Male-specific band in random amplified microsatellite polymorphism fingerprints of Holstein cattle

A total of 65 blood samples collected from Holstein cattle were employed for DNA extraction. Genomic DNA were amplified by means of random amplified polymorphic DNA (RAPD). One hundred and one random primers (Operon kits OPAA, OPAO, OPAV, OPC, OPE and OPA-06) were used for polymerase chain reactions (PCR). The PCR products were size fractionated by means of electrophoresis in agarose gel, transferred to a nylon membrane, and then hybridized with a alpha-32P-labeled microsatellite probe for random amplified microsatellite polymorphism (RAMPO) analysis. All the primers produced many amplification patterns in RAMPO fingerprints, but only OPE-01 (5'-CCCAAGGTCC-3') yielded a male-specific strong signal. Among the 65 cases, the specific band could be observed in the RAMPO fingerprints of the 24 male samples but not in the 41 female samples. Therefore, this specific band is peculiar to male cattle.     

SSRD: simple sequence repeats database of the human genome

Simple sequence repeats are predominantly found in most organisms. They play a major role in studies of genetic diversity, and are useful as diagnostic markers for many diseases. The simple sequence repeats database (SSRD) for the human genome was created for easy access to such repeats, for analysis, and to be used to understand their biological significance. The data includes the abundance and distribution of SSRs in the coding and non-coding regions of the genome, as well as their association with the UTRs of genes. The exact locations of repeats with respect to genomic regions (such as UTRs, exons, introns or intergenic regions) and their association with STS markers are also highlighted. The resource will facilitate repeat sequence analysis in the human genome and the understanding of the functional and evolutionary significance of simple sequence repeats. SSRD is available through two websites, http://www.ccmb.res.in/ssr and http://www.ingenovis.com/ssr.     

Molecular characterization and genetic mapping of random amplified microsatellite polymorphism in barley

 This study has analyzed the molecular basis and genetic behaviour of the polymorphism generated by the amplification of barley genomic DNA with primers complementary to microsatellites. Primers anchored at the 5′ end, used alone or in combination with arbitrary sequence primers, generated random amplified microsatellite polymorphisms (RAMPs). Unanchored primers were also used as single primers in a microsatellite primed-PCR (MP-PCR). Twenty six randomly selected RAMP DNA fragments which showed polymorphism between the cultivars Steptoe and Morex were cloned and sequenced. All sequences showed the expected repeated motif at the end of the insert, with the number of repeats ranging from five to ten. Genomic sequences containing low numbers of microsatellite motifs were preferentially amplified; therefore, only a fraction of the polymorphism could be attributed to variation in the number of microsatellite motifs at the priming site. Some sequences contained either cryptic simple sequences or members of families of repeated DNA. Polymorphism at the internal cryptic simple sequences was detected by RAMP bands inherited as co-dominant markers. Four MP-PCR bands were cloned and sequenced. A number of repeats identical to the primer itself were found at each end of the insert. Two allelic bands were polymorphic for an internal microsatellite. The potential use of cloned bands as fingerprinting tools was investigated by employing them as hybridization probes in Southern blots containing digested barley DNA from a sample of cultivars. RAMP probes produced complex hybridization band patterns. MP-PCR probes produced either a highly variable single locus or low-copy number loci. Segregations for 31 RAMPs and three MP-PCR bands were studied in a population of 70 doubled-haploids from the Steptoe/Morex cross. One third of all markers were co-dominantly inherited. Markers were positioned on an RFLP map and found to be distributed in all barley chromosomes. The new markers enlarged the overall length of the map to 1408 cM. Received: 6 May 1998 / Accepted: 20 July 1998     

Mapping and analysis of simple sequence repeats in the Arabidopsis thaliana genome

Simple sequence repeats (SSRs) are becoming standard DNA markers for plant genome analysis and are being used as markers in marker assisted breeding. And hence because of its great significance we have initiated this study to analyze complete genome of Arabidopsis thaliana for the prevalence of mono-, di-, tri-, tetra-, penta- and hexa- mer repeats in the coding and non-coding regions of the chromosome and to map their exact position on the sequence. We have developed a program that can search a repeat of any length, its exact position on the chromosome and also its frequency of occurrence in the genome. Analysis of the results reveal that maximum number of repeats were found in chromosome 1 followed by chromosome 2 and 4 whereas, chromosome 3 and 5 contain relatively less number of these repeats. Among the SSRs, hexamers and dimers were more predominant in the chromosomes. Overall data showed that Chromosome 5 has minimum number of repeats. The abundance or rarity of various simple repeats in different chromosomes is not explained by nucleotide composition of sequence or potential repeated motifs to form alternative DNA structures. This suggests that in addition to nucleotide composition of repeat motifs, characteristic DNA replication / repair / recombination machinery might play an important role in genesis of repeats. The positional information is given at www.geocities.com/amubioinfo/ARD. This positional information can help Arabidopsis researchers to identify new polymorphisms in chromosomal regions of interest based on the SSRs that map in the area.     

Distribution and characterization of simple sequence repeats in Gossypium raimondii genome

Simple sequence repeats (SSRs) can be derived from the complete genome sequence. These markers are important for gene mapping as well as marker-assisted selection (MAS). To develop SSRs for cotton gene mapping, we selected the complete genome sequence of Gossypium raimondii, which consisted of 4447 non-redundant scaffolds. Out of 775.2 Mb sequence examined, a total of 136,345 microsatellites were identified with a density of 5.69 kb per SSR in the G. raimondii genome leading to development of 112,177 primer pairs. The distributions of SSRs in the genome were non-random. Among the different motifs ranging from 1 to 6 bp, penta-nucleotide repeats were most abundant (30.5%), followed by tetra-nucleotide repeats (18.2%) and di-nucleotide repeats (16.9%). Among all identified 457 motif types, the most frequently occurring repeat motifs were poly-AT/TA, which accounted for 79.8% of the total di-nt SSRs, followed by AAAT/TTTA with 51.5% of the total tetra-nucleotede. Further, 18,834 microsatellites were detected from the protein-coding genes, and the frequency of gene containing SSRs was 46.0% in 40,976 genes of G. raimondii. These genome-based SSRs developed in the present study will lay the groundwork for developing large numbers of SSR markers for genetic mapping, gene discovery, genetic diversity analysis, and MAS breeding in cotton.     

Differential distribution of simple sequence repeats in eukaryotic genome sequences.

Complete chromosome/genome sequences available from humans, Drosophila melanogaster, Caenorhabditis elegans, Arabidopsis thaliana, and Saccharomyces cerevisiae were analyzed for the occurrence of mono-, di-, tri-, and tetranucleotide repeats. In all of the genomes studied, dinucleotide repeat stretches tended to be longer than other repeats. Additionally, tetranucleotide repeats in humans and trinucleotide repeats in Drosophila also seemed to be longer. Although the trends for different repeats are similar between different chromosomes within a genome, the density of repeats may vary between different chromosomes of the same species. The abundance or rarity of various di- and trinucleotide repeats in different genomes cannot be explained by nucleotide composition of a sequence or potential of repeated motifs to form alternative DNA structures. This suggests that in addition to nucleotide composition of repeat motifs, characteristic DNA replication/repair/recombination machinery might play an important role in the genesis of repeats. Moreover, analysis of complete genome coding DNA sequences of Drosophila, C. elegans, and yeast indicated that expansions of codon repeats corresponding to small hydrophilic amino acids are tolerated more, while strong selection pressures probably eliminate codon repeats encoding hydrophobic and basic amino acids. The locations and sequences of all of the repeat loci detected in genome sequences and coding DNA sequences are available at http://www.ncl-india.org/ssr and could be useful for further studies.     

Identification of polymorphic simple sequence repeats in the genome of the zebrafish.

The zebrafish has drawn a great deal of attention as a developmental system because it offers the ability to combine excellent embryology and genetics. Here, we report that simple sequence repeats are abundant in the zebrafish genome and are highly polymorphic between two outbred lines, making them useful markers for the construction of a genetic map of this organism.     

Identification of a novel sequence at the 3' end of the GB virus B genome

GB virus B (GBV-B) is a virus of the family Flaviviridae that infects small primates (Saguinus sp. [tamarins]) and shows similarities to hepatitis C virus (HCV) in genome organization, protein function, tissue tropism, and pathogenicity. This suggests the possibility of using tamarins infected by GBV-B or GBV-B/HCV chimeric viruses as a surrogate animal model of HCV infection. To achieve the construction of such chimeric viruses, it is essential to produce a complete and infectious GBV-B genomic RNA. We have identified a novel sequence at the 3' end of the GBV-B genome and show that it can be arranged in a secondary structure resembling that of the 3' end of the HCV genome, which is known to be essential for infectivity.     

Color-coding reveals tandem repeats in the Escherichia coli genome

Genomic DNA contains a wide variety of repetitive sequences. In Escherichia coli, there have been several classes of repetitive sequences reported, some of which cluster as tandem repeats. We propose a novel method for analyzing symbolic sequences by two-dimensional pattern formation with color-coding. We applied this method for searching tandem repeats in the E. coli genome and found approximately 50 repeats with periods longer than 30 bases. The longest repeat has a period of 1267 bases.     

High GC content of simple sequence repeats in Herpes simplex virus type 1 genome

The presence, locations and composition of simple sequence repeats (SSRs) in Herpes simplex virus type 1 (HSV-1) genome were extracted and analyzed by using the software Imperfect Microsatellite Extractor (IMEx). There were 663 mon-, 502 di-, 184 tri-, 20 tetra-, 4 penta- and 4 hexanucleotide SSRs that were observed in different distribution between coding and noncoding regions in the HSV-1 genome. G/C, GC/CG, and (GGC)(n) were predominant in mononucleotide, dinucletide, trinucleotide repeats respectively. Indeed, the results showed that GC content in simple sequence repeats was notably higher than that in entire HSV-1 genome. Our data might be helpful for studying the pathogenesis, genome structure and evolution of HSV-1.     

Simple sequence repeats in the Helicobacter pylori genome

We describe an integrated system for the analysis of DNA sequence motifs within complete bacterial genome sequences. This system is based around ACeDB, a genome database with an integrated graphical user interface; we identify and display motifs in the context of genetic, sequence and bibliographic data. Tomb et al . (1997) previously reported the identification of contingency genes in Helicobacter pylori through their association with homopolymeric tracts and dinucleotide repeats. With this as a starting point, we validated the system by a search for this type of repeat and used the contextual information to assess the likelihood that they mediate phase variation in the associated open reading frames (ORFs). We found all of the repeats previously described, and identified 27 putative phase-variable genes (including 17 previously described). These could be divided into three groups: lipopolysaccharide (LPS) biosynthesis, cell-surface-associated proteins and DNA restriction/modification systems. Five of the putative genes did not have obvious homologues in any of the public domain sequence databases. The reading frame of some ORFs was disrupted by the presence of the repeats, including the alpha(1-2) fucosyltransferase gene, necessary for the synthesis of the Lewis Y epitope. An additional benefit of this approach is that the results of each search can be analysed further and compared with those from other genomes. This revealed that H . pylori has an unusually high frequency of homopurine:homopyrimidine repeats suggesting mechanistic biases that favour their presence and instability.     

Evaluation of amplified fragment length polymorphism and simple sequence repeats for tomato germplasm fingerprinting: utility for grouping closely related traditional cultivars.

Cultivated tomato (Solanum lycopersicum L.) germplasm shows limited genetic variation. Many DNA marker systems have been used for genetic diversity studies in wild and cultivated tomatoes, but their usefulness for characterizing phenotypic differences among very closely related cultivars remains uncertain. We have used 19 selected simple sequence repeat (SSR) markers and 7 amplified fragment length polymorphism (AFLP) primer combinations to characterize 48 cultivars of tomato, mainly traditional cultivars from the south-east of Spain. The main types were Solanum lycopersicum L. 'Muchamiel', 'De la pera', and 'Moruno'. The robustness of the dendrograms and the discrimination power reached with each marker type were similar. Unique fingerprinting even of the most closely related tomato cultivars could be obtained using a combination of some SSR and AFLP markers. A better grouping of the 'Muchamiel' cultivars was observed with SSR markers, whereas the grouping of cultivars of 'De la pera' type was best achieved with AFLPs. However, both types of markers adequately grouped cultivars of the main types, confirming the utility of SSR and AFLP markers for the identification of traditional cultivars of tomato.     

Cleaved amplified polymorphic sequence and amplified fragment length polymorphism markers linked to the fertility restorer gene in chili pepper (Capsicum annuum L.)

Cytoplasmic male sterility (CMS) in plants, which is due to failure to produce functional pollen, is a maternally inherited trait. Specific nuclear genes that sup-press CMS, termed fertility restorer (Rf) genes, have been identified in several plants. In this study, Rf-linked molecular markers in pepper (Capsicum annuum L.) were detected by bulked segregant analysis of eight amplified fragment length polymorphisms (AFLPs). Only AFRF8 was successfully converted to a cleaved amplified polymorphic sequence (CAPS) marker. This was named AFRF8CAPS and genotype determination using it agreed with that obtained with the original AFRF8. A linkage map with a total size of 54.1 cM was constructed with AFRF8CAPS and the seven AFLP markers using the Kosambi function. The AFRF8CAPS marker was shown to be closest to Rf with a genetic distance of 1.8 cM. These markers will be useful for fast and reliable detection of restorer lines during F(1) hybrid seed production and breeding programs in pepper.     

Comparison of simple sequence repeats in 19 Archaea

All organisms that have been studied until now have been found to have differential distribution of simple sequence repeats (SSRs), with more SSRs in intergenic than in coding sequences. SSR distribution was investigated in Archaea genomes where complete chromosome sequences of 19 Archaea were analyzed with the program SPUTNIK to find di- to penta-nucleotide repeats. The number of repeats was determined for the complete chromosome sequences and for the coding and non-coding sequences. Different from what has been found for other groups of organisms, there is an abundance of SSRs in coding regions of the genome of some Archaea. Dinucleotide repeats were rare and CG repeats were found in only two Archaea. In general, trinucleotide repeats are the most abundant SSR motifs; however, pentanucleotide repeats are abundant in some Archaea. Some of the tetranucleotide and pentanucleotide repeat motifs are organism specific. In general, repeats are short and CG-rich repeats are present in Archaea having a CG-rich genome. Among the 19 Archaea, SSR density was not correlated with genome size or with optimum growth temperature. Pentanucleotide density had an inverse correlation with the CG content of the genome.     

The nucleotide sequence of an untranslated but conserved domain at the 3' end of the avian sarcoma virus genome.

The genomes of numerous avian retroviruses contain at their 3' termini a conserved domain denoted "c". The precise boundaries and function of "c" have been enigmas. In an effort to resolve these issues, we determined the sequence of over 900 nucleotides at the 3' end of the genome of the Schmidt-Ruppin subgroup A strain of avian sarcoma virus (ASV). We obtained the sequence from a suitable fragment of ASV DNA that had cloned into the single-stranded DNA phage M13mp2. Computer-assisted analysis of the sequence revealed the following structural features: i) the length of "c" - 473 nucleotides; ii) the 3' terminal domain of src, ending in an amber codon at the 5'boundary of "c"; iii) terminator codons that preclude continuous translation from "c"; iv) suitably located sequences that may serve as signals for the initiation of viral RNA synthesis and for the processing and/or polyadenylation of viral mRNA; v) a repeated sequence that flanks src and that could facilitate deletion of this gene; vi) repeated sequences within "c"; and vii) unexplained homologies between sequences in "c" and sequences in several other nucleic acids, including the 5' terminal domain of the ASV genome, tRNATrp and its inversion, the complement of tRNATrp and its inversion, and the 18S RNA of eukaryotic ribosomes. We conclude that "c" probably does not encode a protein, but its sequence may nevertheless serve several essential functions in viral replication.