Abundance, polymorphism and genetic mapping of microsatellites in rice

Dinucleotide microsatellites have been characterized and used as genetic markers in rice. Screening of a rice genomic library with poly(dG-dA)·(dC-dT) and poly(dG-dT)·(dC-dA) probes indicated that (GA)_n repeats occurred, on average, once every 225 kb and (GT)_n repeats once every 480 kb. DNA sequencing of ten randomly selected microsatellites indicated that the numbers of repeats ranged from 12 to 34 and that the patterns of microsatellites in rice were similar to those of humans and other mammals. Primers to these microsatellite loci as well as to four published microsatellite-containing sequences have been designed and degrees of polymorphism has been examined with 20 rice accessions. Multiple alleles, ranging from 5 to 11, have been observed at all the microsatellite loci in 20 rice accessions. Alleles specific to two cultivated subspecies, indica and japonica, were found in some microsatellite loci. Heterozygosity values of all the microsatellite markers were significantly higher than those of RFLP markers, based upon a parallel comparison. Ten microsatellite loci have been genetically mapped to four rice chromosomes. The genomic distribution of microsatellites appears to be random in rice.     

The incidence of mini- and micro-satellite repetitive DNA in the canine genome

We have estimated the incidence of microand mini-satellites in the dog genome. A genomic phage library from canine liver, with an average insert size of 16 kb, was screened to detect potentially polymorphic microand mini-satellite sequences, which may be useful for the development of markers of inherited diseases, for fingerprinting, or for population genetics. Synthetic oligonucleotide probes were used to search for microsatellite sequences, and minisatellites were investigated with eight heterologous VNTR probes. (CA)_n.(GT)_n sequences were by far the most frequent, with a calculated average distance between consecutive loci of 42 kb. The average distance between loci of tri- or tetra-nucleotide repeats was about 330 kb. Mean inter-locus distances were 320 kb for (GGC)_n, 205 kb for (GTG)_n, 563 kb for (AGG)_n, 320 kb for (TCG)_n, 233 kb for (TTA)_n, 384 kb for (CCTA)_n, 368 kb for (CTGT)_n, 122 kb for (TTCC)_n, 565 kb for (TCTA)_n, and 229 kb for (TAGG)_n. Cross-hybridization with eight human minisatellite probes was found at average distances of 1400 kb; only one did not hybridize at all. We conclude that the di-, tri and tetra-nucleotide short tandem repeats, as well as some minisatellite sequences, are potentially useful as genetic markers, for mapping of the canine genome, and also for paternity testing and the analysis of population characteristics.     

Heterogeneities in the distribution of (GACA)n simple repeats in the karyotypes of primates and mouse

Summary Tandemly organized simple repetitive sequences are widespread in all eukaryotes. The organization of the simple tetrameric (GACA)_n sequences at chromosomal loci has been investigated using in situ hybridization with chemically pure oligonucleotide probes. Both biotin- and digoxigenin-attached (GACA)₄ probes reveal specific hybridization signals over the short arms of all acrocentric autosomes in man. In the other examined primates the NOR-bearing autosomes could be detected by in situ hybridization with (GACA)₄, and a major concentration of the GACA simple repeats could be observed on the Y chromosome in the gibbon and mouse; the hybridization site in the gibbon Y chromosome coincides particularly with the silver-stainable NOR. In the past, accumulations of (GACA)_n sequences were demonstrated mainly on vertebrate sex chromosomes. Therefore, the organization of GACA simple sequences is discussed in the context of their evolutionary potential accumulation and the possible linkage with the primate rDNA loci.     

Polymorphic microsatellite loci of the rat (Rattus norvegicus)

The EMBL and GenBank DNA databases were searched for microsatellite sequences of the rat containing dinucleotide repeats of (CA)_n and (GA)_n. Among those obtained, 23 sequences were analyzed by polymerase chain reaction to examine the size variation of the amplified fragment in inbred rat strains. All of the 23 microsatellite sequences varied in size among the strains tested. The 23 microsatellite loci in a pair of substrains separated from the same progenitor strain were then analyzed. Fragments identical in size were observed in all loci of the two substrains, indicating the stability of the microsatellite over a large number of generations. The microsatellite loci, therefore, should be useful markers for linkage analyses in the rat.     

Polymerase chain reaction primers for polymorphic microsatellite loci from the túngara frog Physalaemus pustulosus

We developed eight PCR?primer pairs of polymorphic microsatellite loci for the túngara frog Physalaemus pustulosus. Genomic libraries were enriched for one of four microsatellite repeat sequences (CA_n, GA_n, ATG_n and TAGA_n). Following characterization of microsatellite loci by sequencing, primers were designed and PCR conditions optimized. Microsatellite PCR‐amplification was tested in 37 frogs from 8 populations in Costa Rica and Panama. Primer sequences, PCR conditions, allelelic diversities and observed as well as expected heterozygosities in the screened populations are described.     

Variable (CA/GT)n simple sequence repeat DNA in the alga Chlamydomonas

A partial genomic DNA library of Chlamydomonas reinhardtii was screened with an (AC)₁₁ probe for the presence of (CA/GT)_n simple sequence repeats (SSRs). Based on the frequency of these repeats in the partial genomic library, we estimate that (CA/GT)_n repeats occur at a rate of about one every 17.7 kb in the C. reinhardtii genome. Ten positive clones were sequenced and four polymerase chain reaction (PCR) primer sets flanking (CA/GT)_n sequences were constructed for four loci. The PCR was used to specifically amplify these regions from multiple isolates of C. reinhardtii. All four loci were highly polymorphic in the C. reinhardtii isolates. A simple Mendelian inheritance pattern was found for all four loci, which showed 2:2 segregation in the tetrads resulting from a cross between C. reinhardtii and C. smithii. Our results suggest that these simple sequence repeat DNA loci will be useful for identity testing, population studies, linkage analysis, and genome mapping in Chlamydomonas.     

Isolation and characterisation of 11 tetranucleotide microsatellite loci in the common genet (Genetta genetta)

We report the isolation and characterization of 11 polymorphic tetranucleotide microsatellite loci in the common genet (Genetta genetta) from genomic libraries enriched for (AAAG) _n and (AGAT) _n repeat sequences. We chose to develop tetranucleotide repeats because they can be scored less ambiguously. In a sample of 25 individuals, we observed between four and thirteen alleles per locus and their observed and expected heterozygosities ranged from 0.60 to 0.84 and from 0.68 to 0.92, respectively. All genotypic frequencies conformed to Hardy–Weinberg equilibrium expectations and there were no instances of linkage disequilibrium detected between pairs of loci. These loci will be of use in studies of population genetics, historical demography, and molecular ecology of the common genet.     

The use of microsatellite DNA markers for soybean genotype identification

Conventional morphological and pigementation traits, as well as disease resistance, have been used to distinguish the uniqueness of new soybean cultivars for purposes of plant variety protection. With increasing numbers of cultivars and a finite number of conventional characters, it has become apparent that such traits will not suffice to establish uniqueness. The objective of this work was to provide an initial evaluation of microsatellite or simple-sequence-repeat (SSR) DNA markers to develop unique DNA profiles of soybean genotypes. Microsatellites are DNA sequences such as (AT) _n /(TA) _n and (ATT) _n /(TAA) _n that are composed of tandemly repeated 2–5-basepair DNA core sequences. The DNA sequences flanking microsatellites are generally conserved allowing the selection of polymerase chain reaction (PCR) primers that will amplify the intervening SSR. Variation in the number of tandem repeats, n, results in PCR product length differences. The SSR alleles present at three (AT) _n /(TA) _n and four (ATT) _n /(TAA) _n loci were determined in each of 96 diverse soybean genotypes. Between 11 and 26 alleles were found at each of the seven loci. Only two genotypes had identical SSR allelic profiles and these had very similar pedigrees. The gene diversity for the seven markers averaged 0.87 for all 96 genotypes and 0.74 for a subset of 26 North American cultivars. These are much higher than soybean gene diversity values obtained using RFLP markers, and are similar to the average values obtained for human microsatellite markers. SSR markers provide an excellent complement to the conventional markers that are currently used to characterize soybean genotypes.     

Short tandem repeat polymorphic markers for the rat genome from marker-selected libraries

In an effort to generate a genome-wide set of high-quality polymorphic markers for the rat, we used the marker-selection method, which has already been proven useful for the development of markers, especially for the human genome. Small-insert (300–900 bp) rat genomic libraries were constructed with an estimated complexity of three genome equivalents and enriched for short tandem repeat sequences (STRs). The enriched libraries were found to contain 45% (CA)_n and 27% (GATA)_n, representing at least a 50-fold enrichment over unselected small insert genomic libraries. A subset of 2160 STR-containing clones, primarily of the (GATA)_n class of repeats, were sequenced. PCR primers flanking the repeats were synthesized from some of the sequences from the (CA)_n and (GATA)_n classes of STRs and tested for polymorphism in a panel of eight inbred rat strains. This strategy yielded 147 polymorphic markers, which mapped with high odds to all chromosomes by linkage in three F₂ populations. The integration of these STR markers with other rat genetic markers and mapping reagents will facilitate the mapping of disease genes in the rat and the identification of loci associated with complex mammalian phenotypes. Received: 6 May 1998 / Accepted: 6 August 1998     

Potential of (GATA)n microsatellites from rice for inter- and intra-specific variability studies

Background

The microsatellite, (GATA)_n has been frequently used for DNA fingerprinting. However, very few attempts have been made to analyze (GATA)_n-containing loci in rice.

Results

Three polymorphic (GATA)_n-harboring loci viz. OS1A6, OS1H10 and OS2E7, containing 7–13 repeat motifs were identified from a genomic library of a cultivated rice, Oryza sativa var. Basmati-370 using oligonucleotide probe (GATA)₄. When (GATA)_n flanking primers were used to screen 26 wilds (representing different genomes of rice), 16 cultivars, 47 Indian elite rice varieties and 37 lines resistant/susceptible to bacterial blight, up to 22 alleles were obtained at an individual locus. Also, interestingly the bacterial blight resistant lines clustered into a separate group from the remaining rice genotypes, when a dendrogram was constructed based on the polymorphism obtained at the three loci. This may be due to the partial homology of the clones OS1H10 and OS2E7 to regions encoding O. longistaminata receptor kinase-like protein and pathogenesis-related protein. The ability of these O. sativa flanking primers to amplify DNA of maize, wheat, barley and oat indicates that these (GATA)_n-containing loci are conserved across different cereal genera.

Conclusions

The large allele number obtained reveals the potential of (GATA)_n-containing loci as powerful tools to detect simple sequence length polymorphism (SSLP). The (GATA)_n-flanking primers were not only useful in distinguishing between closely related genotypes, but could also be used for cross-species amplification and are also conserved across different cereal genera. These loci could also cluster the bacterial blight resistant/susceptible lines into different groups based on the resistance genes present in them.     

Informativeness of human (dC-dA)n · (dG-dT)n polymorphisms

Abundant human interspersed repetitive DNA sequences of the form (dC-dA)_n · (dG-dT)_n have been shown to exhibit length polymorphisms. Examination of over 100 human (dC-dA)_n · (dG-dT)_n sequences revealed that the sequences differed from each other both in numbers of repeats and in repeat sequence type. Using a set of precise classification rules, the sequences were divided into three categories: perfect repeat sequences without interruptions in the runs of CA or GT dinucleotides (64% of total), imperfect repeat sequences with one or more interruptions in the run of repeats (25%), and compound repeat sequences with adjacent tandem simple repeats of a different sequence (11%). Informativeness of (dC-dA)_n · (dG-dT)_n markers in the perfect sequence category was found to increase with increasing average numbers of repeats. PIC values ranged from 0 at about 10 or fewer repeats to above 0.8 for sequences with about 24 or more repeats. (dC-dA)_n · (dG-dT)_n polymorphisms in the imperfect sequence category showed lower informativeness than expected on the basis of the total numbers of repeats. The longest run of uninterrupted CA or GT repeats was found to be the best predictor of informativeness of (dC-dA)_n · (dG-dT)_n polymorphisms regardless of the repeat sequence category.     

Isolation and characterization of microsatellites in Chinese soft‐shelled turtle,Pelodiscus sinensis

Fifteen polymorphic microsatellite loci were developed for the Chinese soft‐shelled turtle (Pelodiscus sinensis) from the (GT)_n microsatellite‐enriched genomic library, using the fast isolation by amplified fragment length polymorphism of sequences containing repeats protocol. The polymorphism of all 15 loci ranged from two to seven alleles with observed heterozygosities ranging from 0.03 to 0.98 (mean 0.43) in one population of 40 individuals. These novel loci will be helpful for understanding the population structure at genetic level and marker‐assisted breeding of this vulnerable species.     

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.     

Replication in mammalian cells recapitulates the locus-specific differences in somatic instability of genomic GAA triplet-repeats

Friedreich ataxia is caused by an expanded (GAA·TTC)_n sequence in intron 1 of the FXN gene. Small pool PCR analysis showed that pure (GAA·TTC)₄₄₊ sequences at the FXN locus are unstable in somatic cells in vivo, displaying both expansions and contractions. On searching the entire human and mouse genomes we identified three other genomic loci with pure (GAA·TTC)₄₄₊ sequences. Alleles at these loci showed mutation loads of <1% compared with 6.3–30% for FXN alleles of similar length, indicating that somatic instability in vivo is regulated by locus-specific factors. Since distance between the origin of replication and the (CTG·CAG)_n sequence modulates repeat instability in mammalian cells, we tested if this could also recapitulate the locus-specific differences for genomic (GAA·TTC)_n sequences. Repeat instability was evaluated following replication of a (GAA·TTC)₁₁₅ sequence in transfected COS1 cells under the control of the SV40 origin of replication located at one of five different distances from the repeat. Indeed, depending on the location of the SV40 origin relative to the (GAA·TTC)_n sequence, we noted either no instability, predominant expansion or both expansion and contraction. These data suggest that mammalian DNA replication is a possible mechanism underlying locus-specific differences in instability of GAA triplet-repeat sequences.     

Survey of plant short tandem DNA repeats

Length variations in simple sequence tandem repeats are being given increased attention in plant genetics. Some short tandem repeats (STRs) from a few plant species, mainly those at the dinucleotide level, have been demonstrated to show polymorphisms and Mendelian inheritance. In the study reported here a search for all of the possible STRs ranging from mononucleotide up to tetranucleotide repeats was carried out on EMBL and GenBank DNA sequence databases of 3026 kb nuclear DNA and 1268 kb organelle DNA in 54 and 28 plant species (plus algae), respectively. An extreme rareness of STRs (4 STRs in 1268 kb DNA) was detected in organelle compared with nuclear DNA sequences. In nuclear DNA sequences, (AT)_n sequences were the most abundant followed by (A)_n · (T)_n, (AG)_n · (CT)_n, (AAT)_n · (ATT)_n, (AAC)_n · (GTT), (AGC)_n · (GCT)_n, (AAG)_n · (CTT)_n, (AATT)_n · (TTAA)_n, (AAAT)_n · (ATTT)_n and (AC)_n · (GT)_n sequences. A total of 130 STRs were found, including 49 (AT)_n sequences in 31 species, giving an average of 1 STR every 23.3 kb and 1 (AT)_n STR every 62 kb. An abundance comparable to that for the dinucleotide repeat was observed for the tri- and tetranucleotide repeats together. On average, there was 1 STR every 64.6 kb DNA in monocotyledons versus 1 every 21.2 kb DNA in dicotyledons. The fraction of STRs that contained G-C basepairs increased as the G+C contents went up from dicotyledons, monocotyledons to algae. While STRs of mono-, di- and tetranucleotide repeats were all located in non coding regions, 57% of the trinucleotide STRs containing G-C basepairs resided in coding regions.     

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.     

Clustering of C2---H2 zinc finger motif sequences within telomeric and fragile site regions of human chromosomes

Ninety-three phage clones identified by hybridization with a C₂---H₂ zinc finger sequence probe have been grouped into 23 genetic loci. Partial sequencing verified that each locus belonged to the zinc finger family. Oligonucleotide primer pairs were developed from these sequences to serve as STS markers for these loci. One or more clones from each locus was mapped onto human metaphase chromosomes by fluorescence in situ hybridization. Several loci map to identical chromosomal regions, indicating the possible presence of multigene clusters. Zinc finger loci were found to reside predom nantly either in telomeric regions or in chromosomal bands known to exhibit chromosome fragility. Chromosome 19 carries a disproportionate fraction (10 of 23) of the mapped zinc finger loci.     

Instability of (GATA)<Subscript> n</Subscript> microsatellite loci in the parthenogenetic Caucasian rock lizard<Emphasis Type="Italic"> Darevskia unisexualis</Emphasis> (Lacertidae)

Mini- and microsatellites, comprising tandemly repeated short nucleotide sequences, are abundant dispersed repetitive elements that are ubiquitous in eukaryotic genomes. In humans and other bisexual species hypervariable mini- and microsatellite loci provide highly informative systems for monitoring of germline and somatic instability. However, little is known about the mechanisms by which these loci mutate in species that lack effective genetic recombination. Here, multilocus DNA fingerprinting was used to study M13 minisatellite and (GATA) _n microsatellite instability in the parthenogenetic Caucasian rock lizard Darevskia unisexualis (Lacertidae). DNA fingerprinting of 25 parthenogenetic families, from six isolated populations in Armenia (comprising a total of 84 siblings), using the oligonucleotide (GATA)₄ as a hybridization probe, revealed mutant fingerprinting phenotypes in 13 siblings that differed from their mothers in several restriction DNA fragments. In three families (8 siblings), the mutations were present in the germline. Moreover, the mutant fingerprint phenotypes detected in siblings were also present in population DNA samples. No intrafamily variations in DNA fingerprint patterns were observed with the M13 minisatellite probe. Estimates of the mutation rate for (GATA) _n microsatellite loci in D. unisexualis showed that it was as high as that seen in some bisexual species, reaching 15% per sibling or 0.95% per microsatellite band. Furthermore, in one case, a somatic (GATA) _n microsatellite mutation was observed in an adult lizard. These findings directly demonstrate that mutations in (GATA) _n microsatellite loci comprise an important source of genetic variation in parthenogenetic populations of D. unisexualis.Communicated by G. P. Georgiev     

Isolation and characterization of polymorphic microsatellites in a Yangtze River fish,brass gudgeon (Coreius heterodon Bleeker)

The brass gudgeon (Coreius heterodon) is a fish of economic importance in the Yangtze River. From a (GATA)_n‐enriched genomic library, 25 microsatellites were developed by employing the fast isolation by AFLP of sequences containing repeats (FIASCO) protocol. Nine loci exhibited polymorphism with two to 12 alleles (mean 3.9 alleles/locus) in a test population, and observed heterozygosity ranged from 0.1111 to 0.9630 (mean 0.4426). Three of the nine loci showed polymorphism in a congeneric species, the largemouth bronze gudgeon Coreius guichenoti. These loci should provide sufficient level of genetic diversity to evaluate the fine‐scale population structure of C. heterodon.     

An SSR-based genetic linkage map for perennial ryegrass (<Emphasis Type="Italic">Lolium perenne</Emphasis> L.)

A simple sequence repeat (SSR)-based linkage map has been constructed for perennial ryegrass (Lolium perenne L.) using a one-way pseudo-testcross reference population. A total of 309 unique perennial ryegrass SSR (LPSSR) primer pairs showing efficient amplification were evaluated for genetic polymorphism, with 31% detecting segregating alleles. Ninety-three loci have been assigned to positions on seven linkage groups. The majority of the mapped loci are derived from cloned sequences containing (CA)_n-type dinucleotide SSR arrays. A small number (7%) of primer pairs amplified fragments that mapped to more than one locus. The SSR locus data has been integrated with selected data for RFLP, AFLP and other loci mapped in the same population to produce a composite map containing 258 loci. The SSR loci cover 54% of the genetic map and show significant clustering around putative centromeric regions. BLASTN and BLASTX analysis of the sequences flanking mapped SSRs indicated that a majority (84%) are derived from non-genic sequences, with a small proportion corresponding to either known repetitive DNA sequence families or predicted genes. The mapped LPSSR loci provide the basis for linkage group assignment across multiple mapping populations.