: Acyrthosiphum, Brevicoryne, Diuraphis, Illinoia, Macrosiphoniella, Macrosiphum, Metopeurum, Metapolophium, Myzus, Phorodon, Sitobion and Uroleucon and Neuquenaphidinae: Neuquenaphis. Our results show cross‐species application of known microsatellite loci is a highly promising source of codominant markers for population genetic and evolutionary studies in aphids.

Transferability of hexaploid wheat (Triticum aestivum) microsatellite markers to tetraploid species (T. turgidum) carrying A and B genomes

Emmer is the tetraploid wheat (genome constitution: AABB) from which hexaploid or common wheat (Triticum aestivum L., genome:AABBDD) was derived. We examined transferability of 180 microsatellite markers from hexaploid wheat to wild emmer wheat [T. turgidum ssp. dicoccoides (Korn. Ex Asch. & Graebner) Thell.] and cultivated emmer wheat [T. turgidum ssp. dicoccum (Schrank ex Shobler) Thell.]. Among the 180 primer pairs, 121 (67.2%) amplified microsatellite loci from the ssp. dicoccoides genome and 122 (67.8%) from the ssp. dicoccum genome. Between these two subspecies, polymorphic banding patterns were observed at 97 (53.5%) microsatellite loci. Five primer pairs (2.8%) produced amplification products only from the ssp. dicoccoides genome while 7 other primer pairs (3.9%) produced products only from the ssp. dicoccum genome. Fifty microsatellite markers (27.8%) failed to amplify any products in these two subspecies of T. turgidum. The microsatellite markers we identified to show cross-species transferability should be useful in studies of gene mapping and genome evolution in tetraploid wheat.

Association between Chromosome 1p and 19q Loss and Outcome in Pediatric Malignant Gliomas: Results from the CCG-945 Cohort

Background: Results of recent molecular studies on malignant gliomas in adults showed that deletions within the short arm of chromosome 1 and/or the long arm of chromosome 19 identified a prognostically favorable subgroup of tumors that often respond to conventional chemotherapy. To determine if this association applies to pediatric malignant gliomas, we examined the correlation between 1p and 19q deletions and outcome in the cohort derived from the Children’s Cancer Group study 945, the largest randomized trial of such tumors completed to date. Methods: Archival histopathologic material yielded tissue of sufficient quality and quantity for genotyping in 121 specimens. Sections were examined histologically and targets that contained malignant glioma were isolated by microdissection and subjected to polymerase-chain-reaction-based amplification of microsatellite loci using fluorochrome-labeled primers, followed by capillary electrophoresis, and fluorescent fragment analysis. One hundred and seven specimens had 2 alleles for at least 1 of the 3 1p loci examined, and were therefore informative for determining loss of heterozygosity, defined as at least a twofold difference in fluorescence intensity between the paired allelic bands; 99 were informative at 19q. Results: Thirty-two tumors (29.9%) had loss of heterozygosity involving 1p, 27 (28%) involving 19q, and 13 involving both, an incidence consistent with previous reports involving adult malignant gliomas. However, outcome analyses of the entire cohort found no favorable association between 1p loss, 19q loss, or the combination, and survival. Subset analysis disclosed no association with outcome in either arm of the study (which compared efficacy of 2 chemotherapeutic regimens) or in subgroups stratified by age, tumor location, or tumor histology. Conclusion: In contrast to recent observations of adult malignant gliomas, deletions involving chromosomes 1p or 19q did not predict a survival advantage in this series of pediatric high-grade gliomas, which might indicate that frequencies of various histologic and molecular subtypes of malignant gliomas differ between children and adults, and supports further efforts to identify prognostic indicators relevant to pediatric gliomas.

Fast isolation of microsatellite loci of very diverse repeat motifs by library enrichment in echinoderm species, Amphipholis squamata and Echinocardium cordatum

AbstractWe conceived a microsatellite enrichment protocol in which probes of several repeat motifs are mixed during the whole procedure. The repeats found generally differed from those of the probes and were very diverse, from mononucleotides to pentanucleotides. We tested several modifications with alternative: (i) digestion/ligation buffers; (ii) polymerases; and (iii) purification methods. The simplest methods always worked as well as classical ones or even better, resulting in probably the simplest protocol for isolating microsatellites of diverse motifs from genomes with low microsatellite frequency. The proportion of positive clones, polymorphism levels and cross‐specific amplification of microsatellite loci significantly vary between species.

Amplification of DNA markers from scat samples of the least weaselMustela nivalis nivalis

To test the feasibility of using field-collected scats as a source of DNA in the study of the least weaselMustela nivalis nivalis Linnaeus, 1766, DNA was extracted from scat samples collected from captive weasels using a modified extraction protocol. Using universal primers, the control region of the mitochondrial genome was successfully amplified from scat-extracted DNA. This amplification resulted in two products; one equivalent in size and sequence to the product obtained from tissue-extracted weasel DNA, and the other slightly larger and equivalent in size and sequence to the domestic house mouseMus musculus, the food source of the captive weasels. This demonstrates the reliability of DNA extraction from scats, as well as the possibility, under favourable circumstances, of identifying the prey species from the same samples. In addition, we attempted to amplify microsatellite loci from both tissue and scat-extracted DNA using six primer pairs designed for other mustelids, the American minkMustela vison and the wolverineGulo gulo. While three loci, Mvi57 (American mink), Ggu216 and Ggu234 (wolverine), were found to be polymorphic in the least weasel, amplification of these loci from the scat extracted DNA was only successful for approximately half of the samples. Although further work is needed, the present results suggest that it is possible to use scats as a source of DNA in field studies of the least weasel.

Sequencing of simple sequence repeat anchored polymerase chain reaction amplification products of Biomphalaria glabrata.

Simple sequence repeat anchored polymerase chain reaction amplification (SSR-PCR) is a genetic typing technique based on primers anchored at the 5' or 3' ends of microsatellites, at high primer annealing temperatures. This technique has already been used in studies of genetic variability of several organisms, using different primer designs. In order to conduct a detailed study of the SSR-PCR genomic targets, we cloned and sequenced 20 unique amplification products of two commonly used primers, CAA(CT)6 and (CA)8RY, using Biomphalaria glabrata genomic DNA as template. The sequences obtained were novel B. glabrata genomic sequences. It was observed that 15 clones contained microsatellites between priming sites. Out of 40 clones, seven contained complex sequence repetitions. One of the repeats that appeared in six of the amplified fragments generated a single band in Southern analysis, indicating that the sequence was not widespread in the genome. Most of the annealing sites for the CAA(CT)6 primer contained only the six repeats found within the primer sequence. In conclusion, SSR-PCR is a useful genotyping technique. However, the premise of the SSR-PCR technique, verified with the CAA(CT)6 primer, could not be supported since the amplification products did not result necessarily from microsatellite loci amplification.

PHYLOGEOGRAPHIC STUDIES IN THE TROPICAL SEAWEED CLADOPHOROPSIS MEMBRANACEA (CHLOROPHYTA, ULVOPHYCEAE) REVEAL A CRYPTIC SPECIES COMPLEX1

The seaweed Cladophoropsis membranacea (Hofman Bang ex. C. Agardh) Børgesen is a widely distributed species on coral reefs and along rocky coastlines throughout the tropics and subtropics. In a recent population‐level survey openface>1600 individuals with eight microsatellite loci, a number of isolates from biogeographically disjunct locations could not be amplified for any of the loci. Nonamplifiable and amplifiable isolates co‐occurred within the Canary Islands, Cape Verde Islands, and in the Caribbean. These unexpected results led to question whether or not C. membranacea is a single species. Phylogenetic relationships were evaluated using rDNA ITS1 and ITS2 sequence comparisons from 42 isolates sampled from a subset of 30 of the 66 locations. Four well‐supported clades were identified. Sequence divergence within clades was <1%, whereas between‐clade divergence was 2%–3%. Intraindividual variation was extremely low with no effects on the analysis. A strong, but imperfect, correspondence was found between ITS clades and amplifiable microsatellite loci. It is concluded that C. membranacea consists of three cryptic species. Using Pacific isolates as an outgroup, the most basal clade included the Central Canary Islands, Cape Verde, and Bonaire (Caribbean) isolates and thus spanned the widest latitude. Two derived sister clades consisted of a southern transtropical group stretching across the SE Caribbean to the Cape Verde Islands and African coast (but not the Canary Islands) and a NE‐Canary Island‐Mediterranean clade that also included the Red Sea. The detection of overlapping biogeographic distributions highlights the importance of ecotypic differentiation with respect to temperature and the importance of shifting sea surface isotherms that have driven periodic extinctions and recolonizations of the Canary Islands—a crossroads of marine floral exchange—since the last glacial maximum.

PHYLOGEOGRAPHIC STUDIES IN THE TROPICAL SEAWEED CLADOPHOROPSIS MEMBRANACEA (CHLOROPHYTA, ULVOPHYCEAE) REVEAL A CRYPTIC SPECIES COMPLEX1

The seaweed Cladophoropsis membranacea (Hofman Bang ex. C. Agardh) Børgesen is a widely distributed species on coral reefs and along rocky coastlines throughout the tropics and subtropics. In a recent population-level survey openface>1600 individuals with eight microsatellite loci, a number of isolates from biogeographically disjunct locations could not be amplified for any of the loci. Nonamplifiable and amplifiable isolates co-occurred within the Canary Islands, Cape Verde Islands, and in the Caribbean. These unexpected results led to question whether or not C. membranacea is a single species. Phylogenetic relationships were evaluated using rDNA ITS1 and ITS2 sequence comparisons from 42 isolates sampled from a subset of 30 of the 66 locations. Four well-supported clades were identified. Sequence divergence within clades was <1%, whereas between-clade divergence was 2%–3%. Intraindividual variation was extremely low with no effects on the analysis. A strong, but imperfect, correspondence was found between ITS clades and amplifiable microsatellite loci. It is concluded that C. membranacea consists of three cryptic species. Using Pacific isolates as an outgroup, the most basal clade included the Central Canary Islands, Cape Verde, and Bonaire (Caribbean) isolates and thus spanned the widest latitude. Two derived sister clades consisted of a southern transtropical group stretching across the SE Caribbean to the Cape Verde Islands and African coast (but not the Canary Islands) and a NE-Canary Island-Mediterranean clade that also included the Red Sea. The detection of overlapping biogeographic distributions highlights the importance of ecotypic differentiation with respect to temperature and the importance of shifting sea surface isotherms that have driven periodic extinctions and recolonizations of the Canary Islands—a crossroads of marine floral exchange—since the last glacial maximum.

Polymorphic microsatellite repeats are not conserved between Leishmania donovani and Leishmania major

AbstractThirteen sets of polymerase chain reaction (PCR) primers were designed to amplify microsatellite loci identified in the genome sequence of Leishmania major. Polymorphisms were detected in L. major at all loci. In Leishmania donovani only two of these loci were informative for classification purposes with this data set. The PCR products of all loci from one L. donovani strain were sequenced and it was found that the number of repeats in the microsatellite loci were either substantially reduced with respect to L. major or absent altogether. Consequently it is unlikely to be possible to use the genome sequence of L. major to identify polymorphic microsatellite loci in other Leishmania species.

Isolation and characterization of microsatellites in Echinochloa (L.) Beauv. spp.

AbstractFive primer pairs were developed that amplify microsatellite loci in three agronomically important Echinochloa (L.) Beauv. species: E. colona (L.) Link, E. crus‐galli (L.) Beauv. and E. crus‐pavonis (Kunth) Schultes. The microsatellites were tested on 24 individuals representing three species collected in rice fields from different geographical regions and revealed 3–7 alleles per microsatellite. Gene diversity [1 − Σpij2] for four polymorphic loci within E. crus‐galli ranged from 0.12 to 0.61. Alleles at a fifth locus were useful in discriminating the species. The microsatellites should provide useful markers for intraspecific diversity studies and aid classification of species within this complex genus.

Introgression of rye chromatin on chromosome 2D in the Portuguese wheat landrace 'Barbela'

The old Portuguese wheat landrace aggregate known as 'Barbela' shows good productivity under the low-fertility conditions often associated with acid soils. The use of genomic rye DNA, in combination with 45S rDNA and the repetitive sequences dpTal and pScl 19.2 as probes, in two sequential in situ hybridization steps enabled the identification of all chromosomes in the 'Barbela' wheat lines and the detection of the introgression of rye-origin chromatin onto wheat chromosome arm 2DL in two of the lines. Amplification of microsatellite loci using published primer pairs showed that the distal segment of wheat chromosome 2DL, which was involved in the rye translocation, was deleted. The identification and characterization of small recombinant chromosome segments in wheat-rye lines may allow their use in plant breeding programmes. Their presence in farmer-maintained material demonstrates the importance of maintaining, characterizing, and collecting landrace material before valuable genetic combinations are lost as uniform commercial crops are introduced.

Polymorphic microsatellite markers identified in individual Plasmodium falciparum oocysts from wild-caught Anopheles mosquitoes.

The oocysts of Plasmodiom falciparam that form on the midgut wall of anopheline vectors represent the meiotic products of the sexual cycle of the parasite and are useful for genetic analyses. Recognizing the limitations of current markers for the analysis of genetic variation, we describe a protocol for the DNA isolation and PCR amplification of microsatellite loci from individual oocysts. We present a simple method for the storage and isolation of individual Plasmodium oocysts from wild-caught mosquitoes and demonstrate that it is possible to extract sufficient oocyst DNA for approximately 50 amplifications. Genotyping was successful for 10 microsatellite loci and, using Genescan technology, we found extensive microsatellite polymorphism in an analysis of a dozen oocysts from each of 2 Anopheles gambiae female specimens collected within 6 months at the same location in northern Tanzania. All of the loci exhibited at least 2 alleles, with a maximum of 5. Homozygous and heterozygous oocysts could easily be discerned, but most of the oocysts were homozygous. The procedure has great potential for shedding light on genetic mechanisms operating during meiosis in mosquito vectors and the influence this may have on the genetic structure of natural populations of P. falciparum.

Heterologous amplification of microsatellite loci from mouse and rat in oryzomyine and Thomasomyine South American rodents.

Eleven heterologous primers were investigated in 119 individuals of 11 species of rodents of the Oryzomyine and Thomasomyine groups. The animals were collected at four sites of the "Cerrado," a dryland biome located on the Brazilian Plateau, all of them being karyotyped and taxonomically allocated according to the karyotype. Four of these primers, R47, R65, R75 (from Rattus), and ATP (from Mus) cross-amplified in at least one of these taxa, giving products of seven, nine, one, and three bands, respectively. These values are of the same order as others obtained when heterologous primers were amplified in other orders of mammals. Of the 20 products amplified in these two rodent groups by these four primers, only 7 of the bands were seen in a heteromorphic state (one individual presenting two bands), in two species (Rhipidomys aff. leucodactylus and Oryzomys megacephalus). The others occurred as monomorphic bands.

Allelotype of Uterine Leiomyomas

Uterine leiomyomas are the most common benign tumor that arise from smooth muscle cells of the myometrium. Little is known about the etiology and pathogenesis of this tumor. To investigate the molecular pathogenesis of these tumors, we have conducted an allelotype of 102 leiomyomas from 12 patients, using 67 fluorescently-tagged oligonucleotide primers amplifying microsatellite loci covering all autosomes. No areas of the genome showed frequent loss of heterozygosity (LOH); however, the highest rate of LOH (9%) was observed on 7q, consistent with previous cytogenetic observations. Uterine leiomyomas are sometimes multiple. In general, multiplicity of other types of neoplasm is associated with genetic predisposition to the disease. Because multiple tumors were available from each of the 12 patients studied, we looked for evidence of allele-specific LOH, which might indicate the presence of an underlying predisposition gene. However, no evidence for allele-specific LOH was detected, indicating that if cases of multiple uterine leiomyoma are due to an underlying predisposition gene, it is unlikely to be a recessive oncogene.

Alleletyping of an oligodendrocyte-type-2 astrocyte lineage derive from a human glioblastoma multiforme.

We have conducted alleletyping of two novel cell lines derived from glioblastoma multiforme, which appear to have arisen from different glial lineages, by using 76 fluorescently labeled oligonucleotide primers amplifying microsatellite loci covering the entire human genome. One cell line, Hu-O-2A/Gb1, expresses antigens and metabolic profiles characteristic of the oligodendrocyte-type-2 astrocyte (0-2A) lineage of the rat central nervous system. This cell line generated, in vitro, cells with characteristics of 0-2A progenitor cells, oligodendrocytes and astrocytes. The second cell line, IN1434, is derived from an astrocyte or a precursor cell restricted to astrocytic differentiation. Hu-O-2A/Gbl cells show allelic losses of loci on chromosomes 2, 5, 6, 7, 8, 9, 10, 11, 13, 15, 16, 17, 20 and 21. IN1434 cells are likely to have allelic losses of loci on chromosomes 1, 3, 8 and 10, although no control DNA is available for this cell line. These results, for the first time, provide a detailed information of the molecular genetic defects occurring in Hu-O-2A/Gb1 and IN1434.