Use of 5´-anchored primers for the enhanced recovery of specific microsatellite markers in Brassica napus L.

Microsatellite markers have assumed great significance in biological research. The isolation and characterisation of microsatellites involves DNA library construction and screening, DNA sequencing, primer design and PCR optimisation. When a microsatellite is situated close to the beginning or end of a cloned fragment, specific primers cannot be designed for one of the flanking sequences, thus hindering the utilisation of such microsatellites as markers. The present approach was to use one 5′-anchored primer complementary to the microsatellite sequence in combination with one specific Cy5- labelled primer with a view to retrieving useful microsatellites, which would otherwise be lost. Six pairs of a 5′ anchored primer and a specific primer were used across a set of 31 Brassica napus winter cultivars and one accession each of five additional Brassica species. Using laser fluorometry a single labelled product was observed after amplification with each of four primer pairs, and one primer pair gave two labelled products. Three products corresponded in size with the products expected if 5′ anchoring was effective, indicating the amplification of locus-specific full-length products including all of the microsatellite repeats. All six primer pairs showed polymorphisms across the Brassica species examined, but only one primer pair showed polymorphisms within B. napus, making it useful for genetic analysis in rapeseed cultivars. The other primer pairs could be useful in studying gene introgression into B. napus or for investigating interspecific crosses involving different Brassica species. Received: 5 August 1999 / Accepted: 1 November 1999     

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.     

Highly Informative Single-Copy Nuclear Microsatellite DNA Markers Developed Using an AFLP-SSR Approach in Black Spruce (Picea mariana) and Red Spruce (P. rubens)

Background

Microsatellites or simple sequence repeats (SSRs) are highly informative molecular markers for various biological studies in plants. In spruce (Picea) and other conifers, the development of single-copy polymorphic genomic microsatellite markers is quite difficult, owing primarily to the large genome size and predominance of repetitive DNA sequences throughout the genome. We have developed highly informative single-locus genomic microsatellite markers in black spruce (Picea mariana) and red spruce (Picea rubens) using a simple but efficient method based on a combination of AFLP and microsatellite technologies.

Principal Findings

A microsatellite-enriched library was constructed from genomic AFLP DNA fragments of black spruce. Sequencing of the 108 putative SSR-containing clones provided 94 unique sequences with microsatellites. Twenty-two of the designed 34 primer pairs yielded scorable amplicons, with single-locus patterns. Fourteen of these microsatellite markers were characterized in 30 black spruce and 30 red spruce individuals drawn from many populations. The number of alleles at a polymorphic locus ranged from 2 to 18, with a mean of 9.3 in black spruce, and from 3 to 15, with a mean of 6.2 alleles in red spruce. The polymorphic information content or expected heterozygosity ranged from 0.340 to 0.909 (mean = 0.67) in black spruce and from 0.161 to 0.851 (mean = 0.62) in red spruce. Ten SSR markers showing inter-parental polymorphism inherited in a single-locus Mendelian mode, with two cases of distorted segregation. Primer pairs for almost all polymorphic SSR loci resolved microsatellites of comparable size in Picea glauca, P. engelmannii, P. sitchensis, and P. abies.

Significance

The AFLP-based microsatellite-enriched library appears to be a rapid, cost-effective approach for isolating and developing single-locus informative genomic microsatellite markers in black spruce. The markers developed should be useful in black spruce, red spruce and other Picea species for various genetics, genomics, breeding, forensics, conservation studies and applications.     

Microsatellites in the estrogen receptor (ESR1, ESR2) and androgen receptor (AR) genes and breast cancer risk in African American and Nigerian women

Genetic variants in hormone receptor genes may be crucial predisposing factors for breast cancer, and microsatellites in the estrogen receptor (ESR1, ESR2) and androgen receptor (AR) genes have been suggested to play a role. We studied 258 African-American (AA) women with breast cancer and 259 hospital-based controls, as well as 349 Nigerian (NG) female breast cancer patients and 296 community controls. Three microsatellites, ESR1_TA, ESR2_CA and AR_CAG, in the ESR1, ESR2 and AR genes, respectively, were genotyped. Their repeat lengths were then analyzed as continuous and dichotomous variables. Analyses of continuous variables showed no association with breast cancer risk in either AA or NG at ESR1_TA; AA cases had shorter repeats in the long allele of ESR2_CA than AA controls (Mann-Whitney P  = 0.036; logistic regression P  = 0.04, OR  = 0.91, 95% CI 0.83–1.00), whereas NG patients had longer repeats in the short allele than NG controls (Mann-Whitney P  = 0.0018; logistic regression P  = 0.04, OR  = 1.06, 95% CI 1.00–1.11); and AA cases carried longer repeats in the short allele of AR_CAG than AA controls (Mann-Whitney P  = 0.038; logistic regression P  = 0.03, OR  = 1.08, 95% CI 1.01–1.15). When allele sizes were categorized as dichotomous variables, we discovered that women with two long alleles of ESR2_CA had increased risk of breast cancer (OR  = 1.38, 95% CI 1.10–1.74; P  = 0.006). This is the first study to investigate these three microsatellites in hormonal receptor genes in relation to breast cancer risk in an indigenous African population. After adjusting for multiple-testing, our findings suggest that ESR2_CA is associated with breast cancer risk in Nigerian women, whereas ESR1_TA and AR_CAG seem to have no association with the disease among African American or Nigerian women.     

Eleven new microsatellites for hop (Humulus lupulus L.)

We present a new set of 11 polymorphic microsatellite primer sequences for use with Humulus lupulus. Microsatellite‐enriched libraries for GA_n and GT_n types of repeats were produced. Sequencing of 72 clones revealed 42 unique inserts containing microsatellites, out of which 19 primer pairs were designed and microsatellite amplification was tested on 39 wild hops and cultivars. Eleven primer pairs showed single locus amplification with 2–13 alleles, average 7.2, of which 17 unique alleles were discovered. One primer pair amplified too strong stutter bands, one locus was monomorphic and multilocus amplification was obtained with the remaining six primer pairs.     

Isolation and characterization of 20 polynucleotide microsatellite markers in a vulnerable Korean snail, <Emphasis Type="Italic">Ellobium chinense,</Emphasis> using 454 pyrosequencing

A small and air-breathing snail, Ellobium chinense (Ellobiidae), is a vulnerable species by International Union for Conservation of Nature (IUCN). To protect and manage habitat and population of E. chinense, microsatellite markers were developed using 454 pyrosequencing and 20 polymorphic microsatellite markers were identified. A total of 146,704 sequences containing a minimum of four repeat motifs (mean, 631 base pairs) were identified from 499,505 reads. Among 80 loci containing more than nine repeat units, 34 primer sets (42.5 %) produced strong PCR products, of which 20 were polymorphic among 48 samples of E. chinense. All loci exhibited high genetic variability, with an average of 18.9 alleles per locus, and the mean observed and expected heterozygosities were 0.65 and 0.90, respectively. In addition, cross-amplification was tested for all 20 loci in the same family species, Melampus sincaporensis. None of the primer pairs resulted in effective amplification, which might be due to their high mutation rates. Our work demonstrated the utility of next-generation 454 sequencing as a method for the rapid and cost-effective identification of microsatellites. The high degree of polymorphism exhibited by the 20 newly developed microsatellites will be useful in future conservation genetic studies of this species.     

Microsatellites from Clarias batrachus and their polymorphism in seven additional catfish species

We isolated 18 novel microsatellite loci from the walking catfish (Clarias batrachus), and examined their cross‐amplification in seven additional catfish species from three families. Sixteen of the 18 microsatellites were polymorphic in the source species (allele number: 2–10/locus and expected heterozygosity: 0.30–0.87). Moreover, nine of these 18 primer pairs cross‐amplified specific and polymorphic products from the genome of at least six of the seven other catfish species tested. However, the success rate of cross‐species amplification varied from locus to locus, indicating that cross‐species amplification of microsatellites is locus‐dependent.     

Genome-Based Microsatellite Development in the Culex pipiens Complex and Comparative Microsatellite Frequency with Aedes aegypti and Anopheles gambiae

Background

Mosquitoes in the Culex pipiens complex are among the most medically important vectors for human disease worldwide and include major vectors for lymphatic filariasis and West Nile virus transmission. However, detailed genetic studies in the complex are limited by the number of genetic markers available. Here, we describe methods for the rapid and efficient identification and development of single locus, highly polymorphic microsatellite markers for Cx. pipiens complex mosquitoes via in silico screening of the Cx. quinquefasciatus genome sequence.

Methodology/Principal Findings

Six lab colonies representing four Cx. pipiens and two Cx. quinquefasciatus populations were utilized for preliminary assessment of 38 putative loci identified within 16 Cx. quinquefasciatus supercontig assemblies (CpipJ1) containing previously mapped genetic marker sequences. We identified and validated 12 new microsatellite markers distributed across all three linkage groups that amplify consistently among strains representing the complex. We also developed four groups of 3–5 microsatellite loci each for multiplex-ready PCR. Field collections from three cities in Indiana were used to assess the multiplex groups for their application to natural populations. All were highly polymorphic (Mean  = 13.0 alleles) per locus and reflected high polymorphism information content (PIC) (Mean  = 0.701). Pairwise F_ST indicated population structuring between Terre Haute and Fort Wayne and between Terre Haute and Indianapolis, but not between Fort Wayne and Indianapolis. In addition, we performed whole genome comparisons of microsatellite motifs and abundance between Cx. quinquefasciatus and the primary vectors for dengue virus and malaria parasites, Aedes aegypti and Anopheles gambiae, respectively.

Conclusions/Significance

We demonstrate a systematic approach for isolation and validation of microsatellites for the Cx. pipiens complex by direct screen of the Cx. quinquefasciatus genome supercontig assemblies. The genome density of microsatellites is greater in Cx. quinquefasciatus (0.26%) than in Ae. aegypti (0.14%), but considerably lower than in An. gambiae (0.77%).     

Development of microsatellite markers for muskellunge (Esox masquinongy) and cross‐species amplification in two other esocids

We report the development of seven polymorphic microsatellite loci in muskellunge (Esox masquinongy) using an unenriched subgenomic library. Polymorphic loci exhibited 2–11 alleles with observed heterozygosities ranging from 0.190 to 0.917 (n = 24). All seven loci amplified by their respective primer pairs resulted in monomorphic products in northern pike (E. lucius) whereas three loci amplified monomorphic products in grass pickerel (E. americanus americanus); these results imply conservation of flanking sequence but loss of polymorphism between these closely related species. Only one of six microsatellite primers developed in a previous study in northern pike amplified polymorphic products in muskellunge.     

Isolation and characterization of expressed sequence tag-linked microsatellite loci for the European eel (Anguilla anguilla)

A European eel (Anguilla anguilla) expressed sequence tag database consisting of 795 contigs and 4008 singletons was screened for microsatellites sequences. Primers were designed to amplify 96 repeats, of which 86 gave good quality amplification products. Twenty-eight microsatellites were selected for further microsatellite genotyping. Only two loci were found to be monomorphic; out of the 26 polymorphic loci, number of alleles per locus ranged from two to 14, while the observed and expected heterozygosities ranged from 0.05 to 0.93, and from 0.05 to 0.95, respectively. All 28 primer sets tested revealed positive amplification in American eel (Anguilla rostrata).     

Effectiveness of bovine microsatellites in resolving paternity cases in American bison, Bison bison L.

A set of 33 cattle microsatellite primer pairs was tested with the DNA of American bison from a captive population in Belgium and evaluated for usefulness in parentage testing. Two primer sets did not amplify and three were monomorphic. Among the polymorphic markers, the number of alleles ranged from two to nine. Heterozygosity, polymorphism information content (PIC) and probability of exclusion (PE) values were low by comparison with those obtained with the same markers in cattle. Two methods of estimating PE were used, one which assumed equal allele frequencies between parental sexes and another which took into account differences in allele frequencies between parental sexes. An internationally accepted set of nine microsatellites gives cumulative PE values of 0·98 and 0·97, respectively, for the two methods. The potential of this marker set to identify bison × cattle hybrids is discussed. Because bison and cattle have a common ancestor, these microsatellites are a useful way to establish genetic distances and can lead to the construction of phylogenetic trees.     

Rapid development of 36 polymorphic microsatellite markers for Tetranychus truncatus by transferring from Tetranychus urticae

Tetranychus truncatus Ehara is a phytophagous spider mite that is now one of the most important pests of agricultural and economic crops in East and Southeast Asia. However, population genetics and other studies of T. truncatus have been impeded by the lack of microsatellite markers, which are expensive and time-consuming to identify. Previous studies indicated a high potential of cross-amplification of microsatellites in Tetranychus species, meaning that the microsatellite flanking sequences are sufficiently homologous among Tetranychus species that the primers for one species may work in another species. Here, we tested 205 primer pairs designed from the whole genome sequence of Tetranychus urticae Koch, a sister species of T. truncatus, for microsatellite markers in three populations of T. truncatus in China (N = 94). About half (102) of these primer pairs yielded the desired PCR products, 36 of which revealed polymorphism in T. truncatus. Each of the 36 markers harbored between 2 and 23 alleles, with a mean polymorphic information content of 0.589 (0.119–0.922 range). The mean observed and expected heterozygosity across loci and the three populations were 0.468 and 0.628, respectively. Of the 36 primer pairs, 22 also worked in Tetranychus piercei, but only a few of them worked in T. ludeni and T. phaselus. Cross-amplification is thus a cost-effective way to develop microsatellite markers, which can be of great value in population genetics studies.     

GenBank数据库中鳜鱼微卫星位点的筛选及特征分析

微卫星(Microsatellite)是一类由2-6个核苷酸经多次单位串联组成的高度变异重复DNA序列(Schlotterer and Tautz,1992)。它具有按照孟德尔方式分离、突变快、多态信息含量丰富、呈共显性遗传等特点,其核心序列在同一物种中具有保守性,因此,可以根据微卫星的侧翼序列设计合适的引     

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.     

Single locus microsatellites isolated using 5' anchored PCR.

Microsatellites are widely used as genetic markers because they are co-dominant, multiallelic, easily scored and highly polymorphic. A major drawback of microsatellite markers is the time and cost required to characterise them. We have developed a novel technique to reduce this cost by producing a microsatellite-rich PCR profile from genomic DNA which was cloned to yield a genomic library enriched for microsatellites. Sequence data and subsequent allele scoring within pedigrees revealed that these microsatellites retained their original repeat length and segregated normally. This technique permits genomic amplification with only one specific primer. Together with enrichment, the savings in primer costs reduces the cost of microsatellite characterisation considerably.     

Development of microsatellite markers in potato and their transferability in some members of Solanaceae

We have developed thirty new microsatellite markers in potato by screening genomic libraries and ESTs. Genomic libraries of potato cultivar Kufri Bahar were screened for sequences containing microsatellite motifs GA, GT, ACA, ATC, GAA, TAA and GATA. Using flanking sequences, PCR primers were designed for microsatellites identified from genomic libraries and ESTs. Sixteen new primer pairs from genomic libraries and fourteen from ESTs along with seven previously published primer pairs amplified PCR products in the selected genotypes comprising of 65 Solanum tuberosum lines and 14 other species of the potato gene pool. Neighbor-joining tree based on genetic distance matrix developed using microsatellite markers successfully distinguished all these genotypes in the expected size range. Seventeen microsatellites could also be cross-amplified in at least one of the five members of solanaceae, namely tomato, eggplant, pepper, petunia and tobacco. The new microsatellite markers obtained in this study will be useful in various genetic and taxonomic studies in potato and related genomes.     

Isolation,characterization and cross‐species amplification of microsatellite DNA loci in the tropical American yam Dioscorea trifida

We developed microsatellite markers in American yam (Dioscorea trifida). A microsatellite sequence‐enriched genomic library was screened, and after sequencing, primers were designed for 20 microsatellites. Among these, eight primer pairs yielded amplification products that were both interpretable and polymorphic in 24 yam cultivars. The number of alleles per locus ranged from two to 13 and the overall expected heterozygosity was around 0.5. Six of the eight Dioscorea trifida microsatellite loci gave amplification products in other Dioscorea species.     

Japanese quail microsatellite loci amplified with chicken-specific primers

Forty-eight primer pairs for chicken (Gallus gallus) microsatellite loci were tested in polymerase chain reaction (PCR) amplification of Japanese quail (Coturnix japonica) genomic DNA. Amplification products were obtained from 28 primer-pairs (58.3%) after optimizing the PCR conditions. Eleven (22.9%) of these generated specific products and 17 yielded non-specific amplification products. Eight markers (ADL0037, ADL0038, ADL0142, ADL0143, ADL0206, ADL0315, ADL0366, and HUJ0006) were polymorphic and three were monomorphic (ADL0023, ADL0024, and ADL0257) in four Japanese quail populations. Specific amplification products from each of the 11 PCR primers were sequenced. Seven of the eight polymorphic and one of three monomorphic markers contained simple tandem repeats. Six of these microsatellite loci (ADL0037, ADL0315, ADL0142, ADL0143, ADL0366 and ADL0257) may be homologous to the corresponding chicken loci from which the markers were developed.     

Genetic and functional analysis of the DLG4 gene encoding the post-synaptic density protein 95 in schizophrenia

Hypofunction of N-methyl-D-aspartate (NMDA) receptor-mediated signal transduction has been implicated in the pathophysiology of schizophrenia. Post-synaptic density protein 95 (PSD95) plays a critical role in regulating the trafficking and activity of the NMDA receptor and altered expression of the PSD95 has been detected in the post-mortem brain of patients with schizophrenia. The study aimed to examine whether the DLG4 gene that encodes the PSD95 may confer genetic susceptibility to schizophrenia. We re-sequenced the core promoter, all the exons, and 3′ untranslated regions (UTR) of the DLG4 gene in 588 Taiwanese schizophrenic patients and conducted an association study with 539 non-psychotic subjects. We did not detect any rare mutations at the protein-coding sequences of the DLG4 gene associated with schizophrenia. Nevertheless, we identified four polymorphic markers at the core promoter and 5′ UTR and one single nucleotide polymorphism (SNP) at the 3′UTR of the DLG4 gene in this sample. Genetic analysis showed an association of a haplotype (C–D) derived from 2 polymorphic markers at the core promoter (odds ratio = 1.26, 95% confidence interval = 1.06–1.51, p = 0.01), and a borderline association of the T allele of the rs13331 at 3′UTR with schizophrenia (odds ratio = 1.19, 95% confidence interval = 0.99–1.43, p = 0.06). Further reporter gene assay showed that the C-D-C-C and the T allele of the rs13331 had significant lower activity than their counter parts. Our data indicate that the expression of the DLG4 gene is subject to regulation by the polymorphic markers at the core promoter region, 5′ and 3′UTR of the gene, and is associated with the susceptibility of schizophrenia.