Comparative analysis of polymorphism and chromosomal location of tomato microsatellite markers isolated from different sources

Previously isolated tomato (Lycopersicon esculentum) microsatellite markers were mainly clustered in the centromeric heterochromatin and not located in euchromatic regions. To achieve a more-uniform distribution of microsatellite markers for genome mapping purposes, a set of tomato microsatellite markers containing dinucleotide simple sequence repeats were developed by screening genomic libraries enriched for single-copy sequences, and screening the tomato EST database. The tomato microsatellites isolated in these ways were characterized by combinations of different types of repeated motifs and they were polymorphic in a set of L. esculentum varieties detecting up to four alleles. A total of 20 markers were placed on the genetic map of tomato. Interestingly, all markers isolated from genomic libraries enriched for single-copy sequences by PstI-pre-digestion mapped into the centromeric regions. The majority of markers derived from EST sequences contained predominantly AT microsatellites and were located in euchromatic regions. Received: 22 December 2000 / Accepted: 4 May 2001     

Microsatellite-based deletion bin system for the establishment of genetic-physical map relationships in wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

Because of polyploidy and large genome size, deletion stocks of bread wheat are an ideal material for physically allocating ESTs and genes to small chromosomal regions for targeted mapping. To enhance the utility of deletion stocks for chromosome bin mapping, we characterized a set of 84 deletion lines covering the 21 chromosomes of wheat using 725 microsatellites. We localized these microsatellite loci to 94 breakpoints in a homozygous state (88 distal deletions, 6 interstitial), and 5 in a heterozygous state representing 159 deletion bins. Chromosomes from homoeologous groups 2 and 5 were the best covered (126 and 125 microsatellites, respectively) while the coverage for group 4 was lower (80 microsatellites). We assigned at least one microsatellite in up to 92% of the bins (mean 4.97 SSR/bin). Only a few discrepancies concerning marker order were observed. The cytogenetic maps revealed small genetic distances over large physical regions around the centromeres and large genetic to physical map ratios close to the telomeres. As SSRs are the markers of choice for many genetic and breeding studies, the mapped microsatellite loci will be useful not only for deletion stock verifications but also for allocating associated QTLs to deletion bins where numerous ESTs that could be potential candidate genes are currently assigned.     

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.     

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.     

Molecular mapping of the centromeres of tomato chromosomes 7 and 9

The centromeres of two tomato chromosomes have been precisely localized on the molecular linkage map through dosage analysis of trisomic stocks. To map the centromeres of chromosomes 7 and 9, complementary telo-, secondary, and tertiary trisomic stocks were used to assign DNA markers to their respective chromosome arms and thus to localize the centromere at the junction of the short and long arms. It was found that both centromeres are situated within a cluster of cosegregating markers. In an attempt to order the markers within the centric clusters, genetic maps of the centromeric regions of chromosomes 7 and 9 were constructed from F₂ populations of 1620Lycopersicon esculentum × L. pennellii (E × P) plants and 1640L. esculentum × L. pimpinellifolium (E × PM) plants. Despite the large number of plants analyzed, very few recombination events were detected in the centric regions, indicating a significant suppression of recombination at this region of the chromosome. The fact that recombination suppression is equally strong in crosses between closely related (E × PM) and remotely related (E × P) parents suggests that centromeric suppression is not due to DNA sequence mismatches but to some other mechanism. The greatest number of centromeric markers was resolved in theL. esculentum × L. pennellii F₂ population. The centromere of chromosome 7 is surrounded by eight cosegregating markers: three on the short arm, five on the long arm. Similarly, the centric region of chromosome 9 contains ten cosegregating markers including one short arm marker and nine long arm markers. The localization of centromeres to precise intervals on the molecular linkage map represents the first step towards the characterization and ultimate isolation of tomato centromeres.     

Isolation and mapping of microsatellite markers specific for the D genome of bread wheat.

The potential of Aegilops tauschii, the diploid progenitor of the D genome of wheat, as a source of microsatellite markers for hexaploid bread wheat was investigated. By screening lambda phage and plasmid libraries of Ae. tauschii genomic DNA, dinucleotide microsatellites containing GA and GT motifs were isolated and a total of 65 functional microsatellite markers were developed. All primer pairs that were functional in Ae. tauschii amplified well in hexaploid wheat. Fifty-five loci amplified by 48 primer sets were placed onto a genetic framework map of the reference population of the International Triticeae Mapping Initiative (ITMI) 'Opata 85' x 'W7984'. The majority of microsatellite markers could be assigned to the chromosomes of the D genome of wheat. The distribution of the markers along the chromosomes is random. Chromosomal location of 22 loci nonpolymorphic in the reference population was determined using nullitetrasomic lines of Triticum aestivum 'Chinese Spring'. The results of this study demonstrate the value of microsatellite markers isolated from Ae. tauschii for the study of bread wheat. The microsatellite markers developed improve the existing wheat microsatellite map and can be used in a wide range of genetic studies and breeding programs.     

Characterization and genetic mapping of simple repeat sequences in the tomato genome

Tomato genomic libraries were screened for the presence of simple sequence repeats (SSRs) with seventeen synthetic oligonucleotide probes, consisting of 2- to 5-basepair motifs repeated in tandem. GAn and GTn sequences were found to occur most frequently in the tomato genome (every 1.2 Mb), followed by ATTn and GCCn (every 1.4 Mb and 1.5 Mb, respectively). In contrast, only ATn and GAn microsatellites (n > 7) were found to be frequent in the GenBank database, suggesting that other motifs may be preferentially located away from genes. Polymorphism of microsatellites was measured by PCR amplification of individual loci or by Southern hybridization, using a set of ten tomato cultivars. Surprisingly, only two of the nine microsatellite clones surveyed (five GTn, three GAn and one ATTn), showed length variation among these accessions. Polymorphism was also very limited betweenLycopersicon esculentum andL. pennelli, two distant species. Southern analysis using the seventeen oligonucleotide probes identified GATAn and GAAAn as useful motifs for the detection of multiple polymorphic fragments among tomato cultivars. To determine the structure of microsatellite loci, a GAn probe was used for hybridization at low stringency on a small insert genomic library, and randomly selected clones were analyzed. GAn based motifs of increasing complexity were found, indicating that simple dinucleotide sequences may have evolved into larger tandem repeats such as minisatellites as a result of basepair substitution, replication slippage, and possibly unequal crossing-over. Finally, we genetically mapped loci corresponding to two amplified microsatellites, as well as nine large hypervariable fragments detected by Southern hybridization with a GATA8 probe. All loci are located around putative tomato centromeres. This may contribute to understanding of the structure of centromeric regions in tomato.     

JunctionViewer: customizable annotation software for repeat-rich genomic regions

Background  

Repeat-rich regions such as centromeres receive less attention than their gene-rich euchromatic counterparts because the former are difficult to assemble and analyze. Our objectives were to 1) map all ten centromeres onto the maize genetic map and 2) characterize the sequence features of maize centromeres, each of which spans several megabases of highly repetitive DNA. Repetitive sequences can be mapped using special molecular markers that are based on PCR with primers designed from two unique "repeat junctions". Efficient screening of large amounts of maize genome sequence data for repeat junctions, as well as key centromere sequence features required the development of specific annotation software.     

Microsatellite markers for genome analysis in Brassica. II. Assignment of rapeseed microsatellites to the A and C genomes and genetic mapping in Brassica oleracea L.

Microsatellites are highly polymorphic and efficient markers for the analysis of plant genomes. Primer specificity, however, may restrict the applicability of these markers even between closely related species for comparative mapping studies. We have demonstrated that the majority of microsatellites identified in oilseed rape (Brassica napus L; AC genome) correspond to loci which can be easily assigned to the A and C progenitor genomes. A study with 63 primer pairs has shown that 54% detect two loci, one from each genome, while 25% and 21%, respectively, are either A or C genome-specific. The distribution of rapeseed microsatellites in the C genome was investigated by genetic mapping in Brassica oleracea L. Ninety two dinucleotide microsatellites were screened for polymorphism in an F₂ population derived from a cross between collard and cauliflower, for which an RFLP map has been constructed previously. Thirty three primer pairs (35.7%) have yielded either unspecific or no PCR products whereas the remaining primer pairs amplified one or more distinct loci. The level of polymorphism found in the mapping population was 49.2%. A total of 29 primer pairs disclosed 34 loci of which 31 are evenly distributed on 8 of the 9 B. oleracea linkage groups. For the remaining three markers linkage could not be established. Our results showed that microsatellite markers from the composite genome of B. napus can serve as a useful marker system in genetic studies and for plant-breeding objectives in B. oleracea. Received: 14 April 2000 / Accepted: 3 July 2000     

Bioinformatic Mining of Type I Microsatellites from Expressed Sequence Tags of Channel Catfish (<Emphasis Type="Italic">Ictalurus punctatus</Emphasis>)

Gene-derived markers are pivotal to the analysis of genome structure, organization, and evolution and necessary for comparative genomics. However, gene-derived markers are relatively difficult to develop. This project utilized the genomic resources of channel catfish expressed sequence tags (ESTs) to identify simple sequence repeats (SSRs), or microsatellites. It took the advantage of ESTs for the establishment of gene identities, and of microsatellites for the acquisition of high polymorphism. When microsatellites are tagged to genes, the microsatellites can then be used as gene markers. A bioinformatic analysis of 43,033 ESTs identified 4855 ESTs containing microsatellites. Cluster analysis indicated that 1312 of these ESTs fell into 569 contigs, and the remaining 3534 ESTs were singletons. A total of 4103 unique microsatellite-containing genes were identified. The dinucleotide CA/TG and GA/TC pairs were the most abundant microsatellites. AT-rich microsatellite types were predominant among trinucleotide and tetranucleotide microsatellites, consistent with our earlier estimation that the catfish genome is highly AT-rich. Our preliminary results indicated that the majority of the identified microsatellites were polymorphic and, therefore, useful for genetic linkage mapping of catfish. Mapping of these gene-derived markers is under way, which will set the foundation for comparative genome analysis in catfish.     

Characterization and comparison of microsatellites derived from repeat-enriched libraries and expressed sequence tags

The construction of high-density linkage maps for use in identifying loci underlying important traits requires the development of large numbers of polymorphic genetic markers spanning the entire genome at regularly spaced intervals. As part of our efforts to develop markers for rainbow trout (Oncorhynchus mykiss), we performed a comparison of allelic variation between microsatellite markers developed from expressed sequence tag (EST) data and anonymous markers identified from repeat-enriched libraries constructed from genomic DNA. A subset of 70 markers (37 from EST databases and 33 from repeat enriched libraries) was characterized with respect to polymorphism information content (PIC), number of alleles, repeat number, locus duplication within the genome and ability to amplify in other salmonid species. Higher PIC was detected in dinucleotide microsatellites derived from ESTs than anonymous markers (72.7% vs. 54.0%). In contrast, dinucleotide repeat numbers were higher for anonymous microsatellites than for EST derived microsatellites (27.4 vs.18.1). A higher rate of cross-species amplification was observed for EST microsatellites. Approximately half of each marker type was duplicated within the genome. Unlike single-copy markers, amplification of duplicated microsatellites in other salmonids was not correlated to phylogenetic distance. Genomic microsatellites proved more useful than EST derived microsatellites in discriminating among the salmonids. In total, 428 microsatellite markers were developed in this study for mapping and population genetic studies in rainbow trout.     

A PCR-Based Genetic Map for Human Chromosome 3

Oligonucleotide primers for 125 simple sequence repeat microsatellite-based genetic markers have been assayed by polymerase chain reaction (PCR) in the CEPH reference family panel. These microsatellites include 101 dinucleotide repeats as well as 24 new tetranucleotide repeats. The average heterozygosity of this marker set was 72.4%. Genetic data were analyzed with the genetic mapping package LINKAGE. A subset of these microsatellite markers define a set of 56 uniquely ordered loci (>1000:1 against local inversion) that span 271 cM. Sixty-seven additional loci were tightly linked to markers on the uniquely ordered map, but could not be ordered with such high precision. These markers were positioned by CMAP into confidence intervals. One hundred thirteen of the microsatellite markers were also tested on a chromosome 3 framework somatic cell hybrid panel that divides this chromosome into 23 cytogenetically defined regions, integrating the genetic and physical maps of this chromosome. The high density, high heterozygosity, and PCR format of this genetically and physically mapped set of markers will accelerate the mapping and positional cloning of new chromosome 3 genes.     

Distribution of dinucleotide microsatellites in the Drosophila melanogaster genome.

Microsatellites, a special class of repetitive DNA, have become one of the most popular genetic markers. The progress of various genome projects has made it possible to study the genomic distribution of microsatellites and to evaluate the potential influence of several parameters on their genesis. We report the distribution of dinucleotide microsatellites in the genome of Drosophila melanogaster. When considering only microsatellites with five or more repeat units, the average length of dinucleotide repeats in D. melanogaster is 6.7 repeats. We tested a wide range of parameters which could potentially influence microsatellite density, and we did not detect a significant influence of recombination rate, number of exons, or total length of coding sequence. In concordance with the neutral expectation for the origin of microsatellites, a significant positive correlation between AT content and (AT/TA)n microsatellite density was detected. While this pattern may indicate that microsatellite genesis is a random process, we also found evidence for a nonrandom distribution of microsatellites. Average microsatellite density was higher on the X chromosome, but extreme heterogeneity was observed between different genomic regions. Such a clumping of microsatellites was also evident on a more local scale, as 38.9% of the contiguous sequences analyzed showed a deviation from a random distribution of microsatellites.     

Development of microsatellite markers targeting (GATA) n motifs in sorghum [Sorghum bicolor (L.) Moench]

Microsatellite markers targeting (GATA) _n motifs are known to be highly polymorphic. Genome-wide development of such markers has not been reported in sorghum. The main objective of this study was to identify Class I microsatellites with (GATA) _n motifs in the sorghum genome through in silico analysis and assess their potential as molecular markers. The study identified a total of 128 such motifs, of which 14, 16 and 98 motifs were present in the genic, upstream and non-genic regions, respectively. The majority of the (GATA) _n motifs were found in the non-genic regions of the genome while 23.44 % of them were found within the genes and upstream of genes. About 110 PCR-based markers were developed targeting these microsatellites and 50 of them distributed across the genome were validated in 24 diverse sorghum genotypes representing different racial groups. Thirty-eight markers were polymorphic, with average polymorphism information content value of 0.69, and the sorghum genotypes could be grouped into two major clusters. These markers with robust amplification combined with good allelic diversity represent a new set of microsatellite markers in sorghum reported for the first time that will be highly useful for various genetics and molecular breeding applications.     

Chromosomal localization and molecular characterization of 53 cosmid-derived bovine microsatellites

Gene mapping in cattle has progressed rapidly in recent years largely owing to the introduction of powerful genetic markers, such as the microsatellites, and through advances in physical mapping techniques such as synteny mapping and fluorescence in situ hybridization (FISH). Microsatellite markers are often not physically mapped because they are generally isolated from small insert plasmid libraries, which makes their chromosomal localization inefficient. In this report we describe the FISH mapping of a large group of cosmid-derived bovine microsatellite markers, as our contribution to the European mapping initiative, BovMap. One objective of BovMap is to develop a set of anchored loci for the cattle genome map.Two cosmid libraries were screened with probes corresponding to the (AC) _n microsatellite motif. Positive clones were mapped by FISH, and then a subset was further analyzed by sequencing the region flanking the microsatellite repeat. In total, 58 clones were hybridized with chromosomes and identified loci on 22 of the 31 different bovine chromosomes. Three clones contained satellite DNA. Two or more markers were placed on 12 chromosomes. Sequencing of the microsatellites and flanking regions was performed directly from 43 cosmids, as previously reported (Ferretti et al. Anim. Genet. 25, 209–214, 1994). Primers were developed for 39 markers and used to describe the polymorphism associated with the corresponding loci.     

A high density genetic map of tobacco (<Emphasis Type="Italic">Nicotiana tabacum</Emphasis> L.) obtained from large scale microsatellite marker development

Tobacco (Nicotiana tabacum L.) is a species in the large family of the Solanaceae and is important as an agronomic crop and as a model system in plant biotechnology. Despite its importance, only limited molecular marker resources are available that can be used for genome analysis, genetic mapping and breeding. We report here on the development and characterization of 5,119 new and functional microsatellite markers and on the generation of a high-resolution genetic map for the tetraploid tobacco genome. The genetic map was generated using an F2 mapping population derived from the intervarietal cross of Hicks Broadleaf × Red Russian and merges the polymorphic markers from this new set with those from a smaller set previously used to produce a lower density map. The genetic map described here contains 2,317 microsatellite markers and 2,363 loci, resulting in an average distance between mapped microsatellite markers which is less than 2 million base pairs or 1.5 cM. With this new and expanded marker resource, a sufficient number of markers are now available for multiple applications ranging from tobacco breeding to comparative genome analysis. The genetic map of tobacco is now comparable in marker density and resolution with the best characterized genomes of the Solanaceae: tomato and potato.     

Centromere-linked microsatellite markers for linkage groups 3, 4, 6, 7, 13, and 20 of zebrafish (Danio rerio)

A large number of interesting mutations affecting development and organogenesis have been identified through genetic screens in zebrafish. Mapping of these mutations to a chromosomal region can be rapidly accomplished using half-tetrad analysis. However, knowledge of centromere-linked markers on every chromosome is essential to this mapping method. Centromeres on all 25 linkage groups have been mapped on the RAPD zebrafish genetic map. However, species specificity and the lack of codominance make RAPD markers less practical for mapping than microsatellite-based markers. On the microsatellite-based genetic map, centromere-linked markers have been identified for 19 linkage groups. No direct evidence has been published linking microsatellite markers to the centromeres of linkage groups 3, 4, 6, 7, 13, and 20. Therefore, we compared the microsatellite-based genetic map with the RAPD map to identify markers most likely linked to the centromeres of these 6 linkage groups. These candidate markers were tested for potential centromere linkage using four panels of half-tetrad embryos derived by early-pressure treatment of eggs from four different female zebrafish. We have identified microsatellite markers for linkage groups 3, 4, 6, 7, 13, and 20 to within 1.7 cM of their centromeres. These markers will greatly facilitate the rapid mapping of mutations in zebrafish by half-tetrad analysis.     

Assignment of non-informative turkey genetic markers through comparative approaches

Molecular markers such as microsatellites, provide genetic signposts for navigating genomes. In general, genetic markers that are monomorphic or non-informative in mapping populations typically remain unmapped and as such are less likely to be included in future studies. The use of hybrid cell panels and in silico mapping via whole genome sequences allow for positional mapping of non-segregating markers. This study utilizes the INRA ChickRH6 whole-genome radiation hybrid panel and chicken whole-genome shotgun sequence to map microsatellite markers from the turkey (Meleagris gallopavo). Thirty-three of the 41 markers typed on the RH panel had significant linkage to at least one other marker and 83 of 100 sequences returned significant BLAST similarities. Positioning of these markers provides additional sequence tagged sites in the turkey genome and increases the potential use of these markers for future genetic studies.     

Pig microsatellites isolated from cosmids revealing polymorphism and localized on chromosomes

One of the most widely studied simple sequences in the mammalian genome is the (TG)_n dinucleotide sequence. Because these microsatellites are highly polymorphic, we chose to study microsatellites from cosmids to provide genetic markers for the porcine genome. After screening a porcine cosmid library with a (CA)₁₀ probe, 20 cosmids containing microsatellites were subcloned and 17 microsatellites identified by sequencing. Oligonucleotide primers flanking the repeat were designed for seven (TG)_n microsatellites with n > 14. These seven microsatellites revealed polymorphism and were regionally assigned to chromosomes by fluorescent in situ hybridization of initial cosmids. These seven loci will be useful for both the construction of the genetic map and as landmark loci on the physical map of the porcine genome.