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Frequent in-frame length variations are found in the diverged simple repeat sequences of the protein-coding regions of two putative protein kinase genes of Brassica napus.

Two putative protein kinase cDNA clones were isolated from Brassica napus by screening with a putative protein kinase cDNA clone of Arabidopsis thaliana. The deduced amino acid sequences show a distinct modular composition, consisting of a possible protein kinase catalytic region at the amino terminus and a highly acidic region encoded from diverged simple repeat sequences at the carboxy terminus. Comparison of the nucleotide sequences encoding this acidic region revealed a high rate of in-frame length variation, while preserving the acidic characteristics. Similar variation is also found in the noncoding regions of these clones.

Amino Acid Sequence↗

Characterization and genetic mapping of simple sequence repeats in the rice genome.

We searched partial sequences of over 22,706 rice cDNA and 1220 genomic DNA clones to find and characterize simple sequence repeats (SSRs) in the rice genome. The most frequently found repeated SSR motif in both cDNA and genomic DNA sequences was d(CCG/CGG)n. The second most frequently found SSR was d(AG/CT)n. In contrast with mammalian genomes, in which d(AC/GT)n sequences are the most abundant, d(AG/GT)n sequences were not frequently observed in rice. Sequences containing d(CCG/CGG)n, d(AG/CT)n repeats, and other SSRs were chosen for polymorphism detection. It was predicted that 17 of 20 SSRs in cDNA sequences were located in 5'-untranslated regions near initiation codons. Twenty-two loci can be mapped on our RFLP linkage map by these SSRs. Six markers were tested with 16 japonica rice varieties as templates for PCR. Two markers exhibited amplified fragment length polymorphism among these rice varieties, implying that SSRs are polymorphic among rice varieties which have similar genetic backgrounds. Even these polymorphic SSRs are located within or around genes which code ubiquitous proteins.

Base Sequence↗

Characterization and phylogenetic relationships among microsporidia infecting silkworm, Bombyx mori, using inter simple sequence repeat (ISSR) and small subunit rRNA (SSU-rRNA) sequence analysis.

This study is the first report on the genetic characterization and relationships among different microsporidia infecting the silkworm, Bombyx mori, using inter simple sequence repeat PCR (ISSR-PCR) analysis. Six different microsporidians were distinguished through molecular DNA typing using ISSR-PCR. Thus, ISSR-PCR analysis can be a powerful tool to detect polymorphisms and identify microsporidians, which are difficult to study with microscopy because of their extremely small size. Of the 100 ISSR primers tested, only 28 primers had reproducibility and high polymorphism (93%). A total of 24 ISSR primers produced 55 unique genetic markers, which could be used to differentiate the microsporidians from each other. Among the 28 SSRs tested, the most abundant were (CA)n, (GA)n, and (GT)n repeats. The degree of band sharing was used to evaluate genetic similarity between different microsporidian isolates and to construct a phylogenetic tree using Jaccard's similarity coefficient. The results indicate that the DNA profiles based on ISSR markers can be used as diagnostic tools to identify different microsporidia with considerable accuracy. In addition, the small subunit ribosomal RNA (SSU-rRNA) sequence gene was amplified, cloned, and sequenced from each of the 6 microsporidian isolates. These sequences were compared with 20 other microsporidian SSU-rRNA sequences to develop a phylogenetic tree for the microsporidia isolated from the silkworms. This method was found to be useful in establishing the phylogenetic relationships among the different microsporidians isolated from silkworms. Of the 6 microsporidian isolates, NIK-1s revealed an SSU-rRNA gene sequence similar to Nosema bombycis, indicating that NIK-1s is similar to N. bombycis; the remaining 5 isolates, which differed from each other and from N. bombycis, were considered to be different variants belonging to the species N. bombycis.

Animals↗

Microsatellite instability in Japanese hereditary non-polyposis colorectal cancer does not induce mutation of a simple repeat sequence of the bax gene.

The identification of frameshift mutations in the coding region of the bax gene in Western cases of hereditary non-polyposis colorectal cancer provided evidence that the disease could be caused by the disruption of a tumor suppressor gene associated with microsatellite instability. In order to determine whether this observation could be extended to Japanese colorectal cancers, we analyzed the simple repeat sequence of (G)8 in the bax gene of tumor genomes from 15 cases of Japanese hereditary non-polyposis colorectal cancer that showed microsatellite instability in at least one of four examined microsatellite loci. None of 15 tumor DNA samples had a frameshift mutation in this region of the bax gene. Our results suggest that direct disruption of the bax gene is not a major factor responsible for carcinogenesis in Japanese hereditary non-polyposis colorectal cancers even though microsatellite instability is present.

Colorectal Neoplasms, Hereditary Nonpolyposis↗

Chromosome localization and orientation of the simple sequence repeat of human satellite I DNA.

The predominant chromosomal locations of human satellite I DNA were detected using fluorescent in situ hybridization (FISH). Synthetic deoxyoligonucleotides designed from consensus sequences of the simple sequence repeats of satellite 1 were used as probes. The most abundant satellite I repeat, the -A-B-A-B-A- form, is located at the pericentromeric regions of chromosomes 3, 4, 13, 14, 15, 21, and 22. The less abundant -B-B-B-form was not detected on chromosome 4, but was present at all the other locations. A variation of FISH that allows strand-specific hybridization of single-stranded probes (CO-FISH) determined that the human satellite I sequences are predominantly arranged in head-to-tail fashion along the DNA strand.

Base Sequence↗

Simple sequence repeats in Escherichia coli: abundance, distribution, composition, and polymorphism.

Computer-based genome-wide screening of the DNA sequence of Escherichia coli strain K12 revealed tens of thousands of tandem simple sequence repeat (SSR) tracts, with motifs ranging from 1 to 6 nucleotides. SSRs were well distributed throughout the genome. Mononucleotide SSRs were over-represented in noncoding regions and under-represented in open reading frames (ORFs). Nucleotide composition of mono- and dinucleotide SSRs, both in ORFs and in noncoding regions, differed from that of the genomic region in which they occurred, with 93% of all mononucleotide SSRs proving to be of A or T. Computer-based analysis of the fine position of every SSR locus in the noncoding portion of the genome relative to downstream ORFs showed SSRs located in areas that could affect gene regulation. DNA sequences at 14 arbitrarily chosen SSR tracts were compared among E. coli strains. Polymorphisms of SSR copy number were observed at four of seven mononucleotide SSR tracts screened, with all polymorphisms occurring in noncoding regions. SSR polymorphism could prove important as a genome-wide source of variation, both for practical applications (including rapid detection, strain identification, and detection of loci affecting key phenotypes) and for evolutionary adaptation of microbes.

Base Composition↗

Sequence variations of simple sequence repeats on chromosome-4 in two subspecies of the Asian cultivated rice.

Computational screening of the chromosome-4 sequence of the rice cultivar Nipponbare ( Oryza sativa L. japonica) revealed 1,844 tandem simple sequence repeats (SSRs) or microsatellites with SSR motifs >/=20 bp and repeated unit length of 1-6 base pairs. Thus SSRs occur once in every 18.8 kb, on the average, on the chromosome with one SSR per 23.8 kb and 16 kb on the short and long arms, respectively. No SSR was detected in the core region of the centromere. Poly(AT)(n) repeats represented the most abundant and length polymorphic class of SSRs on the chromosome, but it did not occur in the exons. GC-rich trinucleotide repeats were most abundant in the coding regions, representing 71.69% of the SSRs identified in the exons. Two hundred and twenty four SSRs were associated with the repetitive DNA sequences, most of them were poly(AT)(n) tracts. Sequence variations of SSRs between two cultivars, representing the two subspecies of the Asian cultivated rice indica and japonica, were identified, revealing that divergence and convergence of the two subspecies could be traced by the analysis of SSRs. These results provide a great opportunity for SSR-based marker development and comparative genome analysis of the two subspecies of the Asian cultivated rice.

Base Composition↗

Genetic diversity in Arabidopsis thaliana L. Heynh. investigated by cleaved amplified polymorphic sequence (CAPS) and inter-simple sequence repeat (ISSR) markers.

In this study, we investigated genetic diversity among 37 accessions in Arabidopsis thaliana from Eurasia, North Africa and North America using morphological traits and two polymerase chain reaction (PCR)-based marker systems: cleaved amplified polymorphic sequences (CAPS) and inter-simple sequence repeats (ISSR). Cluster analysis based on genetic similarities calculated from CAPS data grouped the accessions roughly according to their geographical origin: one large group contained accessions from Western, Northern and Southern Europe as well as North Africa, a second group consisted of Eastern European and Asian continental accessions. North American accessions were interspersed into these groups. Contrary to the CAPS analysis, the dendrogram obtained from the ISSR data did not reflect the geographical origin of the accessions, and the calculated genetic distances did not match the CAPS results. This could be attributable to an uneven genomic distribution of ISSR markers as substantiated by a database search for ISSR binding sites in A. thaliana genomic DNA sequence files, or to the ISSR's different mode of evolution. We recommend CAPS markers for diversity analysis in A. thaliana because a careful selection of markers can ascertain an even representation of the entire genome.

Arabidopsis↗

Identification of cecidophyopsis mites (Acari: Eriophyidae) based on variable simple sequence repeats of ribosomal DNA internal transcribed spacer-1 sequences via multiplex PCR.

A PCR multiplex technique was developed for identifying Cecidophyopsis mites using species-specific differences in rDNA ITS-1 sequences. Four PCR primers derived from ITS-1 were used for the simultaneous amplification (multiplex PCR) of interspecifically variable simple sequence repeats (vSSRs). Mites were identified by electrophoresing PCR products alongside those obtained from plasmids containing ITS copies of known mite species. The multiplex PCR assay was rapid, reproducible and had a sensitivity comparable to sequencing. It was used to identify mite specimens on Ribes from around the world. It also identified a profile from mites on R. rubrum that had no equivalent amongst the known Cecidophyopsis species. Sequence and ecological analysis of this mite suggest that it is a new species of nongall-forming Cecidophyopsis mite.

Animals↗

Detection and quantification of in vitro-culture induced chimerism using simple sequence repeat (SSR) analysis in Theobroma cacao (L.).

Mutation rates are often elevated in plants regenerated from in vitro culture, giving rise to so-called 'somaclonal variation'. Detailed characterisation of mutation profiles that arise during culture should improve our understanding of processes influencing mutation and allow the selection of protocols yielding the fewest/least severe changes. Somatic mutations will usually produce genetic chimeras where unchanged alleles are retained by some cells. Such chimeras are difficult to detect but likely to form a significant proportion of any regenerant population. We present a simple protocol that enables the provisional diagnosis of both homogenous and chimeric mutants among large regenerant populations, together with a semi-quantitative means of estimating the proportion of mutant cells. The assay exploits consistent differential amplification of alternate simple sequence repeat alleles at heterozygous loci. Calibration of the relative amplification of alleles from two genotypes-and the synthetic chimeras created from them-revealed a strong linear relationship between 'peak heights' representing alternate alleles following capillary electrophoresis. The assay predicts chimeric composition to a reasonable level of confidence (+/-5%) so long as the infrequent allele exceeds 15% of the template. The system was applied to 233 regenerants of cocoa somatic embryogenesis and identified 72 (31%) putative chimeric mutants for slippage mutation or allele loss across two loci.

Alleles↗

Instability of simple sequence repeats in a mammalian cell line.

Short tandem repeat sequences in the mammalian genome are considered to be unstable, since many of them are polymorphic in length; however, the extent of this instability has been difficult to quantitate. We have directly determined the rate of mutation of a simple sequence repeat in a mammalian cell line. A plasmid containing a dinucleotide repeat [poly(CA/GT)] that disrupts the reading frame of a downstream gene was integrated into the genome of a mouse cell line, and spontaneous revertants were selected. Reversion rates were more than 100 times higher in cells carrying the repeated sequence than in control cells that carried the same fusion gene with a 4-bp out-of-frame deletion. Revertant clones derived from the lines carrying the dinucleotide repeat had insertions or deletions of one or more repeat units.

Animals↗

Analysis of eriophyid mite rDNA internal transcribed spacer sequences reveals variable simple sequence repeats.

Ribosomal DNA internal transcribed spacers of the eriophyid mites Cecidophyopsis ribis, C. selachodon, C. spicata, C. alpina, C. aurea, C. grossulariae and Phylocoptes gracillis were amplified using PCR, cloned and sequenced. Sequences for the ITS1 of Cecidophyopsids were 92-99% homologous. Cecidophyopsis inter-specific differences were found in seventeen simple sequence repeats (vSSRs), fourteen point mutations and two indels. No intra-specific variation in vSSRs was detected. A hypothetical structure for ITS1 was obtained and vSSRs were mapped onto this. Changes in vSSRs were compensated for by changes in complementary vSSRs or through multiple point mutations. A comparison with vSSRs of other arthropods suggested that the levels of intra-specific variation in Cecidophyopsis mites was less than in organisms which do not use arrhentoky for male determination.

Animals↗

Study of simple sequence repeat (SSR) markers from wheat expressed sequence tags (ESTs).

The increasing availability of expressed sequence tags (ESTs) in wheat ( Triticum aestivum) and related cereals provides a valuable resource of non-anonymous DNA molecular markers. We examined 170,746 wheat ESTs from the public (International Triticeae EST Cooperative) and Génoplante databases, previously clustered in contigs, for the presence of di- to hexanucleotide simple sequence repeats (SSRs). Analysis of 46,510 contigs identified 3,530 SSRs, which represented 7.5% of the total number of contigs. Only 74% of the sequences allowed primer pairs to be designed, 70% led to an amplification product, mainly of a high quality (68%), and 53% exhibited polymorphism for at least one cultivar among the eight tested. Even though dinucleotide SSRs were less represented than trinucleotide SSRs (15.5% versus 66.5%, respectively), the former showed a much higher polymorphism level (83% versus 46%). The effect of the number and type of repeats is also discussed. The development of new EST-SSRs markers will have important implications for the genetic analysis and exploitation of the genetic resources of wheat and related species and will provide a more direct estimate of functional diversity.

Breeding↗

Diversity and selection in sorghum: simultaneous analyses using simple sequence repeats.

Although molecular markers and DNA sequence data are now available for many crop species, our ability to identify genetic variation associated with functional or adaptive diversity is still limited. In this study, our aim was to quantify and characterize diversity in a panel of cultivated and wild sorghums (Sorghum bicolor), establish genetic relationships, and, simultaneously, identify selection signals that might be associated with sorghum domestication. We assayed 98 simple sequence repeat (SSR) loci distributed throughout the genome in a panel of 104 accessions comprising 73 landraces (i.e., cultivated lines) and 31 wild sorghums. Evaluation of SSR polymorphisms indicated that landraces retained 86% of the diversity observed in the wild sorghums. The landraces and wilds were moderately differentiated (F st=0.13), but there was little evidence of population differentiation among racial groups of cultivated sorghums (F st=0.06). Neighbor-joining analysis showed that wild sorghums generally formed a distinct group, and about half the landraces tended to cluster by race. Overall, bootstrap support was low, indicating a history of gene flow among the various cultivated types or recent common ancestry. Statistical methods (Ewens-Watterson test for allele excess, lnRH, and F st) for identifying genomic regions with patterns of variation consistent with selection gave significant results for 11 loci (approx. 15% of the SSRs used in the final analysis). Interestingly, seven of these loci mapped in or near genomic regions associated with domestication-related QTLs (i.e., shattering, seed weight, and rhizomatousness). We anticipate that such population genetics-based statistical approaches will be useful for re-evaluating extant SSR data for mining interesting genomic regions from germplasm collections.

Cluster Analysis↗

An in silico mining for simple sequence repeats from expressed sequence tags of zebrafish, medaka, Fundulus, and Xiphophorus.

Teleost fish genome projects involving model species are resulting in a rapid accumulation of genomic and expressed DNA sequences in public databases. The expressed sequence tags (ESTs) collected in the databases can be mined for the analysis of both structural and functional genomics. In this study, we in silico analyzed 49,430 unigenes representing a total of 692,654 ESTs from four model fish for their potential use in developing simple sequence repeats (SSRs), or microsatellites. After bioinformatical mining, a total of 3,018 EST derived SSRs (EST-SSRs) were identified for 2,335 SSR containing ESTs (SSR-ESTs). The frequency of identified SSR-ESTs ranged from 1.5% for Xiphophorus to 7.3% for zebrafish. The dinucleotide repeat motif is the most abundant SSR, accounting for 47%, 52%, 64%, and 78% for medaka, Fundulus, zebrafish, and Xiphophorus, respectively. Simulation analysis suggests that a majority of these EST-SSRs have sufficient flanking sequences for polymerase chain reaction (PCR) primer design. Comparative DNA sequence analyses of SSR-ESTs identified several cross-species SSRs and sequences that may be used as cross-reference genes in comparative studies. For example, the flanking sequences of one SSR (CTG)n within the pituitary tumor-transforming gene (PTTG) 1 interacting protein (PTTGIP), showed conservation spanning the medaka, Fundulus, human, and mouse genomes. This study provides a large body of information on EST-SSRs that can be useful for the development of polymorphic markers, gene mapping, and comparative genome analysis. Functional analysis of these SSR-ESTs may reveal their role in metabolism and gene evolution of these model species.

Amino Acid Sequence↗

Data mining for simple sequence repeats in expressed sequence tags from barley, maize, rice, sorghum and wheat.

Plant genomics projects involving model species and many agriculturally important crops are resulting in a rapidly increasing database of genomic and expressed DNA sequences. The publicly available collection of expressed sequence tags (ESTs) from several grass species can be used in the analysis of both structural and functional relationships in these genomes. We analyzed over 260000 EST sequences from five different cereals for their potential use in developing simple sequence repeat (SSR) markers. The frequency of SSR-containing ESTs (SSR-ESTs) in this collection varied from 1.5% for maize to 4.7% for rice. In addition, we identified several ESTs that are related to the SSR-ESTs by BLAST analysis. The SSR-ESTs and the related sequences were clustered within each species in order to reduce the redundancy and to produce a longer consensus sequence. The consensus and singleton sequences from each species were pooled and clustered to identify cross-species matches. Overall a reduction in the redundancy by 85% was observed when the resulting consensus and singleton sequences (3569) were compared to the total number of SSR-EST and related sequences analyzed (24 606). This information can be useful for the development of SSR markers that can amplify across the grass genera for comparative mapping and genetics. Functional analysis may reveal their role in plant metabolism and gene evolution.

Computational Biology↗

A comparative approach to physical and linkage mapping of genes on canine chromosomes using gene-associated simple sequence repeat polymorphisms illustrated by studies of dog chromosome 9.

We describe and illustrate a comparative approach to creating physical and linkage maps of genes on dog chromosomes. The approach is particularly useful in species, like the dog, which have a rudimentary gene map not integrated with microsatellite loci. Human or mouse cDNAs for genes to be mapped are used to isolate cosmid or phage clones from dog genomic libraries. Clones verified to contain the homologous canine gene coding sequences are screened for "gene-associated" simple sequence repeat polymorphisms (SSRPs). The unique sequences flanking the repeats are used to design PCR primers to amplify the repeat and gene-associated SSR length differences that are informative for linkage analysis used in canine pedigrees to study linkage between loci or with diseases. The same canine clones are employed as probes in fluorescence in situ hybridization (FISH) studies to physically map the loci to specific sites on dog chromosomes. This approach creates a combined gene and gene-associated microsatellite anchor locus framework map. In this article we review our recent use of this approach to map a series of genes found on human chromosome 17 (HSA17) to two dog chromosomes. Canine chromosome 9 (CFA9) contains 11 loci found on HSA17q, while two genes from HSA17p map to CFA5, demonstrating disruption of HSA17 synteny at the centromere. The order of 11 HSA17q genes on CFA9 was conserved in the dog, but the entire group is inverted with respect to the centromere when compared to human and mouse. Maps created by this approach can be used to advantage for integrating anonymous microsatellites with gene maps, including microsatellites found in genome scans to be linked to canine diseases. This makes it possible to identify the homologous chromosomal region in the human or mouse genome and to make use of this information in formulating hypotheses regarding candidate genes, as has recently been illustrated by other investigators.

Animals↗

Simple sequence repeat map of the sunflower genome.

Several independent molecular genetic linkage maps of varying density and completeness have been constructed for cultivated sunflower ( Helianthus annuus L.). Because of the dearth of sequence and probe-specific DNA markers in the public domain, the various genetic maps of sunflower have not been integrated and a single reference map has not emerged. Moreover, comparisons between maps have been confounded by multiple linkage group nomenclatures and the lack of common DNA markers. The goal of the present research was to construct a dense molecular genetic linkage map for sunflower using simple sequence repeat (SSR) markers. First, 879 SSR markers were developed by identifying 1,093 unique SSR sequences in the DNA sequences of 2,033 clones isolated from genomic DNA libraries enriched for (AC)(n) or (AG)(n) and screening 1,000 SSR primer pairs; 579 of the newly developed SSR markers (65.9% of the total) were polymorphic among four elite inbred lines (RHA280, RHA801, PHA and PHB). The genetic map was constructed using 94 RHA280 x RHA801 F(7) recombinant inbred lines (RILs) and 408 polymorphic SSR markers (462 SSR marker loci segregated in the mapping population). Of the latter, 459 coalesced into 17 linkage groups presumably corresponding to the 17 chromosomes in the haploid sunflower genome ( x = 17). The map was 1,368.3-cM long and had a mean density of 3.1 cM per locus. The SSR markers described herein supply a critical mass of DNA markers for constructing genetic maps of sunflower and create the basis for unifying and cross-referencing the multitude of genetic maps developed for wild and cultivated sunflowers.

Journal Article↗