Microsatellite genotyping by primer extension and MALDI-ToF mass spectrometry

A novel method for genotyping microsatellite alleles using primer extensions and mass spectrometry analysis has been developed. Following PCR amplification of the target region, a genotyping primer, with its 3′ end directly flanking the microsatellite repeats, was extended by a mixture of dNTPs complementary to the nucleotides composing the microsatellite. The length and molecular weight of extended primers vary with the number of repeats present in the allele(s) under examination. The weights of extension products were determined using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-ToF MS) and used to identify genotypes on the basis of differential primer extension. This is a platform that is not gel based and is amenable to multiplexing and automation. The technique enables identification of heterozygous progeny in which alleles differ by a single trinucleotide repeat. The method is illustrated by genotyping a polymorphic microsatellite identified in an intron of the barleyMlo gene.     

The use of unilateral PCR to identify prominent heteroduplexes formed during PCR of the mouse microsatellite locus D17Mit23

Microsatellite markers are useful tools for understanding the evolutionary history of discrete segments of the mammalian genome. We used the microsatellite marker D17Mit23 to study the portion of the mouse genome known as the t complex, a naturally occurring variant of Chromosome 17. We identified an allelic variant of D17Mit23, which is shared by two forms of the t complex, the t haplotypes t ^w2 and t ^Lub2 . Polymerase chain reaction (PCR) analysis of DNA samples from mice that were heterozygous for either haplotype resulted in gel patterns with prominent bands of higher molecular weight in addition to the bona-fide D17Mit23 alleles. The appearance of these higher molecular weight bands, although consistent with heteroduplex formation, was not diminished through the use of reconditioning PCR. We used a modified form of asymmetric PCR, called “unilateral PCR”, to show that the higher molecular weight bands are heterodu-plexes and to identify their constituent strands. Certain microsatellite motifs may be especially prone to the production of prominent heteroduplex products, and this may lead to the erroneous genotyping of DNA samples.     

Polarity of mutations in tumor-associated microsatellite instability

Many factors have been implicated in influencing the rate of microsatellite mutations, including the length and base composition of the repeat motif, number of repeats, base composition of flanking sequences and, perhaps most importantly, degree of perfection of the repeats. The latter is of clinical relevance, since in both spino-cerebellar ataxia and fragile X syndrome, alleles with imperfect repeats appear to be much more stable than perfect ones. As yet, the relative importance of increased replication slippage and decreased mismatch repair efficiency in the preference of mutations to occur within perfect repeats has not been fully determined. D13S308E is an asymmetric trinucleotide repeat microsatellite with the sequence (CAT)₃CAC(CAT)CAC(CAT)₂CAC(CAT)CAC(CAT) ₁₅ , thus containing two parts: an 11-repeat imperfect portion (underlined above) and a 15-repeat perfect one (bold). We sequenced eight new mutant alleles of D13S308E from three human gastric tumors with instability in this and other microsatellites. In all mutations the size variation occurred exclusively in the perfect part of the microsatellite. These results constitute direct evidence that the molecular basis of microsatellite alterations seen in normal cells is similar to those that occur in human tumors with extensive microsatellite instability. The investigation of mechanisms involved in microsatellite mutations has been handicapped by the fact that they are rare events. The microsatellite instability observed in malignant tumors provides us with a useful general system to study these mechanisms. Received: 24 June 1997 / Accepted 7 October 1997     

Two length variants of the microsatellite FH2295 as markers for body size of female Portuguese water dogs

Genetic studies in purebred Portuguese water dogs (PWD) have previously identified genetic loci controlling skeleton size. The FH2295 genetic marker was reported to control 43.6% of the size variation in this breed. In the present study, we amplified and sequenced the genomic DNA from female PWD of different sizes in the region of the FH2295 genetic marker. Polymerase chain reaction (PCR) products of 700 and 800 bp were generated and sequencing revealed the presence of a microsatellite marker including either 5 or 24 repeats of the tetranucleotide sequence “CTTT”. Dogs were divided into groups based on their genotypes: homozygote for the short allele (II) or homozygote for the long allele (BB) or heterozygote (IB). The smallest dogs were homozygous with 24 repeats and the largest dogs were homozygous with five repeats. Genetic transmission of the microsatellite marker appears to follow Mendelian laws since all puppies born to a homozygous small dog genotyped “BB” included one or two “B” allele. We applied a PCR method to characterize the sequence of the previously indentified dog genetic marker FH2295 and propose that the length of the microsatellite identified could be used as a predictor for the body size of female PWD.     

Abundance and polymorphism of microsatellite markers in the tea tree (Melaleuca alternifolia, Myrtaceae)

 The sequencing of 831 clones from an enriched microsatellite library of Melaleuca alternifolia (Myrtaceae) yielded 715 inserts containing repeat motifs. The majority of these (98%) were dinucleotide repeats or trinucleotide repeats averaging 22 and 8 repeat motifs respectively. The AG/GA motif was the most common, accounting for 43% of all microsatellites. From a total of 139 primer pairs designed, 102 produced markers within the expected size range. The majority of these (93) were polymorphic. Primer pairs were tested on five selected M. alternifolia genotypes. Loci based on dinucleotide repeats detected on average a greater number of alleles (4.2) than those based on trinucleotide repeats (2.9). The loci described will provide a large pool of polymorphisms useful for population studies, genetic mapping, and possibly application in other Myrtaceae. Received: 28 July 1998 / Accepted: 8 October 1998     

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     

Two polymorphic microsatellites in a coding segment of the canine androgen receptor gene

A 0.6-kb segment of exon 1 of the canine androgen receptor gene contains two polymorphic CAG tandem repeats which encode strings of glutamine homopolymers. The number of CAGs in each tandem repeat was determined by (1) polymerase chain reaction (PCR) amplification of a gene segment containing both repeats, (2) cleavage between repeats with restriction enzyme EcoO109I and (3) fractionation of the restriction fragments containing individual CAG repeats by denaturing polyacrylamide gel electrophoresis (PAGE). Individual genomic DNA samples from 80 unrelated dogs (53 males plus 27 females for a total of 107 X chromosomes) contained 10–12 CAGs in the 5′ repeats and 10–13 CAGs in the 3′ repeats. Thirteen distinct androgen receptor genotypes were identified. Eleven (or 41%) of the 27 unrelated females were heterozygous in one or both repeat regions, whereas all male samples produced single bands as expected for X chromosome markers. A total of seven distinct haplotypes contributed to the 13 genotypes. The ‘polymorphism information content’ or PIC for this seven-allele X chromosome marker was 0.67.     

Length polymorphism and allele structure of trinucleotide microsatellites in natural accessions of Arabidopsis thaliana

 The objective of this work was to assess the degree of trinucleotide microsatellite length polymorphism in the selfing species Arabidopsis thaliana. PCR amplifications of 12 microsatellite loci among 49 natural populations revealed between one to eight length variants (alleles) for each locus. The average number of alleles per locus was four and the average genetic diversity index was 0.43. Divergence between length variants was investigated at the nucleotide level. Several observations emerge from the sequence data: (1) for most loci, length polymorphism results only from variations in the number of trinucleotide repeats; (2) for a few others, some variability was noted in the flanking sequences; (3) for compound and interrupted loci containing two arrays of trinucleotide repeats, length variations preferentially affect the longest one. Five of the Arabidopsis thaliana accessions were clearly composed of two sublines. In 2 other accessions, some heterozygous individual plants, probably resulting from recent outcrosses, were found. A phylogenetic tree constructed on the basis of trinucleotide microsatellite allelic diversity shows that genetic relationships among the accessions are not correlated with their geographic origin. Received: 4 November 1997 / Accepted: 3 March 1998     

Genotyping HapSTR loci: phase determination from direct sequencing of PCR products

HapSTRs combine information from a microsatellite (or simple tandem repeat, STR) with one or more single nucleotide polymorphisms in the DNA sequence immediately flanking the STR. These loci may offer increased power for the estimation of demographic parameters, but also present some challenges for data collection and analysis. We describe a process for inferring HapSTR alleles, including the flanking haplotypes, STR alleles and their phase relative to each other, directly from DNA sequence electropherograms of PCR products from heterozygous individuals. Our approach eliminates the need for more costly and time-consuming processes, such as cloning or acrylamide gel electrophoresis to separate alleles prior to sequencing.     

Microsatellites reveal high genetic diversity within colonies of Camponotus ants

In order to characterize the sociogenetic structure of colonies in the carpenter ants Camponotus herculeanus and C. ligniperda, we have developed microsatellite markers. The three loci studied were either fixed for different alleles in the two species or showed different patterns of polymorphisms. Genotyping of workers and males showed that the broods of C. ligniperda include several matrilines, a rare phenomenon in the genus. Five alleles from a locus polymorphic in both species were sequenced from the respective PCR-products. A part of the length variation appeared to be due to changes outside the repeat sequence, and some PCR products of an equal length had a different number of dinucleotide repeats.     

Microsatellite markers from sugarcane (Saccharum spp.) ESTs cross transferable to erianthus and sorghum

Analysis of a sugarcane (Saccharum spp.) EST (expressed sequence tag) library of 8678 sequences revealed approximately 250 microsatellite or simple sequence repeats (SSRs) sequences. A diversity of dinucleotide and trinucleotide SSR repeat motifs were present although most were of the (CGG)_n trinucleotide motif. Primer sets were designed for 35 sequences and tested on five sugarcane genotypes. Twenty-one primer pairs produced a PCR product and 17 pairs were polymorphic. Primer pairs that produced polymorphisms were mainly located in the coding sequence with only a single pair located within the 5′ untranslated region. No primer pairs producing a polymorphic product were found in the 3′ untranslated region. The level of polymorphism (PIC value) in cultivars detected by these SSRs was low in sugarcane (0.23). However, a subset of these markers showed a significantly higher level of polymorphism when applied to progenitor and related genera (Erianthus sp. and Sorghum sp.). By contrast, SSRs isolated from sugarcane genomic libraries amplify more readily, show high levels of polymorphism within sugarcane with a higher PIC value (0.72) but do not transfer to related species or genera well.     

Direct PCR sequencing of dystrophin polymorphic CACA alleles after purification to remove shadow bands.

A method is described that allows the sequencing of polymerase chain reaction (PCR) products containing CACA repeats. The method was tested using a DNA polymorphism that exists at the 3' end of the dystrophin gene. This polymorphism consists of a variation in the length of a CACA dinucleotide repeat. Four alleles from a total of 16 individuals were sequenced at this locus after the DNA sequence had been amplified by the PCR. Five examples of each of the common alleles were sequenced. For each allele all five sequences were the same. The only example of a rare allele was also sequenced. The PCR products of DNA sequences containing dinucleotide repeats consist of a number of bands differing by 2 bp below the most intense main band. Previously, direct sequencing of the PCR products lead to ambiguities and smearing at and above the CACA repeat. In this paper, the main PCR band was cut out of a sequencing gel and directly sequenced to give a clear DNA sequence. Our results indicate that for a particular allele, all individuals had exactly the same DNA sequence. This implies that with the appropriate choice of oligonucleotide primers, polymorphisms could be detected without electrophoresis.     

Development of SNP markers and haplotype analysis of the candidate gene for rhg1, which confers resistance to soybean cyst nematode in soybean

Soybean cyst nematode (SCN; Heterodera glycines Ichinohe) is one of the most destructive pests in the cultivation of soybean (Glycine max (L.) Merr.) worldwide. Markers based on the SCN resistance gene will enable efficient marker-assisted selection (MAS). We sequenced the candidate gene rhg1 in six resistant and two susceptible soybean genotypes and identified 37 SNPs (single nucleotide polymorphisms) among the sequences, of which 11 were in the coding region. Seven of these 11 SNPs led to changes in the amino acid sequence of the gene. The amino acid sequence we obtained differs from the previously published one by a stretch of 26–27 amino acids. Six codominant allele-specific SNP markers based on agarose gel detection were developed and tested in 70 genotypes, among which occurred only nine different haplotypes. Two neutrality tests (Tajima’s D and Fu and Li’s F) were significant for the six SNP loci in the 70 genotypes, which is consistent with intensive directional selection. A strong LD pattern was detected among five SNPs except 2868T > C. Two SNPs (689C > A and 757C > T) formed one haplotype (689C-757C) that was perfectly associated with SCN resistance. The new allele-specific PCR markers located in the alleged sequence of the rhg1 candidate gene, combined with the microsatellite marker BACR-Satt309, will significantly improve the efficiency of MAS during the development of SCN-resistant cultivars.     

Inter-simple sequence repeat (ISSR) amplification for analysis of microsatellite motif frequency and fingerprinting in rice (Oryza sativa L.)

 Inter-simple sequence repeat (ISSR) amplification was used to analyze microsatellite motif frequency in the rice genome and to evaluate genetic diversity among rice cultivars. A total of 32 primers, containing different simple sequence repeat (SSR) motifs, were tested for amplification on a panel of 59 varieties, representative of the diversity of cultivated rice (Oryza sativa L.). The ISSR analysis provided insights into the organization, frequency and levels of polymorphism of different simple sequence repeats in rice. The more common dinucleotide motifs were more amenable to ISSR analysis than the more infrequent tri-, tetra- and penta-nucleotide motifs. The ISSR results suggested that within the dinucleotide class, the poly(GA) motif was more common than the poly(GT) motif and that the frequency and clustering of specific tri- and tetra-nucleotide simple sequence repeats was variable and motif-specific. Furthermore, trinucleotide ISSR markers were found to be less polymorphic than either dinucleotide or certain tetranucleotide ISSR markers, suggesting which motifs would be better targets for microsatellite marker development. The ISSR amplification pattern was used to group the rice genotypes by cluster analysis. These results were compared to surveys of the same varieties for amplified fragment length polymorphism (AFLP), restriction fragment length polymorphism (RFLP) and isozyme markers. The ISSR fingerprint could be used to differentiate the genotypes belonging to either Japonica or Indica sub species of cultivated rice and to dissect finer levels of diversity within each subspecies. A higher percentage of polymorphic bands was produced with the ISSR technique than the AFLP method, based on a similar PCR reaction. Therefore, ISSR amplification proved to be a valuable method for determining genetic variability among rice varieties and for rapidly identifying cultivars. This efficient genetic fingerprinting technique would be useful for characterizing the large numbers of rice accessions held in national and international germplasm centers. Received: 25 May 1998 / Accepted: 17 September 1998     

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     

Evaluating the potential of SSR flanking regions for examining taxonomic relationships in the Vitaceae

Three EST-derived microsatellite loci from Vitis vinifera were amplified and sequenced across eight species of Vitaceae from four different genera. Phylogenetic analysis of the microsatellite’s flanking regions produced informative results in congruence with previous studies. Generic relationships were respected and the data produced sufficient inter-specific variation to distinguish between Cayratia acris and Cayratia saponaria, two very closely related species. Overall, the sequence alignments showed that priming sites were conserved, whereas microsatellite repeats were present in most cases but structurally variable. The sequence data provided information on the evolutionary patterns of various microsatellite repeats and their correlation to evolutionary relationships among taxa. Received: 15 December 2000 / Accepted: 12 April 2001     

Congruence between environmental parameters, morphology and genetic structure in Australia’s most widely distributed eucalypt, Eucalyptus camaldulensis

Eucalyptus camaldulensis is one of the most widely utilised eucalypts. It is also the only eucalypt that occurs across the Australian continent, playing a key ecological role as fauna habitat and in riverbank stabilisation. Despite its ecological and economic importance, uncertainty remains regarding the delineation of genetic and morphological variants. Nine hundred and ninety trees from 97 populations, representing the species’ geographic range were genotyped using 15 microsatellite loci and patterns of diversity compared with restriction fragment length polymorphisms in 29 of these populations. Both markers showed that despite having a riverine distribution, downstream seed dispersal has had less influence than geographic distance on dispersal patterns. Spatial patterns in the distribution of microsatellite genotypes were compared with environmental parameters and boundaries defined by river systems, drainage basins and proposed subspecies. Significant genetic differences among populations within river systems indicated that rivers should not be treated as a single genetic entity in conservation or breeding programmes. Strong geographic trends were evident with 40% of variation in genetic diversity explained by latitude and moisture index. Isolation by distance and significant correlations between genetic distance and environmental parameters for most loci suggest historical factors have had more influence than selection on current patterns of distribution of genetic diversity. Geographic structuring of molecular variation, together with congruence between genetic and morphological variation indicate that E. camaldulensis should be treated as a number of subspecies rather than a single variable taxon. High levels of genetic diversity and geographic trends in the distribution of variation provide a firm basis for further exploration of the species’ genetic resources.     

Reevaluation of the exact CAG repeat length in hereditary cerebellar ataxias using highly denaturing conditions and long PCR

Hereditary cerebellar ataxias, including spinocerebellar ataxia type I (SCA1), dentato-rubro-pallidoluysian atrophy (DRPLA), and Machado-Joseph disease (MJD), have been associated with unstable CAG repeats. The length of the CAG repeat is a major factor in determining the age of onset of these diseases. In electrophoresis through acrylamide gels with formamide, the CAG repeat length following the polymerase chain reaction (PCR) coincides with the sequence-determined repeat length after subcloning. However, without formamide, PCR products with long CAG repeats appear 1–4 repeats shorter than when electrophoresed with formamide, and the repeat lengths are variable. In addition, the larger the CAG repeats are, the more difficult are the PCR reactions. A mixture containing thermostable Taq and Pwo DNA polymerases (so-called “long PCR”) is much more sensitive than that with Taq polymerase alone in detecting expanded CAG repeats. Therefore, highly denaturing conditions, especially formamide gel electrophoresis, and the “long PCR” protocol should be used to evaluate the exact CAG repeat length. We have used these principles to detect unstable CAG repeats. The normal ranges are 14–34 repeats for MJD, 6–31 repeats for DRPLA, and 21–32 repeats for SCA1. Received: 29 August 1995 / Revised: 12 October 1995     

Nature of polymorphisms in 16S-23S rRNA gene intergenic transcribed spacer fingerprinting of Bacillus and related genera

The intergenic transcribed spacers (ITS) between the 16S and 23S rRNA genetic loci are frequently used in PCR fingerprinting to discriminate bacterial strains at the species and intraspecies levels. We investigated the molecular nature of polymorphisms in ITS-PCR fingerprinting of low-G+C-content spore-forming bacteria belonging to the genera Bacillus, Brevibacillus, Geobacillus, and Paenibacillus: We found that besides the polymorphisms in the homoduplex fragments amplified by PCR, heteroduplex products formed during PCR between amplicons from different ribosomal operons, with or without tRNA genes in the ITS, contribute to the interstrain variability in ITS-PCR fingerprinting patterns obtained in polyacrylamide-based gel matrices. The heteroduplex nature of the discriminating bands was demonstrated by fragment separation in denaturing polyacrylamide gels, by capillary electrophoresis, and by cloning, sequencing, and recombination of purified short and tRNA gene-containing long ITS. We also found that heteroduplex product formation is enhanced by increasing the number of PCR cycles. Homoduplex-heteroduplex polymorphisms (HHP) in a conserved region, such as the 16S and 23S rRNA gene ITS, allowed discrimination of closely related strains and species undistinguishable by other methods, indicating that ITS-HHP analysis is an easy and reproducible additional tool for strain typing.     

Family and population studies on the human pepsinogen A multigene family

Summary Human pepsinogen A (PGA) displays highly polymorphic isozymogen patterns after polyacrylamide gel electrophoresis and activity staining. The patterns differ with respect to the presence and the relative intensity of the individual fractions. Family studies strongly suggest that these isozymogen patterns are encoded by allelic haplotypes, encompassing different numbers and types of PGA genes. In this paper, we confirm the essential features of this multigene model. We establish the relationship between the haplotypes and the corresponding isozymogen patterns by determination of the PGA polymorphism at both the DNA and the protein level in 117 Dutch individuals, 60 of whom were unrelated. The combination of HindIII and EcoRI restriction fragment length polymorphisms (RFLPs) has enabled us to define different haplotypes, which are shown to segregate within families. Most genes are characterized by their specific EcoRI fragments. The HindIII RFLP is in strong linkage disequilibrium with PGA genes showing strong expression of the relevant isozymogen. Although a general picture of the relationship between genotypes and phenotypes is emerging, there are exceptions, suggesting that rare haplotypes evolve by unique crossover events.