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

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

A highly informative CA/GT repeat polymorphism in intron 38 of the human neurofibromatosis type 1 (NF1) gene

We describe a polymorphic microsatellite in intron 38 of the neurofibromatosis type 1 (NF1) gene. The microsatellite consists of a CA/GT dinucleotide repeat detecting 8 alleles; it has a heterozygosity of 82 %.     

Coding versus intron variability: extremely polymorphic HLA-DRB1 exons are flanked by specific composite microsatellites, even in distant populations

Although microsatellite typing is the dominant method in genome research and indirect gene diagnosis, precise relationships of exonic and adjacent simple repeat polymorphisms are not known. We investigated exon 2 sequences of HLA-DRB1 genes and their neighbouring (GT)_n(GA)_m repeats including the intervening single copy spacer. DRB1 is the most polymorphic protein-coding locus in man and all vertebrates investigated. The entire DRB1 variability exists in exon 2. DRB1 genes in different haplotype groups (DR1, DR51, DR52, DR8 and DR53) are accompanied by characteristic modifications of the (GT)_n(GA)_m block (3′ to group-specific single copy spacers). Among more than 520 alleles analysed, > 100 different types of microsatellites were observed. The perfect (GT)_n and (GA)_m blocks vary in length and may be partly ‘degenerated’, mostly in a subgroup-specific manner. Interestingly, the extent of microsatellite diversity varies in given DRB1 alleles. While the microsatellites of the DR7, DR9 alleles and in the DR1 group are virtually invariant, in DR4 and DR13, in particular, simple repeats appear hypervariable with at least 15 or 17 different length alleles, respectively. Comparing Caucasians, Bushmen and South American Indians, the microsatellite variation in identical DRB1 alleles (e.g. DRB1*0102, 03 011, 1302) is smaller than within any of the DR groups in Caucasians. Taken together, extremely polymorphic DRB1 exons evolve in concert with certain variants of an exceptionally well-preserved microsatellite. Received: 8 October 1996     

Abundant (A)n·(T)n mononucleotide repeats in the pig genome: linkage mapping of the porcine APOB, FSA, ALOX12, PEPN and RLN loci

A computer analysis revealed that the mononucleotide repeat (A)_n-(T)_n is about five times as common as (CA)_n-(GT)_n repeats in the porcine genome, with frequency estimates of one every 7kb and 30kb, respectively. Seven mononucleotide repeats with n= 12–25 located close to coding sequences were analysed for polymorphism using polymerase chain reaction (PCR) amplification and polyacrylamide gel electrophoresis. All loci were variable with 3–6 alleles and heterozygosities of 0.26–0.69 based on investigation of 10 unrelated pigs (two wild boars and eight domestic sows). Repeat length correlated with degree of polymorphism. A comparison with (CA)_n-(GT)_n polymorphisms suggested that the number of repeat units rather than the total length of the repeat region is the common denominator that governs polymorphism at both mono- and dinucleotide repeat loci. (A)_n-(T)_n polymorphisms allowed linkage mapping of relaxin to chromosome 1, apolipoprotein B to chromosome 3, aminopepti-dase N to chromosome 7, arachidonate 12-lipoxygenase to chromosome 12, and follistatin to chromosome 16. The rich abundance of potentially informative (A)_n-(T)_n repeats will increase the chances of finding a PCR-based marker near any DNA sequence of interest.     

Novel members and germline polymorphisms in the human T-cell receptor Vb6 family

The human T-cell receptor (Tcr) Vb6 family has been scrutinized for polymorphisms, both in coding as well as in intronic sequences by polymerase chain reaction (PCR), subsequent multiple electroblot hybridizations, and sequence analysis. Multiplex PCR is an efficient means of screening for Tcr variability. Four novel loci could be distinguished and several new alleles are described including two pseudogenes. The Vb6 family is characterized by an intronic stretch of simple repetitive (gt)_n sequences. These elements are hypervariable, especially in the Vb6.7 subfamily, where they are particularly long. The unexpected persistence of simple repetitive sequences in Tcr and major histocompatibility complex (MHC) class II genes over extended periods of the vertebrate evolutionary history can be interpreted in parallel terms in both gene families.The nucleotide sequence data reported in this paper have been submitted to the nucleotide sequence database GenBank and have been assigned the accession numbers M97503–97505.     

Vb6 T-cell receptor elements in artiodactyls: conservation and germline polymorphisms

By use of primers specific for human T-cell receptor (Tcr) Vb6 gene elements, a typing system for Tcr Vb variability in several artiodactyl species has been established. The amplified polymorphic locus is homologous to the human Vb6 gene element. Like the human counterpart, the artiodactyl Vb6 element contains a polymorphic intronic simple (gt)_n repeat stretch. Extensive length polymorphism of this simple repeat sequence in some artiodactyls should allow efficient association studies in a multiplex approach, especially including MHC class II genes. On the protein level the Vb regions display little variability in the inter-species comparison among artiodactyls. The amino acid substitutions are not concentrated in the putative complementarity determining regions, suggesting evolutionary conservation. In addition, the simple repetitive element has been preserved in the same genomic location for more than 7×10⁷ years. Similar evolutionary persistence has already been demonstrated for a (gt)_n(ga)_m repeat stretch in the second intron of the MHC-DRB locus. The reasons for these parallel developments in evolution are so far not clear, but they may point to a biological meaning if not function of the intronic simple repeat element.     

Polymorphic microsatellite DNA amplification customized for chimpanzee paternity testing

DNA segments containing GT/AC dinucleotide repeats in the chimpanzee (Pan troglodytes) genome were screened. Thirteen transformedE. coli colonies were identified with the (GT)₁₀ probe to have chimpanzee DNA fragments containing (GT)_n repeats. These potentially polymorphic (variable n) DNA segments were sequenced. Primers for the polymerase chain reaction (PCR) amplifying these DNA segments were designed. Six pairs of primers yielded polymorphic PCR products. Three of them revealed considerable length polymorphisms and heterozygosities in a group of captive chimpanzees. For studies on chimpanzees in the wild and in captivity, these primers should be useful for paternity testing, for investigating genetic variations, and for improving the genetic maintenance of breeding colonies. The strategy adopted in the present study to obtain PCR primers amplifying polymorphic microsatellite DNA segments may well be applicable to almost all eukaryotic organisms.     

Microsatellite DNA loci for Western Hemlock [Tsuga heterophylla (Raf.) Sarg]

Polymorphic microsatellite loci were developed for Western Hemlock [Tsuga heterophylla (Raf.) Sarg], a prominent forest tree species in Western North America. Microsatellite‐enriched libraries were screened for (CA)_n dinucleotide repeats from which 33 positive clones were sequenced. Polymerase chain reaction (PCR) primers for 16 microsatellite loci were prepared and tested against DNA from unrelated Western Hemlock trees. The 12 most informative microsatellite loci are reported here. From four to 22 alleles per locus were observed, with an average expected heterozygousity of 0.799.     

Different instability of the CAG microsatellite in two haplotype groups of human mitochondrial DNA polymerase gamma

Two single nucleotide polymorphisms of the mitochondrial DNA polymerase gamma gene (POLG1), rs2238296 (T/C) and rs758130 (T/C), were analyzed in individuals of different ethnicity (Russians and Buryats) with known genotypes of the CAG microsatellite located in the same gene. It was shown that microsatellite alleles with repeat numbers other than 10 were significantly more frequent within the TT haplotype. A phylogenetic analysis of human and chimpanzee POLG1 intron 2 sequences suggested that the haplotype TT, which is more heterogeneous regarding the CAG repeat polymorphism, is evolutionally younger than the haplotype CC. These data may be useful in the further research of the association between the CAG microsatellite polymorphism of POLG1 and male infertility.     

Hypervariability of intronic simple (gt)n(ga)m repeats in HLA-DRB genes

We have investigated the extent of DNA variability in intronic simple (gt)_n(ga)_m repeat sequences and correlated this to sequence polymorphisms in the flanking exon 2 of HLA-DRB genes. The polymerase chain reaction (PCR) was used to amplify a DNA fragment containing exon 2 and the repeat region of intron 2. The PCR products were separated on sequencing gels in order to demonstrate length hypervariability of the (gt)_n(ga)_m repeats. In a parallel experiment, the PCR products were cloned and sequenced (each exon 2 plus adjacent simple repeats) to characterize the simple repeats in relation to the HLA-DRB sequences. In a panel of 25 DRB1, DRB4, and DRB5 alleles new sequences were not detected. Restriction fragment length polymorphism (RFLP) subtyping of serologically defined haplotypes corresponds to translated DNA sequences in 85% of the cases, the exceptions involving unusual DR/DQ combinations. Many identical DRB1 alleles can be distinguished on the basis of their adjacent simple repeats. We found group-specific organization of the repeats: the DRw52 supergroup repeats differ from those of DRB1*0101, DRB4*0101, and DRB5*0101 alleles and from those of pseudogenes. Finally, we amplified baboon DNA and found a DRB allele with extensive similarity to DRB1 sequences of the DRw52 supergroup. The simple repeat of the baboon gene, however, resembles that of human pseudogenes. In addition to further subtyping, the parallel study of polymorphic protein and hypervariable DNA alleles may allow conclusions to be drawn on the relationships between the DRB genes and perhaps also on the theory of trans-species evolution.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number M 34258.     

Population structure of nuclear and mitochondrial DNA variation among humpback whales in the North Pacific

The population structure of variation in a nuclear actin intron and the control region of mitochondrial DNA is described for humpback whales from eight regions in the North Pacific Ocean: central California, Baja Peninsula, nearshore Mexico (Bahia Banderas), offshore Mexico (Socorro Island), southeastern Alaska, central Alaska (Prince Williams Sound), Hawaii and Japan (Ogasawara Islands). Primary mtDNA haplotypes and intron alleles were identified using selected restriction fragment length polymorphisms of target sequences amplified by the polymerase chain reaction (PCR–RFLP). There was little evidence of heterogeneity in the frequencies of mtDNA haplotypes or actin intron alleles due to the year or sex composition of the sample. However, frequencies of four mtDNA haplotypes showed marked regional differences in their distributions (Φ_ST = 0.277; P < 0.001; n = 205 individuals) while the two alleles showed significant, but less marked, regional differences (Φ_ST = 0.033; P < 0.013; n = 400 chromosomes). An hierarchical analysis of variance in frequencies of haplotypes and alleles supported the grouping of six regions into a central and eastern stock with further partitioning of variance among regions within stocks for haplotypes but not for alleles. Based on available genetic and demographic evidence, the southeastern Alaska and central California feeding grounds were selected for additional analyses of nuclear differentiation using allelic variation at four microsatellite loci. All four loci showed significant differences in allele frequencies (overall F_ST = 0.043; P < 0.001; average n = 139 chromosomes per locus), indicating at least partial reproductive isolation between the two regions as well as the segregation of mtDNA lineages. Although the two feeding grounds were not panmictic for nuclear or mitochondrial loci, estimates of long-term migration rates suggested that male-mediated gene flow was several-fold greater than female gene flow. These results include and extend the range and sample size of previously published work, providing additional evidence for the significance of genetic management units within oceanic populations of humpback whales.     

A microsatellite within the bovine K-casein gene reveals a polymorphism correlating strongly with polymorphisms previously described at the protein as well as the DNA level

The polymorphism of a (TA)n(CA)n repeat microsatellite present in the third intaron of the bovine K-casein gene (CASK) has been investigated. The existence of six alleles differing only in the number of dinucleotide repeats has been established. A total of 330 animals belonging to nine different pure bred Bos Taurus French breeds or to a cross-bred Bos taurus*Bos indicus population (Créole) were genotyped. The distribution of the microsatellite alleles was examined and clear breed differences were noted. Genotyping of animals by isoelectric focusing (IEF) or restriction fragment length polymorphism (RFLP) (TaqI) was performed, in order to examine the relationship of the microsatellite polymorphism to other previously described CASK polymorphisms, at the protein and DNA levels. Strong correlation was seen, indicating that evolution of the various polymorphisms was not independent, and nine CASK haplotypes were observed.     

Use of short microsatellites from database sequences to generate polymorphisms among Lycopersicon esculentum cultivars and accessions of other Lycopersicon species

A search of nearly 2000 sequences from Solanaceae species in the EMBL and Genbank databases yielded 220 microsatellites. Among these were 80 microsatellites from 675 Lycopersicon entries. Dinucleotide repeats, as well as (CAA)_n and (TAA)_n repeats, were over-represented in non-coding DNA. The other trinucleotide repeats were predominantly found in exonic DNA. PCR analysis of 44 of the microsatellite-containing Lycopersicon loci identified 36 primer pairs that yielded well-scorable fragments, or groups of fragments, in L. esculentum cultivars and accessions of Lycopersicon species. Twenty-nine of these amplified bands that were polymorphic among the four Lycopersicon species. Ten primer pairs generated polymorphic bands among seven tomato cultivars. Upon examining the number of microsatellites and the degree of polymorphisms in relation to the repeat type and motif, the type of DNA the microsatellite resided in, the length of the microsatellite, and the presence of imperfections in the microsatellite, only two significant correlations were found. (i) Imperfect repeats were less polymorphic among species than perfect repeats. (ii) The percentage of loci polymorphic among cultivars increased from 6% for the shortest loci (with eight or less repeat units) to 60% for the group with the longest repeats (12 repeat units or longer). Among the species, however, all length classes contained about 83% polymorphic loci. In general, 2–4 alleles were found for each locus among the samples of the test set. In a few cases, up to eight alleles were found. A combination of these microsatellite loci can therefore be useful in distinguishing cultivars of tomato, which are genetically very closely related to each other. Received: 9 August 1996 / Accepted: 23 August 1996     

Allelic variation at a hypervariable compound microsatellite locus in the ascomycete Ascochyta rabiei

The genome of the fungal chickpea pathogen Ascochyta rabiei was screened for polymorphisms by microsatellite-primed PCR. While ethidium-bromide staining of electrophoretically separated amplification products showed only limited polymorphism among 24 Tunisian A. rabiei isolates, Southern hybridization of purified PCR fragments to restriction digests of fungal DNA revealed polymorphic DNA fingerprints. One particular probe that gave rise to a hypervariable single-locus hybridization signal was cloned from the Syrian isolate AA6 and sequenced. It contained a large compound microsatellite harbouring the penta- and decameric repeat units (CATTT)_n, (CATTA)_n, (CATATCATTT)_n and (TATTT)_n. We call this locus ArMS1 (Ascochyta rabiei microsatellite 1). Unique flanking sequences were used to design primer pairs for locus- specific microsatellite amplification and direct sequencing of additional ArMS1 alleles from Tunisian and Pakistani isolates. A high level of sequence variation was observed, suggesting that multiple mutational mechanisms have contributed to polymorphism. Hybridization and PCR analyses were performed on the parents and 62 monoascosporic F₁ progeny derived from a cross between two different mating types of the fungus. Progeny alleles could be traced back to the parents, with one notable exception, where a longer than expected fragment was observed. Direct sequencing of this new length allele revealed an alteration in the copy number of the TATTT repeat [(TATTT)₅₃ to (TATTT)₆₅], while the remainder of the sequence was unchanged. Received: 11 March 1997 / Accepted: 21 June 1997     

The bovine genome contains polymorphic microsatellites

Dinucleotide repeats constitute so-called microsatellites of the human and other eukaryotic genomes. Microsatellite polymorphisms can be identified through the amplification of the microsatellite DNA using the polymerase chain reaction (PCR), followed by resolution of the amplified DNA fragments on a polyacrylamide sequencing gel. We performed a preliminary sequence database search to identify bovine sequences containing (CA)n, (AC)n, (GT)n, or (TG)n blocks, with n greater than or equal to 6. This search yielded 10 sequences containing one or two of the specified repeat blocks and often additional dinucleotide repeat blocks. One of the microsatellite-containing regions has been sequenced twice from independent clones and the reported sequences showed variation in the number of repeats. PCR-amplified fragments of another sequence, the gene for steroid 21-hydroxylase, ranged from 186 to 216 nucleotides in 43 unrelated animals. The database search, as well as the hypervariable microsatellite in the bovine steroid 21-hydroxylase gene, indicates that dinucleotide blocks may be an abundant source of DNA polymorphism in cattle.     

The use of microsatellite markers for detection of genetic diversity in barley populations

 A barley lambda-phage library was screened with (GA)n and (GT)n probes for developing microsatellite markers. The number of repeats ranged from 2 to 58 for GA and from 2 to 24 for GT. Fifteen selected microsatellite markers were highly polymorphic for barley. These microsatellite markers were used to estimate the genetic diversity among 163 barley genotypes chosen from the collection of the IPK Genebank, Germany. A total of 130 alleles were detected by 15 barley microsatellite markers. The number of alleles per microsatellite marker varied from 5 to 15. On average 8.6 alleles per locus were observed. Except for GMS004 all other barley microsatellite markers showed on average a high value of gene diversity ranging from 0.64 to 0.88. The mean value of gene diversity in the wild forms and landraces was 0.74, and even among the cultivars the gene diversity ranged from 0.30 to 0.86 with a mean of 0.72. No significant differences in polymorphism were detected by the GA and GT microsatellite markers. The estimated genetic distances revealed by the microsatellite markers were, on average , 0.75 for the wild forms, 0.72 for landraces and 0.70 among cultivars. The microsatellite markers were able to distinguish between different barley genotypes. The high degree of polymorphisms of microsatellite markers allows a rapid and efficient identification of barley genotypes. Received: 26 November 1997 / Accepted: 19 January 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     

Comparative analysis of estrogen receptor gene polymorphisms in apes

Polymorphic microsatellite repeats in the promoter region of estrogen receptor α gene (ESRα and the intron 6 region of estrogen receptor β gene (ESRβ) have been reported in human populations. To examine the evolutional state of both repeats, we surveyed the corresponding regions in DNA sequences from the following great apes and gibbons: 56 chimpanzees, 3 bonobos, 16 gorillas, 20 orangutans and 60 gibbons (four species: 17 of Hylobates agilis, 11 of H. lar, 15 of H. muelleri, and 17 of H. syndactylus). In the corresponding region of the TA repeat of human ESRα, chimpanzees and bonobos had two motifs in the repeat tract, (TA)_7–9 and (CA)_4–6. Gorillas had the (TA)_9–10 repeat tracts and orangutans had monomorphic (TA)₇ repeats. Although all great apes maintained the TA expansion, all gibbon sequences contained (TA)₂, implying that the CA dinucleotide expansion arose in the ancestor of chimpanzees and bonobos. The nucleotide sequences of ESRβ showed a very complex repeat pattern in apes. The human sequences had a non-variable preceding sequence at (CA) _n , (GA)₂(TA)₈(CA)₄(TA). In apes that region included {(TA) _n (CA) _n } _n . Gibbon sequences included (TATG) _n and (TATC) _n and no regular construction was observed. A deletion event in the reverse primer site seems to have occurred in the orangutan lineage. In addition, a great diversity of allele length was detected in each gibbon species.     

Characterization of highly variable (GA/CT) n microsatellites in the bur oak,Quercus macrocarpa

The objective of this study was to ascertain the usefulness of polymerase chain reaction (PCR)-based microsatellite analysis for studying pollination and parentage in a wind-pollinated temperate tree. A small insert genomic library of the bur oak (Quercus macrocarpa) was constructed and screened for the presence of (CA/GT) _n and (GA/CT) _n repeats. The proportion of positive clones yielded estimates of 3×10⁵ such dinucleotide repeats per genome, roughly comparable to abundances reported in other eukaryotic genomes. Thirteen positive clones were sequenced. In contrast to mammalian genomes, the (GA/CT) _n motif was more abundant than the (CA/GT) _n motif in these clones. The (GA/CT) _n repeats also showed longer average repeat length (mean n=16.2 versus 7.3), suggesting that they are better candidates for yielding polymorphic genetic markers in oak genomes. Indeed, a survey of adult bur oaks and offspring in a small stand in northern Illinois at 3 of these (GA/CT) _n microsatellite loci revealed Mendelian inheritance and extremely high levels of polymorphism, with the number of alleles at each locus ranging from 11–20 and heterozygosity ranging from 0.66 to 0.75. These results, indicating that (GA/CT) _n microsatellites are both abundant and highly polymorphic in the bur oak genome, suggest that such genetic markers have tremendous potential for applications for studies of parentage, pollination and dispersal in temperate trees.     

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.