Characterisation of polymorphic microsatellite markers from Aegilops tauschii and transferability to the D-genome of bread wheat

Microsatellites were isolated from a Aegilops tauschii (the D-genome donor of bread wheat) library enriched for various motifs. Primers generated from the flanking region of the microsatellites were used successfully to amplify the corresponding loci in the D genome of bread wheat. Additional amplification sometimes also occurred from the A and B genomes. The majority of the microsatellites contained (GA)(n) and (GT)(n) motifs. GA and GT repeats appeared to be both more abundant in this library and more polymorphic than other types of repeats. The allele number for both types of dinucleotide repeats fitted a Poisson distribution. Deviance analysis showed that GA and GT were more polymorphic than other motifs in bread wheat. Within each motif type (di-, tri- and tetra-nucleotide repeats), repeat number has no influence on polymorphism. The microsatellites were mapped using the Triticum aestivum Courtot x Chinese Spring mapping population. A total of 100 markers was developed on this intraspecific map, mainly on the D genome. For polyploid species, isolation of microsatellites from an ancestral diploid donor seems to be an efficient way of developing markers for the corresponding genome in the polyploid plant.     

Mismatch repair-driven mutational bias in D. melanogaster

We have used microsatellite sequences to evaluate the influence of the mismatch repair system on mutation bias in D. melanogaster. While mismatch-proficient cells have the highest mutation rate at (GT)(n) repeats, (AT)(n) repeats were the least stable ones in spel1(-/-) flies lacking functional mismatch repair. Furthermore, the mutation spectrum of long microsatellite alleles in spel1(-/-) was slightly upward biased, resulting in a gain of repeats, whereas wild-type flies have a strong downward bias. Interestingly, this mismatch repair-mediated downward mutation bias is reflected in the genome composition of D. melanogaster. When compared to other species, D. melanogaster has significantly shorter microsatellites. Our results suggest that the mismatch repair system may have an important role in shaping genome composition.     

Characterization of (GT)n and (CT)n microsatellites in two insect species: Apis mellifera and Bombus terrestris.

A set of 52 (CT)n and 23 (GT)n microsatellites in honeybee, 24 (CT)n and 2 (GT)n microsatellites in bumble-bee (n > 6) have been isolated from partial genomic libraries and sequenced. On average, (CT)n and (GT)n microsatellites occur every 15 kb and 34 kb in honeybee and every 40 kb and 500 kb in bumble-bee, respectively. The prevailing categories are imperfect repeats for (CT)n microsatellites in bumble-bee, and perfect repeats for both (CT)n and (GT)n microsatellites in honey-bee. Comparisons with data available in vertebrates indicate a lower proportion of perfect repeats in bees but length distributions are very similar regardless the phylum. This result extends to insects the concept of an evolutionary conservation for quantitative and qualitative characteristics of (CT)n and (GT)n microsatellites. Many (CT)n and (GT)n repeats are surrounded with various types of microsatellites, revealing an associative distribution of short repeat sequences. As expected, a high level of intrapopulational polymorphism has been found with one tested honeybee microsatellite. Also, flanking regions of this microsatellite are similar enough to allow PCR amplification in several other species of Apis and Bombus.     

有尾噬菌体目中微卫星和复合型微卫星的发生及分析

简单重复序列亦称微卫星,被成功应用于许多真核生物、原核生物和病毒的基因组和进化研究,但是噬菌体中的微卫星目前很少被研究。因此对60条尾病毒目基因组中的微卫星和和复合型微卫星(由两个或两个以上直接相邻的微卫星组成)做综合性分析,在这60个基因组中总共观察到11 874个微卫星和449个复合型微卫星。相关性分析表明微卫星个数与基因组大小成正线性相关(ρ=0.899, P<0.01)。参考序列中的微卫星个数少于对应的随机序列中微卫星个数,这种反常现象主要是因为参考序列含有较少的单核苷酸和二核苷酸重复。A/T和AT/TA重复是单核苷酸和二核苷酸重复中最主要的类型,因此单核苷酸重复中的GC含量明显低于相应的序列中的GC含量;相比之下,微卫星中的二核苷酸和三核苷酸重复的GC含量与对应的参考序列的GC含量无明显区别。尾病毒目基因组中的这些结果与其它生物体基因组存在一定的差别。有助于了解尾病毒目中微卫星的分布、进化和生物学功能。     

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.     

Distribution of telomeric (TTAGGG)<Subscript>n</Subscript> sequences in avian chromosomes

The physical ends of mammalian and other vertebrate chromosomes consist of tandemly repeated (TTAGGG)(n) hexamers, nucleating a specialized telomeric structure. However, (TTAGGG)(n) sequences can also occur at non-telomeric sites, providing important insights into karyotypic evolution. By fluorescence in situ hybridization (FISH) we studied the chromosomal distribution of (TTAGGG)(n) sequences in 16 bird species, representing seven different orders. Many species, in particular the ratites, display (TTAGGG)(n) hybridization signals in interstitial and centromeric regions of their macrochromosomes in addition to the typical telomeric signals. In some but not all species these non-telomeric sites coincide with C-band-positive heterochromatin. The retention and/or amplification of telomeric (TTAGGG)(n) repeats at interstitial and centromeric sites may indicate the fusion of ancestral chromosomes. Compared with the macrochromosomes, the microchromosomes of most species are enriched with (TTAGGG)(n) sequences, displaying heterogeneous hybridization patterns. We propose that this high density of (TTAGGG)(n) repeats contributes to the exceptionally high meiotic recombination rate of avian microchromosomes.     

Characterization and isolation of novel microsatellites from the Drosophila dunni subgroup

We have isolated and characterized 77 novel microsatellites from two species, Drosophila dunni and Drosophila nigrodunni, which are closely related Caribbean-island endemics from the Drosophila cardini species group. These species are very distantly related to all other Drosophila from which microsatellites have previously been characterized. We find that the average length of microsatellites isolated in these species is quite small, with an overall mean length of 9.8 repeat units for dinucleotide microsatellites in the two study species. The nucleotide composition of dinucleotides differs between the two species: D. nigrodunni has a predominance of (AC/GT)n repeats, whereas D. dunni has equal numbers of (AC/GT)n and (AG/CT)n repeats. Tri- and tetranucleotide repeats are not abundant in either species. We assayed the variability of eight microsatellites in a closely related third species, Drosophila arawakana, using wild-caught individuals from the island of Guadeloupe. We found the microsatellites to be extremely variable in this population, with observed heterozygosities ranging from 0.541 to 0.889. DNA amplification trials suggest that these eight microsatellites are widely conserved across the D. cardini group, with five of the eight producing amplification products in every species tested. However, the loci are very poorly conserved over greater phylogenetic distances. DNA amplification of the microsatellite loci was unreliable in members of the closely related Drosophila quinaria, Drosophila calloptera, Drosophila guarani and Drosophila tripunctata species groups. Furthermore, these microsatellites could not be detected in the genome of Drosophila melanogaster, despite the conservation of microsatellite flanking regions at some loci. These data indicate that Drosophila microsatellite loci are quite short lived over evolutionary timescales relative to many other taxa.     

(TG:CA)(n) repeats in human housekeeping genes

The unravelling of human genome sequence gives a new opportunity to investigate the role of repetitive sequences in gene regulation. Among the various types of repetitive sequences, the dinucleotide (TG:CA)(n) repeats are one of the most abundant in human genome and exhibit polymorphism. Early on, it was observed that the (TG:CA)(n) repeats could modulate gene expression and has the propensity to undergo conformational transitions in in vivo conditions. Recent reports describe the role of polymorphic (TG:CA)(n) repeats in gene regulation in several genes. In this work, we have analysed the distribution of (TG:CA)(n) (n >or= 6) repeats in human 'housekeeping genes' on which recently released Gene Chip data is available. Our results indicate that (i). The number of short intragenic (TG:CA)(n) repeats is significantly higher than the number of long repeats (ii). the proportion of genes with (TG:CA)(n) repeats (n >or= 12 units) had lower mean expression levels compared to those without these repeats, (iii). the genes belonging to the functional class of 'signalling and communication' had a positive association with repeats in contrast to the genes belonging to the 'information' class that were negatively associated with repeats.     

Microsatellite repeats are not randomly distributed within Norway spruce (Picea abies K.) expressed sequences.

A Norway spruce (Picea abies K.) cDNA library obtained from vegetative bud tissue was screened for the presence of (AG)n and (AC)n microsatellite repeats. Ten (AG)n and six (AC)n microsatellites were found, with an average length of 25.5 repeat units. Most of the microsatellites are simple perfect repeats. The microsatellite distribution within the clones is clearly non-random, with different classes of repeats lying in different positions relative to the coding region and in a highly conserved orientation. An estimate of the frequency of dinucleotide microsatellites in expressed regions was obtained, showing that SSRs (simple sequence repeats) are found in genes about 20 times less frequently than in random genomic clones, with (AG)n repeats more frequent than (AC)n repeats. Potential applications of these sequences as expressed region-based molecular markers are shown by developing six SSR markers for the detection of natural variation in Norway spruce populations and testing two of them for the identification of illegitimate progenies from a mapping population.     

Human papillomavirus 1a complete DNA sequence: a novel type of genome organization among papovaviridae

The complete nucleotide sequence of human papillomavirus type 1a (7811 nucleotides) has been established. The overall organization of the viral genome is different from that of other related papovaviruses (SV40, BKV, polyoma). Firstly, genetic information seems to be coded by one strand. Secondly, no significant homology is found with SV40 or polyoma coding sequence for either DNA or deducted protein sequences. The relatedness of human and bovine papillomaviruses is revealed by a conserved coding sequence in the two species. Two regions can be defined on the viral genome: the putative early region contains two large open reading frames of 1446 and 966 nucleotides, together with several split ones, and corresponds to the transforming part of the bovine papillomavirus type 1 genome, and the remaining sequences, which include two open reading frames likely to encode structural polypeptide(s). The DNA sequence is analysed and putative signals for regulation of gene expression, and homologies with the Alu family of human ubiquitous repeats and the SV40 72-bp repeat are outlines.     

Conformational changes induced in the human protein translin and in the single-stranded oligodeoxynucleotides d(GT)(12) and d(TTAGGG)(5) upon binding of these oligodeoxynucleotides by translin

Translin is a human single-stranded DNA and RNA binding protein that has been highly conserved in eukaryotic evolution. It consists of eight subunits having a highly helical secondary structure that assemble into a ring. The DNA and the RNA are bound inside the ring. Recently, some of us demonstrated that the human translin specifically binds the single-stranded microsatellite repeats, d(GT)(n), the human telomeric repeats, d(TTAGGG)(n), and the Tetrahymena telomeric repeats, d(GGGGTT)(n). These data suggested that translin might be involved in recombination at d(GT)(n).d(AC)(n) microsatellites and in telomere metabolism. Other data indicated that translin might stimulate binding of telomerase to single-stranded telomeric overhangs by unwinding secondary structures formed by the telomeric repeats. Here we present a circular dichroism (CD) analysis of complexes formed between the human translin and the microsatellite and telomeric oligodeoxynucleotides d(GT)(12) and d(TTAGGG)(5). We report that conformational changes occur in both the translin and the oligodeoxynucleotides upon formation of the complexes. In translin octamers bound to the oligodeoxynucleotide d(GT)(12), the fraction of alpha-helices decreases from approximately 67% to approximately 50%, while the fraction of turns and of the unordered structure increases from approximately 11% to approximately 17% and from approximately 19% to approximately 24%, respectively. In the bound oligodeoxynucleotide d(GT)(12), we observed CD shifts which are consistent with a decrease of base stacking and a putative anti-syn switch of some guanines. The oligodeoxynucleotide d(TTAGGG)(5) formed intramolecular quadruplexes under the conditions of our assays and translin was found to unfold the quadruplexes into structures consisting of a single hairpin and three unwound single-stranded d(TTAGGG) repeats. We suggest that such unfolding could account for the stimulation of telomerase activity by translin mentioned above.     

The application of SSRs characterized for grape (Vitis vinifera) to conservation studies in Vitaceae

The use of microsatellite loci developed from a single plant species across a number of related taxa is becoming increasingly widespread. This approach can provide highly informative markers even for species for which microsatellites have not been characterized. As a number of expressed sequence tag (EST)-derived and enrichment-derived microsatellites are available for grape (Vitis vinifera), this study set out to assess transferability of nine such loci across 25 species from five different Vitaceae genera. Intergeneric transfer success in Vitaceae was high (51.1%) and EST-derived loci performed better than enrichment-derived loci. Six loci were further tested across two Australian native species, Cissus hypoglauca and C. sterculiifolia, in order to assess the conservation of microsatellite repeats and their flanking sequences. Polymorphism of these selected loci was successfully tested for each species across a small, isolated rain forest population from northern New South Wales (Australia). Results from this preliminary investigation suggest that it is possible to use grape-derived simple sequence repeats (SSR) loci for population studies on Vitaceae. As Vitaceae are an important component of rain forest flora, the availability of such highly informative loci will be beneficial to future studies aimed at determining the genetic consequences of rain forest fragmentation.     

Transition stages of molecular drive in multiple-copy DNA families in Drosophila

Multigene and non-genic DNA families are in a state of turnover and hence are continually being replaced throughout a population by new variant repeats. To quantify such molecular processes, in the absence of selection, it is necessary to find and compare stages of transistion during the homogenization of at least two non-genic families evolving in parallel in a closely related group of species. Detailed sequence analysis of patterns of variation, at each nucleotide position considered independently, amongst repeats of two tandem DNA families from seven related Drosophila species, reveals all stages of transition during the spread of randomly produced variant repeats. Variant repeats are found at different stages of homogenization and fixation in a population, irrespective of the loci, chromosomes or individuals from which they were cloned. Differences between the families in the relatively small number of variants at each transition stage and the greater number of fully homogenized and fixed variants between species of greater divergence indicate that the process of spread (molecular drive) is rapid relative to the mutation rate and occurs at seemingly different constant rates for each family. Occasional gene conversions, in addition to unequal exchanges, have contributed to family turnover. The significance of these results to the evolution of functional multigene families and divergence and conservation of sequences is discussed.     

Construction of a cytogenetically anchored microsatellite map in rabbit

Rabbit (Oryctolagus cuniculus) represents a valuable source of biomedical models and corresponds to a small but active economic sector in Europe for meat and fur. The rabbit genome has not been thoroughly studied until recently, and high-resolution maps necessary for identification of genes and quantitative trait loci (QTL) are not yet available. Our aim was to isolate over 300 new and regularly distributed (TG)n or (TC)n rabbit microsatellites. To achieve this purpose, 164 microsatellite sequences were isolated from gene-containing bacterial artificial chromosome (BAC) clones previously localized by fluorescence in situ hybridization (FISH) on all the rabbit chromosomes. In addition, 141 microsatellite sequences were subcloned from a plasmid genomic library, and for 41 of these sequences, BAC clones were identified and FISH-mapped. TC repeats were present in 62% of the microsatellites derived from gene-containing BAC clones and in 22% of those from the plasmid genomic library, with an average of 42.9% irrespective of the microsatellite origin. These results suggest a higher proportion of (TC)n repeats and a nonhomogeneous distribution of (TG)n and (TC)n repeats in the rabbit genome compared to those in man. Among the 305 isolated microsatellites, 177 were assigned to 139 different cytogenetic positions on all the chromosomes except rabbit Chromosome 21. Sequence similarity searches provided hit locations on the Human Build 35a and hypothetical assignments on rabbit chromosomes for ten additional microsatellites. Taken together, these results report a reservoir of 305 new rabbit microsatellites of which 60% have a cytogenetic position. This is the first step toward the construction of an integrated cytogenetic and genetic map based on microsatellites homogeneously anchored to the rabbit genome.     

The L1Md long interspersed repeat family in the mouse: almost all examples are truncated at one end.

We have characterized a large repetitive element which has been found at seven different locations within the beta globin locus of the BALB/c mouse. This repeat has an unusual structure in that each of the different members has the same end of the element conserved while the other end terminates at a different point in each repeat member. The sequences within the repeats from the beta globin locus have homology with other repetitive families such as the MIF-1, Bam-5, R, and the BamH1 families. These were recently proposed (T. Fanning, (1983) Nucleic Acids Res. 11, 5073-5091) to be part of a structure with the same organization which we found in the globin locus. Probing plaques from a BALB/c genomic library with sequences derived from the repeats in the globin locus shows that virtually all of the repeats from this family are organized in a manner consistent with the proposed structure.     

Sixteen polymorphic microsatellites in bighead carp (Aristichthys nobilis) and cross-amplification in silver carp (Hypophthalmichthys molitrix)

A (GT)(n) enriched partial genomic library of bighead carp (Aristichthys nobilis) was constructed by employing the (fast isolation by AFLP of sequences containing repeats) FIASCO protocol. Sixteen loci exhibited polymorphism with two to seven alleles/locus (mean 3.263) in a test population and the observed heterozygosity ranging from 0.100 to 0.690 (mean 0.392). Eleven of the 16 bighead carp microsatellites were found to be also polymorphic in silver carp. These polymorphic loci should provide sufficient level of genetic diversity to evaluate population structure of bighead carp.