Characterization of microsatellites and development of chromosome specific STMS markers in bread wheat

Microsatellites, or simple sequence repeats (SSRs), have become the markers of choice for genetic studies with many crop species including wheat. Currently an international effort is underway to enrich the repertoire of available sequence tagged microsatellite site (STMS) markers in wheat. As a part of this effort, we have sequenced 43 clones obtained from a microsatellite-enriched wheat genomic library; 34 clones contained 41 different microsatellites. These microsatellites (mono-, di-, tri- nucleotide repeats) were classified as 19 simple perfect, 18 simple imperfect and 4 compound imperfect types. Dinucleotide repeats were the most abundant (70%). Primer pairs for only 16 microsatellites could be designed, since the flanking sequences of the others were either too short or were otherwise not suitable for designing the microsatellite specific primers. Microsatellite loci of the expected size and polymorphism were successfully amplified from 15 of these 16 primer pairs using three wheat varieties. 14 loci detected by 12 out of the 15 functional primer pairs were assigned to 11 specific chromosomes. An erratum to this article is available at .     

cDNA surveying of specific tissue expression of human chromosome 19 sequences.

cDNA surveying is a straightforward approach for identifying sequences in genomic clones expressed in specific tissues. It has been applied to a subchromosomal region of human chromosome 19 (19q13.2-q13.4), a region that contains several known expressed sequences including the locus for myotonic dystrophy (DM). Genomic clones were selected from this region by probing a human placental cosmid library with a chromosome 19q-specific minisatellite sequence, or human genomic clones were isolated from a cosmid library constructed from a human chromosome 19q13.2-q13.3 hamster hybrid cell line using human repetitive DNA as probe. Pooled cDNAs synthesized from RNA of specific tissues characteristically affected in DM were depleted in repetitive sequences and used as hybridization probes against gridded cosmid arrays. DNA from the cDNA-positive cosmid clones was transferred to nylon filters and reprobed with cDNAs to identify restriction fragments that were expressed in these tissues. Hybridizing restriction fragments were subcloned, sequenced, and demonstrated to be nonrepetitive. Primer pairs complementary to subcloned sequences were constructed and used for PCR amplification of cDNA synthesized from RNA of tissues affected in myotonic dystrophy. PCR products were sequenced to verify the identity of expressed genomic DNA and its corresponding cDNA.     

Isolation and characterization of microsatellites in Brassica rapa L

We report here the isolation and characterization of microsatellites, or simple sequence repeats (SSRs), in Brassica rapa. The size-fractionated genomic library was screened with (GA)(15) and (GT)(15) oligonucleotide probes. A total of 58 clones were identified as having the microsatellite repeats, and specific primer pairs were designed for 38 microsatellite loci. All primer pairs, except two, amplified fragments having the sizes expected from the sequences. Of the 36 primer pairs, 35 amplified polymorphic loci in 19 cultivars of B. rapa, while monomorphism was observed in only one primer pair. A total of 232 alleles was identified by the 36 primer pairs in 19 cultivars of B. rapa, and these primer pairs were examined also in nine Brassicaceae species. Most of the 36 primer pairs amplified the loci in the Brassicaceae species. Segregation of the microsatellites was studied in an F(2) population from a cross of doubled-haploid lines DH27 x G309. The microsatellites segregated in a co-dominant manner. These results indicate that the microsatellites isolated in this study were highly informative and could be useful tools for genetic analysis in B. rapa and other related species.     

Physical Mapping of 49 Microsatellite Markers on Chromosome 19 and Correlation with the Genetic Linkage Map

We have regionally localized 49 microsatellite markers developed by Généthon using a panel of previously characterized somatic cell hybrids that retain fragments from chromosome 19. The tight correlation observed between the physical and the genetic orders of the microsatellites provide cytogenetic anchorages to the genetic map data. We propose a position for the centromere just above D19S415, from the study of two hybrids, each of which retains one of the two derivatives of a balanced translocation t(1;19)(q11;q11). Microsatellites, which can be identified by a standard PCR protocol, are useful tools for the localization of disease genes and for the establishment of YAC or cosmid contigs. These markers can also judiciously be used for the characterization of new hybrid cell line panels. We report such a characterization of 11 clones, 8 of which were obtained by irradiation-fusion. Using the whole hybrid panel, we were able to define the order of 12 pairs of genetically colocalized microsatellites. As examples of gene mapping by the combined use of microsatellites and hybrid cell lines, we regionally assigned the PVS locus between the 19q13.2 markers D19S417 and D19S423 and confirmed the locations of fucosyltransferase loci FUT1, FUT2, and FUT5.     

A chromosome 5-specific repetitive DNA sequence in rice (Oryza sativa L)

Repetitive DNA sequences in the rice genome comprise more than half of the nuclear DNA. The isolation and characterization of these repetitive DNA sequences should lead to a better understanding of rice chromosome structure and genome organization. We report here the characterization and chromosome localization of a chromosome 5-specific repetitive DNA sequence. This repetitive DNA sequence was estimated to have at least 900 copies. DNA sequence analysis of three genomic clones which contain the repeat unit indicated that the DNA sequences have two sub-repeat units of 37 bp and 19 bp, connected by 30-to 90-bp short sequences with high similarity. RFLP mapping and physical mapping by fluorescence in situ hybridization (FISH) indicated that almost all copies of the repetitive DNA sequence are located in the centromeric heterochromatic region of the long arm of chromosome 5. The strategy for cloning such repetitive DNA sequences and their uses in rice genome research are discussed.     

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.     

Development of microsatellite markers specific for the short arm of rye (Secale cereale L.) chromosome 1

We developed 74 microsatellite marker primer pairs yielding 76 polymorphic loci, specific for the short arm of rye chromosome 1R (1RS) in wheat background. Four libraries enriched for microsatellite motifs AG, AAG, AC and AAC were constructed from DNA of flow-sorted 1RS chromosomes and 1,290 clones were sequenced. Additionally, 2,778 BAC-end-sequences from a 1RS specific BAC library were used for microsatellite screening and marker development. From 724 designed primer pairs, 119 produced 1RS specific bands and 74 of them showed polymorphism in a set of ten rye genotypes. We show that this high attrition rate was due to the highly repetitive nature of the rye genome consisting of a large number of transposable elements. We mapped the 76 polymorphic loci physically into three regions (bins) on 1RS; 29, 30 and 17 loci were assigned to the distal, intercalary and proximal regions of the 1RS arm, respectively. The average polymorphism information content increases with distance from the centromere, which could be due to an increased recombination rate along the chromosome arm toward’s the telomere. Additionally, we demonstrate, using the data of the whole rice genome, that the intra-genomic length variation of microsatellites correlates (r = 0.87) with microsatellite polymorphism. Based on these results we suggest that an analysis of the microsatellite length variation is conducted for each species prior to microsatellite development, provided that sufficient sequence information is available. This will allow to selectively design microsatellite markers for motifs likely to yield a high level of polymorphism. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Isolation and characterisation of (AC)n microsatellite genetic markers from human chromosome 16.

A cosmid library of human chromosome 16 has been subcloned, and (AC)n microsatellite positive clones have been identified and sequenced. Oligonucleotide primers flanking the repeat were designed and synthesized for (AC)n microsatellites with n greater than 16. These microsatellite loci were then mapped by PCR using a somatic cell hybrid panel of human chromosome 16, and their heterozygosities and allele frequencies determined. Fourteen (AC)n microsatellites were mapped to discrete physical intervals of human chromosome 16 defined by a mouse/human hybrid panel. Nine of these have expected heterozygosities ranging between 0.60 and 0.79, four have expected heterozygosities between 0.02 and 0.49, and one detected three loci where the alleles could not be resolved.     

Generation of a 5.5-Mb BAC/PAC contig of pig chromosome 6q1.2 and its integration with existing RH, genetic and comparative maps

We generated a sequence-ready BAC/PAC contig spanning approximately 5.5 Mb on porcine chromosome 6q1.2, which represents a very gene-rich genome region. STS content mapping was used as the main strategy for the assembly of the contig and a total of 6 microsatellite markers, 53 gene-related STS and 116 STS corresponding to BAC and PAC end sequences were analyzed. The contig comprises 316 BAC and PAC clones covering the region between the genes GPI and LIPE. The correct contig assembly was verified by RH-mapping of STS markers and comparative mapping of BAC/PAC end sequences using BLAST searches. The use of microsatellite primer pairs allowed the integration of the physical maps with the genetic map of this region. Comparative mapping of the porcine BAC/PAC contig with respect to the gene-rich region on the human chromosome 19q13.1 map revealed a completely conserved gene order of this segment, however, physical distances differ somewhat between HSA19q13.1 and SSC6q1.2. Three major differences in DNA content between human and pig are found in two large intergenic regions and in one region of a clustered gene family, respectively. While there is a complete conservation of gene order between pig and human, the comparative analysis with respect to the rodent species mouse and rat shows one breakpoint where a genome segment is inverted.     

Y chromosome microsatellite isolation from BAC clones in the greater white‐toothed shrew (Crocidura russula)

We constructed a microsatellite library from four Crocidura russula Y chromosome‐specific bacterial artificial chromosome (BAC) clones. Only one of eight microsatellites was male‐specific, despite genome walking to obtain more flanking sequence and testing of 93 primer combinations. Potential reasons for this low success are discussed. The male‐specific locus, CRY3, was genotyped in 90 males, including C. russula from across the species range and two related species. The large difference in CRY3 allele size between eastern and western lineages supports earlier reports of high divergence between them. Despite polymorphism of CRY3 in Morocco, only one allele was found throughout the whole of Europe, consistent with previous studies that suggest recent colonization of Europe from a small number of Moroccan founders.     

Microsatellite DNA markers for rice chromosomes

We found 369 complete microsatellites, of which (CGG/GCC)_n was the most frequent, in 11 798 rice sequences in the database. Of these microsatellites, 35 out of 45 could be successfully converted into microsatellite DNA markers using sequence information in their flanking regions. Thus, the time and labor used to develop new microsatellite DNA markers could be saved by using these published sequences. Twenty eight polymorphic markers between Asominori (japonica) and IR24 (indica) have been correctly mapped on the rice genome and microsatellites appear to be randomly distributed in the rice chromosomes. Integration of these markers with the published microsatellite DNA markers showed that about 35% of the rice chromosomes were covered by the 56 microsatellite DNA markers. These microsatellites were hypervariable and were easily to assay by PCR; they were distributed to all chromosomes and therefore, one can easily select plants carrying desired chromosome regions using these microsatellite DNA markers. Thus, microsatellite maps should aid the development of new breeds of rice saving time, labor, and money.     

Using PAC nested deletions to order contigs and microsatellite markers at the high repetitive sequence containing Npr3 gene locus

Highly polymorphic di- and tetranucleotide repeats in and around Npr3, a potential candidate gene for hypertension, have been identified using a novel approach. Because this chromosomal site is rich in repetitive DNA and difficult to sequence, P1 artificial chromosomes were retrofitted with a loxP transposon to map the gene sequence within a clone using a series of nested deletions. Sequences from ends of deletions 1-3 kb apart identified a (CA)(20) and a (TA)(18)-(CA)(8) repeat 8 kb upstream and within an intron of Npr3, respectively. DNA from 17 individuals was analyzed for length polymorphisms in these and eight additional repeats identified in 200 kb of working draft sequence from this region in GenBank. The sequence contigs and microsatellite repeats from GenBank were ordered using the P1-derived artificial chromosome deletion series. Several of these repeats were found to vary considerably in length in the set of genomic DNA tested. Since this site in chromosome 5p has recently been implicated in disease in studies with genetically hypertensive rats, the microsatellite markers reported here will be useful for genetic analysis and may even be implicated in the disease process in humans. We discuss how these types of data are useful for interpreting draft DNA sequence coming out of the genome projects, and the utility of deletion clones as a resource for ordering contigs and gap filling.     

Sequence-tagged site markers for microsatellites: Simplified technique for rapidly obtaining flanking sequences

A sequencing strategy is described for the rapid recovery of DNA sequences flanking repeat sequences of microsatellites in plant nuclear genomes. Primers that represent a perfect microsatellite repeat and end in a 3′ degenerate base have been used to sequence directly from microsatellite repeats in one direction. The procedure allows the design of one flanking primer that is then used to sequence back through the repeat to define the microsatellite site precisely and also provides for the design of the second flanking primer. The strategy is versatile with various repeat sizes and different categories of microsatellites; perfect, imperfect, and compound were found to be suitable templates for analysis.     

Evolution of chloroplast mononucleotide microsatellites in Arabidopsis thaliana

The level of variation and the mutation rate were investigated in an empirical study of 244 chloroplast microsatellites in 15 accessions of Arabidopsis thaliana. In contrast to SNP variation, microsatellite variation in the chloroplast was found to be common, although less common than microsatellite variation in the nucleus. No microsatellite variation was found in coding regions of the chloroplast. To evaluate different models of microsatellite evolution as possible explanations for the observed pattern of variation, the length distribution of microsatellites in the published DNA sequence of the A. thaliana chloroplast was subsequently used. By combining information from these two analyses we found that the mode of evolution of the chloroplast mononucleotide microsatellites was best described by a linear relation between repeat length and mutation rate, when the repeat lengths exceeded about 7 bp. This model can readily predict the variation observed in non-coding chloroplast DNA. It was found that the number of uninterrupted repeat units had a large impact on the level of chloroplast microsatellite variation. No other factors investigated—such as the position of a locus within the chromosome, or imperfect repeats—appeared to affect the variability of chloroplast microsatellites. By fitting the slippage models to the Genbank sequence of chromosome 1, we show that the difference between microsatellite variation in the nucleus and the chloroplast is largely due to differences in slippage rate. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Five polymorphic microsatellite VNTRs on the human X chromosome

The human genome contains approximately 50,000 copies of an interspersed repeat with the sequence (dT.dG/dA.dC)n, where n = approximately 10-60. We and others have found that several of these repeats have variable lengths in different individuals, with allelic fragments varying in size by multiples of 2 bp. These "microsatellite" variable number of tandem repeats (VNTRs) may be scored by PCR, using unique flanking primers to amplify the repeat-containing regions and resolving the products on DNA sequencing gels. Since few VNTRs have been found on the X chromosome, we screened a flow-sorted X chromosome-specific genomic library for microsatellites. Approximately 25% of the phage clones hybridized to a poly (dT-dG).poly(dA-dC) probe. Of seven X-linked microsatellites present in positive phages, five are polymorphic and three have both eight or more alleles and heterozygosities exceeding 75%. Using PCR to amplify genomic DNAs from hybrid cell panels, we confirmed the X localization of these VNTRs and regionally mapped four of them. The fifth VNTR was regionally mapped by virtue of its tight linkage to DXS87 in Centre du Polymorphisme Humain families. We conclude that whatever factors limit the occurrence of "classical" VNTRs and RFLPs on the X chromosome do not appear to operate in the case of microsatellite VNTRs.     

Construction and characterization of a DNA library from mouse chromosomes 19 purified by flow cytometry

In spite of the constant development concerning physical mapping of eukaryotic genomes, the mouse chromosome 19 remains poorly characterized. In order to improve the possibilities for studying this chromosome, we have constructed a chromosome-specific EcoRI DNA fragment library from mouse chromosomes 19 sorted by flow cytometry. The resulting library contains about 3 X 10(4) recombinant clones. The identified inserts range in size from about 0.2-10 kb, with a 4 kb average size and with no observable redundancy. The purity of the library has been analyzed by flow-blot. For that purpose, chromosomes from 2 cell lines, 1 with a normal karyotype and 1 with translocated chromosome 19, were sorted on nylon filters and hybridized with 9 clones of the library. Results show that 5 clones out of the 9 clearly originate from sorted chromosomes 19 and 3 and are likely to be derived from its DNA, thus indicating that the library of chromosome 19 is of high purity.     

Microsatellite polymorphisms in Bolivian squirrel monkeys (Saimiri boliviensis)

Two different approaches were used to identify new microsatellite polymorphisms among captive Bolivian squirrel monkeys (Saimiri boliviensis). In the first case, PCR primers for published human microsatellite loci were screened using genomic DNA from squirrel monkeys. Six polymorphic loci were identified using DNA samples from 19 unrelated individuals. The average heterozygosity among these six loci is 0.73. In the second set of experiments, a DNA library was created from Saimiri genomic DNA, and clones were selected from that library by screening with probes containing di-, tri-, and tetranucleotide repeats. Six novel microsatellites were identified this way, with an average heterozygosity of 0.59. Primer pairs for these six cloned microsatellites were also screened using a series of DNA samples from ten other platyrrhine species to assess the potential utility of these loci in other taxa. This study provides 12 new DNA polymorphisms that will be useful for various studies of this genus and demonstrates that both approaches can be used to develop new DNA polymorphisms in platyrrhine species.     

Sequence tagged microsatellite profiling (STMP): improved isolation of DNA sequence flanking target SSRs

Sequence tagged microsatellite profiling (STMP) enables the rapid development of large numbers of co-dominant DNA markers, known as sequence tagged microsatellites (STMs). Each STM is amplified by PCR using a single primer specific to the conserved DNA sequence flanking the microsatellite repeat in combination with a universal primer that anchors to the 5′-ends of the microsatellites. It is also possible to convert STMs into conventional microsatellite, or simple sequence repeat (SSR), markers that are amplified using a pair of primers flanking the repeat sequence. Here, we describe a modification of the STMP procedure to significantly improve the capacity to convert STMs into conventional SSRs and, therefore, facilitate the development of highly specific DNA markers for purposes such as marker-assisted breeding. The usefulness of this technique was demonstrated in bread wheat.     

Physical mapping of sequences homologous to an endogenous retrovirus LTR on human chromosome 19

The human genome contains multiple copies of sequences related to the HERV-K family of endogenous retroviruses, homologous to the B-type mouse mammary tumour virus. A DNA fragment closely resembling an HERV-K long tandem repeat (LTR) was detected in a library of hncDNA clones enriched for sequences from human chromosome 19. Sites showing homology to the sequence of this fragment have been identified on human chromosome 19 by hybridization to previously mapped chromosome 19 cosmids. Thus the distribution of LTR sequences on a specific human chromosome has been mapped for the first time. We estimate the total number of such sites on human chromosome 19 to be at least 110. Many of these sites are located in the vicinity of known genes. The precise localizations (to specific cosmids) of LTR-homologous sequences on chromosome 19 can serve as a reference source and will automatically provide further insight into LTR-gene relationships as new genes are mapped onto the chromosome.     

Microdissection of the Prader-Willi syndrome chromosome region and identification of potential gene sequences

The Prader-Willi syndrome chromosome region on the long arm of human chromosome 15 was microdissected and microcloned from 20 GTG-banded metaphase chromosomes, and 5000 recombinant clones were obtained. Of these clones, 39% identify single-copy human DNA sequences, most of which map to the dissected chromosome region and are evolutionarily conserved in other species. Three of eleven clones studied in detail are deleted in several patients with Prader-Willi syndrome. The microclones will be useful for the physical characterization of the Prader-Willi syndrome chromosome region and the identification of the affected genes in this disease.