Development of microsatellite markers and characterization of simple sequence length polymorphism (SSLP) in rice (Oryza sativa L.)

Microsatellite markers containing simple sequence repeats (SSR) are a valuable tool for genetic analysis. Our objective is to augment the existing RFLP map of rice with simple sequence length polymorphisms (SSLP). In this study, we describe 20 new microsatellite markers that have been assigned to positions along the rice chromosomes, characterized for their allelic diversity in cultivated and wild rice, and tested for amplification in distantly related species. Our results indicate that the genomic distribution of microsatellites in rice appears to be random, with no obvious bias for, or clustering in particular regions, that mapping results are identical in intersubspecific and interspecific populations, and that amplification in wild relatives ofOryza sativa is reliable in species most closely related to cultivated rice but becomes less successful as the genetic distance increases. Sequence analysis of SSLP alleles in three relatedindica varieties demonstrated the clustering of complex arrays of SSR motifs in a single 300-bp region with independent variation in each. Two microsatellite markers amplified multiple loci that were mapped onto independent rice chromosomes, suggesting the presence of duplicated regions within the rice genome. The availability of increasing numbers of mapped SSLP markers can be expected to increase the power and resolution of genome analysis in rice.     

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.     

The human episome HALF1: Structure of its genomic counterpart

A human episomal sequence (HALF1) has been identified by its ability to restore expression of hepatic functions when used to transfect a rat dedifferentiated cell line. The genomic equivalent of this human episome (gHALF1) and its flanking sequences were analyzed. HALF1 itself does not present the characteristics of a transposable element but half of its sequence corresponds to retroposons, including Alu and L1 repeats and a processed pseudogene, known to transposevia RNA intermediates. The structural characteristics of these different kinds of retroposons and their origin and evolution were analyzed.     

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.     

Recombination between repeats in Escherichia coli by a recA-independent, proximity-sensitive mechanism

We have examined the influence of proximity on the efficiency of recombination between repeated DNA sequences in Escherichia coli. Our experiments have employed a plasmid-based assay to detect deletions between direct repeats of 100 bp. The rate of deletion of the juxtaposed direct repeats was reasonably high at 6 × 10^–5 per cell. A comparison of recA+ and recA mutant strains showed that these deletion events are primarily the result of recA-independent recombination at these homologous sequences. Random restriction fragments of yeast or E. coli genomic DNA were used to separate the two repeats. Deletion rates decreased over two orders of magnitude with increasing separation of up to 7 kb. There was a surprisingly strong effect of even short sequence separations, with insertions of a few hundred base pairs exhibiting 10-fold reductions of deletion rates. No effect of recA on the efficiency of deletion was observed at any distance between repeats.     

Two distinct P element subfamilies in the genome of Drosophila bifasciata

The genome of Drosophila bifasciata harbours two distinct subfamilies of P-homologous sequences, designated M-type and O-type elements based on similarities to P element sequences from other species. Both subfamilies have some general features in common: they are of similar length (M-type: 2935 bp, O-type: 2986 bp), are flanked by direct repeats of 8 by (the presumptive target sequence), contain terminal inverted repeats, and have a coding region consisting of four exons. The splice sites are at homologous positions and the exons have the coding capacity for proteins of 753 amino acids (M-type) and 757 amino acids (O-type). It seems likely that both types of element represent functional transposons. The nucleotide divergence of the two P element subfamilies is high (31%). The main structural difference is observed in the terminal inverted repeats. Whereas the termini of M-type elements consist of 31 by inverted repeats, the inverted repeats of the O-type elements are interrupted by non-complementary stretches of DNA, 12 by at the 5 end and 14 by at the 3 end. This peculiarity is shared by all members of the O-type subfamily. Comparison with other P element sequences indicates incongruities between the phylogenies of the species and the P transposons. M-type and O-type elements apparently have no common origin in the D. bifasciata lineage. The M-type sequence seems to be most closely related to the P element from Scaptomyza pallida and thus could be considered as a more recent invader of the D. bifasciata gene pool. The origin of the O-type elements cannot be unequivocally deduced from the present data. The sequence comparison also provides new insights into conserved domains with possible implications for the function of P transposons.     

A family of related sequences associated with (TTTAGGG)n repeats are located in the interstitial regions of wheat chromosomes

A family of related sequences associated with (TTTAGGG) _n repeats has been cloned from the wheat cultivar Chinese Spring. These sequences reveal a high level of polymorphism between wheat varieties when used as restriction fragment length polymorphism (RFLP) probes. Although this family of sequences contains motifs homologous to the repeats in the telomeres of wheat, they are located at interstitial sites on wheat chromosomes.     

Complex duplications in maize lines

Rp1 is a disease resistance complex and is the terminal morphological marker on the short arm of maize chromosome 10. Several restriction fragment length polymorphisms (RFLPs), which map within 5 map units of Rp1, were examined to determine if they are also complex in structure. Two RFLP loci, which mapped distally to Rp1, BNL3.04 and PIO200075, existed in a single copy in all maize lines examined. These two loci cosegregated perfectly in 130 test cross progeny. Two RFLP loci that map proximally to Rp1 had unusual structures, which have not yet been reported for maize RFLPs; the loci were complex, with variable numbers of copies in different maize lines. One of the loci, NPI285, occasionally recombined in meiosis to yield changes in the number of copies of sequences homologous to the probe. The other proximal locus, detected by the probes NPI422, KSU3, and KSU4, was relatively stable in meiosis and no changes in the number of restriction fragments were observed. The similarity in map position between Rp1 and the complex RFLP loci indicate there may be genomic areas where variable numbers of repeated sequences are common. The structure of these complex loci may provide insight into the structure and evolution of Rp1.