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.Electronic Supplementary Material Supplementary material is available in the online version of this article at .Communicated by Q. Zhang     

Development of simple sequence repeat DNA markers and their integration into a barley linkage map

Simple sequence repeats (SSRs), or microsatellites, are a new class of PCR-based DNA markers for genetic mapping. The objectives of the present study were to develop SSR markers for barley and to integrate them into an existing barley linkage map. DNA sequences containing SSRs were isolated from a barley genomic library and from public databases. It is estimated that the barley genome contains one (GA)_n repeat every 330 kb and one (CA)_n repeat every 620 kb. A total of 45 SSRs were identified and mapped to seven barley chromosomes using doubled-haploid lines and/or wheat-barley addition-line assays. Segregation analysis for 39 of these SSRs identified 40 loci. These 40 markers were placed on a barley linkage map with respect to 160 restriction fragment length polymorphism (RFLP) and other markers. The results of this study demonstrate the value of SSRs as markers in genetic studies and breeding research in barley.     

Single-copy,species-transferable microsatellite markers developed from loblolly pine ESTs

Microsatellites, or simple sequence repeats (SSRs), are usually regarded as the markers of choice in population genetics research because they exhibit high variability. The development cost of these markers is usually high. In addition, microsatellite primers developed for one species often do not cross-amplify in related species, requiring separate development for each species. However, microsatellites found in expressed sequence tags (ESTs) might better cross-amplify as they reside in or near conserved coding DNA. In this study, we identified 14 Pinus taeda (loblolly pine) EST-SSRs from public EST databases and tested for their cross-species transferability to P. contorta ssp. latifolia, P. ponderosa, and P. sylvestris. As part of our development of a P. contorta microsatellite set, we also compared their transferability to that of 99 traditional microsatellite markers developed in P. taeda and tested on P. contorta ssp. latifolia. Compared to traditional microsatellites, EST-SSRs had higher transfer rates across pine species; however, the level of polymorphism of microsatellites derived from ESTs was lower. Sequence analyses revealed that the frequencies of insertions/deletions and base substitutions were lower in EST-SSRs than in other types of microsatellites, confirming that EST-SSRs are more conserved than traditional SSRs. Our results also provide a battery of 23 polymorphic, robust microsatellite primer pairs for lodgepole pine.Communicated by O. Savolainen     

EST-derived microsatellites from<Emphasis Type="Italic"> Actinidia</Emphasis> species and their potential for mapping

To increase the speed and reduce the cost of constructing a genetic map of Actinidia species (kiwifruit), for use in both breeding and functional genomics programmes, we sampled microsatellites from expressed sequence tags (ESTs) to evaluate their frequency of occurrence and level of polymorphism. Perfect dinucleotide repeats were the microsatellites selected, and these were found to be numerous in both the 5 and 3 ends of the genes represented. The microsatellites were of various lengths, the majority being repeats with the pattern (CT) _n /(GA) _n . One hundred and fifty microsatellites, each with more than 10 dinucleotide repeat units, were chosen as possible markers, and when these were amplified, 93.5% were found to be polymorphic and segregating in a mapping population, with 22.6% amplifying more than one locus. Four marker categories were identified. Fully informative markers made up 27% of the total, 36.2% were female informative, 25.8% were male informative and 10% partly informative. The mapping population was an intraspecific cross in the diploid species Actinidia chinensis, with parents chosen for their diversity in fruit and plant characteristics, and for their geographical separation. Linkage was tested using the software Joinmap and a LOD value of 3. The distribution of the EST-based markers over the linkage groups obtained appeared to be random, taking into consideration the small sample size, that the number of linkage groups (31) exceeded the chromosome number of n=29, and that a number of markers were not assigned to any group. Some microsatellite markers which amplified more than one locus mapped to separate linkage groups. According to our study in A. chinensis, EST-derived microsatellites give large numbers of possible markers very quickly and at reasonable cost. The markers are highly polymorphic, segregate in the mapping population, and increase the value of the genomic map by providing some functional information.Communicated by P. Langridge     

Map and analysis of microsatellites in the genome of <Emphasis Type="Italic">Populus</Emphasis>: The first sequenced perennial plant

We mapped and analyzed the microsatellites throughout 284295605 base pairs of the unambiguously assembled sequence scaffolds along 19 chromosomes of the haploid poplar genome. Totally, we found 150985 SSRs with repeat unit lengths between 2 and 5 bp. The established microsatellite physical map demonstrated that SSRs were distributed relatively evenly across the genome of Populus. On average, These SSRs occurred every 1883 bp within the poplar genome and the SSR densities in intergenic regions, introns, exons and UTRs were 85.4%, 10.7%, 2.7% and 1.2%, respectively. We took di-, tri-, tetra-and pentamers as the four classes of repeat units and found that the density of each class of SSRs decreased with the repeat unit lengths except for the tetranucleotide repeats. It was noteworthy that the length diversification of microsatellite sequences was negatively correlated with their repeat unit length and the SSRs with shorter repeat units gained repeats faster than the SSRs with longer repeat units. We also found that the GC content of poplar sequence significantly correlated with densities of SSRs with uneven repeat unit lengths (tri-and penta-), but had no significant correlation with densities of SSRs with even repeat unit lengths (di-and tetra-). In poplar genome, there were evidences that the occurrence of different microsatellites was under selection and the GC content in SSR sequences was found to significantly relate to the functional importance of microsatellites.     

Genome specific,highly repeated sequences of Hordeum vulgare: cloning,sequencing and squash dot test

Summary Highly repeated sequences of nuclear DNA from barley Hordeum vulgare (L.) variety Erfa were cloned. Several clones containing barley specific repeated DNA were analysed by sequence analysis and Southern blot hybridization. The investigated repeats differ from each other in their length, sequence and redundancy. Their length ranges from 36 bp to about 180 bp. The repeats are AT-rich and differ widely in their redundancy within the barley genome. Southern analysis showed that the repeats belong to different repetition complexes. The possibility for utilizing these clones as probes for simple and fast genome analysis is demonstrated in squash dot experiments.     

Mutation rate at swine microsatellite loci

During genotyping of 38 microsatellites for QTL (quantitative trait loci) mapping in three F₂ swine populations, five mutant alleles were detected in a total of 66,436 parent-offspring transfers of microsatellite alleles, which gives an overall mutation rate of 7.52×10^–5 per locus per generation. No significant (P<0.05) association between mutation rates and other factors (i.e., GC contents in the flanking regions, heterozygosity, and repeat number) was revealed. Detailed sequencing showed that four out of five mutant alleles were caused by insertions of one to five repeats, respectively. The other mutant allele was produced by either an insertion of three repeats or a change of 30 base pairs (a deletion of 16 CT repeats and an insertion of one CA repeat). An insertion of one base pair in the flanking region of a microsatellite was also detected. Together, these data indicate that expansions are more common than contractions among microsatellites and that the mutation processes are very complicated, do not fit with the strict stepwise mutation model and may vary from locus to locus.     

Characterization and analysis of simple sequence repeat (SSR) loci in seashore paspalum (Paspalum vaginatum Swartz)

A size-fractionated TaqI genomic library of seashore paspalum (Paspalum vaginatum Swartz) was screened for the presence of (GA) _n and (CA) _n simple sequence repeats (SSRs). A total of 54 clones with a positive signal were detected among 13,000 clones screened. Forty-seven clones having repeats of n 3 were identified, of which 85% were perfect, 13% were imperfect and 2% were compound repeat sequences. Five of ten primer pairs synthesized to amplify selected loci resulted in a product in the expected size range and were subsequently used to examine SSR polymorphisms among 46 ecotypes of P. vaginatum. The number of alleles resolved on agarose or polyacrylamide gels were similar and ranged from 6 to 16 with an average of 14 per locus. Phenetic analysis of SSR polymorphisms revealed genetic relationships among the P. vaginatum ecotypes that were in general agreement with relationships determined previously by RAPD analysis of the same plant materials. Further screening of the genomic library did not identify (AT) _n , trimeric or tetrameric repeats. Hybridization of an (ATT)₈ oligonucleotide probe to genomic DNA isolated from I. batatas, E. coli, Citrullis lanatus and P. vaginatum suggested that the P. vaginatum genome contained significantly fewer ATT repeats than either the I. batatas or C. lanatus genome.     

Characterization and molecular genetic mapping of microsatellite loci in pepper

Microsatellites or simple sequence repeats are highly variable DNA sequences that can be used as informative markers for the genetic analysis of plants and animals. For the development of microsatellite markers in Capsicum, microsatellites were isolated from two small-insert genomic libraries and the GenBank database. Using five types of oligonucleotides, (AT)₁₅, (GA)₁₅, (GT)₁₅, (ATT)₁₀ and (TTG)₁₀, as probes, positive clones were isolated from the genomic libraries, and sequenced. Out of 130 positive clones, 77 clones showed microsatellite motifs, out of which 40 reliable microsatellite markers were developed. (GA) _n and (GT) _n sequences were found to occur most frequently in the pepper genome, followed by (TTG) _n and (AT) _n . Additional 36 microsatellite primers were also developed from GenBank and other published data. To measure the information content of these markers, the polymorphism information contents (PICs) were calculated. Capsicum microsatellite markers from the genomic libraries have shown a high level of PIC value, 0.76, twice the value for markers from GenBank data. Forty six microsatellite loci were placed on the SNU-RFLP linkage map, which had been derived from the interspecific cross between Capsicum annuum TF68 and Capsicum chinense Habanero. The current SNU2 pepper map with 333 markers in 15 linkage groups contains 46 SSR and 287 RFLP markers covering 1,761.5 cM with an average distance of 5.3 cM between markers.Communicated by J. Dvorak     

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。     

Increase in incidence of chromosome instability and non-conservative recombination between repeats in Saccharomyces cerevisiae hpr1Δ strains

Null hprl strains show a large increase (up to 2000-fold) over wild type in the frequency of occurrence of deletions between direct repeats on three different chromosomes. However, we show that hprl mutations have little or no effect on reciprocal exchange, gene conversion or unequal sister chromatid exchange, as determined using intrachromosomal, interchromosomal and plasmid-chromosome assay systems. A novel intrachromosomal recombination system has allowed us to determine that over 95% of deletions in hpr1 strains do not occur by reciprocal exchange. On the other hand, hpr1 strains show chromosome loss frequencies of up to 100 times the wild-type level. Our results suggest that yeast cells have a very efficient non-conservative recombination mechanism, dependent on RADI and RAD52, that causes deletions between direct DNA repeats, and this mechanism is strongly stimulated in hpr1 strains. The results indicate that the Hpr1 protein is required for stability of DNA repeats and chromosomes. We propose that in the absence of the Hprl protein the cell destabilizes the genome by allowing the initiation of events that lead to deletions of sequences between repeats, and to chromosome instability. We discuss the roles that proteins such as Hprl have in maintaining direct repeats and in preventing non-conservative recombination and consider the importance of these functions for chromosome stability.     

Evolving sea urchin histone genes-nucleotide polymorphisms in the H4 gene and spacers ofStrongylocentrotus purpuratus

Summary We present a comparison of spacer and coding sequences of histone gene repeats from fourStronglycocentrotus purpuratus individuals. Sequences of two previously cloned units (pCO2 and pSp2) were compared with three new histone gene clones, two of them from a single individual. Within a 1.7-kb region, 59 polymorphic sites were found in spacers, in mRNA nontranslated stretches, and at silent sites in codons of the H4 gene. The permitted silent-site changes were as frequent as in any other region studied. The most abundant polymorphisms were single-base substitutions. The ratio of transitions: tranversions: single-base-pair insertions/deletions was 322. A number of larger insertions/deletions were found, as well as differences in the length of (CTA)_n and (CT)_n runs. Two of the five cloned repeats contained an insertion of a 195-bp element that is also present at many other sites in the genomes of everyS. purpuratus individual studied. Pairwise comparisons of the different clones indicate that the variation is not uniformly divergent, but ranges from a difference of 0.34% to 3.0% of all nucleotide sites. A parsimonious tree of ancestry constructed from the pariwise comparisons indicates that recombination between the most distantly related repeats has not occurred in the 1–2 million years necessary for accumulation of the variation. The level of sequence variation found within theS. purpuratus population, for both tandemly repeated and single-copy genes, is 25%–50% of that found betweenS. purpuratus andS. drobachiensis.     

Characterization of relic DNA from barley genome

Summary High-molecular-weight relic DNA fraction can be electrophoretically separated from the bulk of barley DNA digested with different restriction enzymes. We have cloned and analyzed a population of relic DNA fragments. The majority of AluI-relic DNA clones contained barley simple sequence satellite DNA and other families of repetitive DNA. One of these families, designated HvRT, has been analyzed in detail. This family is composed of tandemly arranged 118-bp monomers and is present in 7 × 10⁵ copies in the barley genome. Clones representing the HvRT family were sequenced. HvRT repeats were found to contain high levels of methylated cytosine. The HvRT family was found in the genomes of H. vulgare, H. leporinum, H. murinum, H. jubatum, but not in H. marinum, H. geniculatum, and wheat. Different barley species and cultivars show restriction fragment length polymorphism with the HvRT probe. Chromosome-specific subfamilies of HvRT were found to be present on different barley chromosomes, providing the possibility of using the HvRT probe as a chromosome specific marker. HvRT fragments up to 810 kbp in length were resolved by pulsed field gel electrophoresis.     

The use of microsatellite DNA markers for soybean genotype identification

Conventional morphological and pigementation traits, as well as disease resistance, have been used to distinguish the uniqueness of new soybean cultivars for purposes of plant variety protection. With increasing numbers of cultivars and a finite number of conventional characters, it has become apparent that such traits will not suffice to establish uniqueness. The objective of this work was to provide an initial evaluation of microsatellite or simple-sequence-repeat (SSR) DNA markers to develop unique DNA profiles of soybean genotypes. Microsatellites are DNA sequences such as (AT) _n /(TA) _n and (ATT) _n /(TAA) _n that are composed of tandemly repeated 2–5-basepair DNA core sequences. The DNA sequences flanking microsatellites are generally conserved allowing the selection of polymerase chain reaction (PCR) primers that will amplify the intervening SSR. Variation in the number of tandem repeats, n, results in PCR product length differences. The SSR alleles present at three (AT) _n /(TA) _n and four (ATT) _n /(TAA) _n loci were determined in each of 96 diverse soybean genotypes. Between 11 and 26 alleles were found at each of the seven loci. Only two genotypes had identical SSR allelic profiles and these had very similar pedigrees. The gene diversity for the seven markers averaged 0.87 for all 96 genotypes and 0.74 for a subset of 26 North American cultivars. These are much higher than soybean gene diversity values obtained using RFLP markers, and are similar to the average values obtained for human microsatellite markers. SSR markers provide an excellent complement to the conventional markers that are currently used to characterize soybean genotypes.     

Cytogenetic diversity of SSR motifs within and between Hordeum species carrying the H genome: H. vulgare L. and H. bulbosum L.

Non-denaturing FISH (ND-FISH) was used to compare the distribution of four simple sequence repeats (SSRs)—(AG) _n , (AAG) _n , (ACT) _n and (ATC) _n —in somatic root tip metaphase spreads of 12 barley (H. vulgare ssp. vulgare) cultivars, seven lines of their wild progenitor H. vulgare ssp. spontaneum, and four lines of their close relative H. bulbosum, to determine whether the range of molecular diversity shown by these highly polymorphic sequences is reflected at the chromosome level. In both, the cultivated and wild barleys, clusters of AG and ATC repeats were invariant. In contrast, clusters of AAG and ACT showed polymorphism. Karyotypes were prepared after the identification of their seven pairs of homologous chromosomes. Variation between these homologues was only observed in one wild accession that showed the segregation of a reciprocal translocation involving chromosomes 5H and 7H. The two subspecies of H. vulgare analysed were no different in terms of their SSRs. Only AAG repeats were found clustered strongly on the chromosomes of all lines of H. bulbosum examined. Wide variation was seen between homologous chromosomes within and across these lines. These results are the first to provide insight into the cytogenetic diversity of SSRs in barley and its closest relatives. Differences in the abundance and distribution of each SSR analysed, between H. vulgare and H. bulbosum, suggest that these species do not share the same H genome, and support the idea that these species are not very closely related. Southern blotting experiments revealed the complex organization of these SSRs, supporting the findings made with ND-FISH.     

Fingerprinting,embryo type and geographic differentiation in mango (<Emphasis Type="Italic">Mangifera indica</Emphasis> L., Anacardiaceae) with microsatellites

We report the sequence and variability parameters of 16 microsatellite primer pairs obtained from two mango (Mangifera indica L.) genomic libraries after digestion of DNA of the cultivar Tommy Atkins with HaeIII and RsaI and enrichment in CT repeats. Although no significant differences were recorded between the two libraries in the informativeness of the markers obtained, the RsaI library was shown to be more useful than the HaeIII taking into account the efficiency of the library and the feasibility of clone sequencing. The polymorphism revealed by those microsatellites was evaluated in a collection of 28 mango cultivars of different origins. A total of 88 fragments were detected with the 16 simple sequence repeats (SSRs) with an average of 5.5 bands/SSR. Two primer pairs amplified more than a single locus. The mean expected and observed heterozygosities over the 14 single-locus SSRs averaged 0.65 and 0.69 respectively. The total value for the probability of identity was 2.74 × 10⁻⁹. The SSRs studied allowed the unambiguous identification of all the mango genotypes studied and this discrimination can be carried out with just three selected microsatellites. UPGMA cluster analysis and Principal coordinates analysis group the genotypes according to their origin and their classification as monoembryonic or polyembryonic types reflecting the pedigree of the cultivars and the movement of mango germplasm. The results demonstrate the usefulness of microsatellites for studies on identification, variability, germplasm conservation, domestication and movement of germplasm in mango.     

In silico polymorphic novel SSR marker development and the first SSR-based genetic linkage map in pistachio

Simple sequence repeats (SSRs) are co-dominant markers, and are very useful in constructing consensus maps in heterozygous perennial plant species like pistachio. Pistacia vera L. is the only cultivated species in the genus Pistacia. It is dioecious with a haploid chromosome count of n =?15. Saturated genetic linkage maps can be a reference to identify markers linked to economically important phenotypic traits that could be useful for early breeding and selection programs. Therefore, this study aimed to develop polymorphic SSR markers in silico and to construct the first SSR-based genetic linkage map in pistachio. The DNA sequences of three cultivars (Siirt, Ohadi, and Bagyolu) of P. vera and one genotype belonging to P. atlantica (Pa-18) were obtained by next-generation sequencing, and 625 polymorphic SSR loci were identified from 750 screened in silico polymorphic SSR primer pairs. The novel SSRs were used to construct SSR-based genetic linkage maps in pistachio along with published SSRs in Siirt × Bagyolu F1 population. Most (71.4%) of the SSRs were common markers that were used to construct consensus and parental maps spanning 15 linkage groups (LGs). A total of 384, 317, and 341 markers were mapped in the consensus, female, and male genetic maps with total lengths of 1511.3, 1427.0, and 1453.4 cM, respectively. The large number of SSR markers discovered and the first SSR-based genetic linkage map constructed in this study will be useful for anchoring loci for map integration, and will facilitate marker-assisted selection efforts for important horticultural traits in the genus Pistacia.     

The development of oat microsatellite markers and their use in identifying relationships among Avena species and oat cultivars

Microsatellites have many desirable marker properties. There has been no report of the development and utilization of microsatellite markers in oat. The objectives of the present study were to construct oat microsatellite-enriched libraries, to isolate microsatellite sequences and evaluate their level of polymorphism in Avena species and oat cultivars. One hundred clones were isolated and sequenced from three oat microsatellite-libraries enriched for either (AC/TG) _n , (AG/TC) _n or (AAG/TTC) _n repeats. Seventy eight clones contained microsatellites. A database search showed that 42% of the microsatellite flanking sequences shared significant homology with various repetitive elements. Alu and retrotransposon sequences were the two largest groups associated with the microsatellites. Forty four primer sets were used to amplify the DNA from 12 Avena species and 20 Avena sativa cultivars. Sixty two percent of the primers revealed polymorphism among the Avena species, but only 36% among the cultivars. In the cultivars, the microsatellites associated with repetitive elements were less polymorphic than those not associated with repetitive elements. Only 25% of the microsatellites associated with repetitive elements were polymorphic, while 46% of the microsatellites not associated with repetitive elements showed polymorphism in the cultivars. An average of four alleles with a polymorphism information content (PIC) of 0.57 per primer set was detected among the Avena species, and 3.8 alleles with a PIC of 0.55 among the cultivars. In addition, 54 barley microsatellite primers were tested in Avena species and 26% of the primers amplified microsatellites from oat. Using microsatellite polymorphisms, dendrograms were constructed showing phylogenetic relationships among Avena species and genetic relationships among oat cultivars. Received: 1 November 1999 / Accepted: 14 April 2000     

Abundance, variability and chromosomal location of microsatellites in wheat

The potential of microsatellite sequences as genetic markers in hexaploid wheat (Triticum aestivum) was investigated with respect to their abundance, variability, chromosomal location and usefulness in related species. By screening a lambda phage library, the total number of (GA)_n blocks was estimated to be 3.6 x 10⁴ and the number of (GT)_n blocks to be 2.3 x 10⁴ per haploid wheat genome. This results in an average distance of approximately 270 kb between these two microsatellite types combined. Based on sequence analysis data from 70 isolated microsatellites, it was found that wheat microsatellites are relatively long containing up to 40 dinucleotide repeats. Of the tested primer pairs, 36% resulted in fragments with a size corresponding to the expected length of the sequenced microsatellite clone. The variability of 15 microsatellite markers was investigated on 18 wheat accessions. Significantly, more variation was detected with the microsatellite markers than with RFLP markers with, on average, 4.6 different alleles per microsatellite. The 15 PCR-amplified microsatellites were further localized on chromosome arms using cytogenetic stocks of Chinese Spring. Finally, the primers for the 15 wheat microsatellites were used for PCR amplification with rye (Secale cereale) and barley accessions (Hordeum vulgare, H. spontaneum). Amplified fragments were observed for ten primer pairs with barley DNA and for nine primer pairs with rye DNA as template. A microsatellite was found by dot blot analysis in the PCR products of barley and rye DNA for only one primer pair.