Abundance, polymorphism and genetic mapping of microsatellites in rice

Dinucleotide microsatellites have been characterized and used as genetic markers in rice. Screening of a rice genomic library with poly(dG-dA)·(dC-dT) and poly(dG-dT)·(dC-dA) probes indicated that (GA)_n repeats occurred, on average, once every 225 kb and (GT)_n repeats once every 480 kb. DNA sequencing of ten randomly selected microsatellites indicated that the numbers of repeats ranged from 12 to 34 and that the patterns of microsatellites in rice were similar to those of humans and other mammals. Primers to these microsatellite loci as well as to four published microsatellite-containing sequences have been designed and degrees of polymorphism has been examined with 20 rice accessions. Multiple alleles, ranging from 5 to 11, have been observed at all the microsatellite loci in 20 rice accessions. Alleles specific to two cultivated subspecies, indica and japonica, were found in some microsatellite loci. Heterozygosity values of all the microsatellite markers were significantly higher than those of RFLP markers, based upon a parallel comparison. Ten microsatellite loci have been genetically mapped to four rice chromosomes. The genomic distribution of microsatellites appears to be random 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.     

Characterization and genetic mapping of a short, highly repeated, interspersed DNA sequence from rice (Oryza sativa L.).

Summary A short, highly repeated, interspersed DNA sequence from rice was characterized using a combination of techniques and genetically mapped to rice chromosomes by restriction fragment length polymorphism (RFLP) analysis. A consensus sequence (GGC)_n, where n varies from 13–16, for the repeated sequence family was deduced from sequence analysis. Southern blot analysis, restriction mapping of repeat element-containing genomic clones, and DNA sequence analysis indicated that the repeated sequence is interspersed in the rice genome, and is heterogeneous and divergent. About 200000 copies are present in the rice genome. Single copy sequences flanking the repeat element were used as RFLP markers to map individual repeat elements. Eleven such repeat elements were mapped to seven different chromosomes. The strategy for characterization of highly dispersed repeated DNA and its uses in genetic mapping, DNA fingerprinting, and evolutionary studies are discussed.     

High-resolution mapping adjacent to the Pc71 crown-rust resistance locus in hexaploid oat

The D526-derived BC₁F₂ population of hexaploid oat segregates for resistance to crown rust isolate 345. A mapping population consisting of 440 F2 individuals was used to develop a high-resolution RFLP map of the area of the genome where Pc71, the locus conferring this resistance, is located. Twelve RFLP markers have been identified within ca. 6 cM of Pc71, with cosegregating loci Xcdo1502 and Xcdo783 positioned 0.2 cM from the locus. All of the RFLP markers map to the same side of the locus, suggesting either that the Pc71 resistance locus resides at the end of a linkage group, or that there is no detectable heterozygosity on the opposite side. Some degree of microcollinearity between rice and oat is present in this area, as the two markers, Xcdo783 and Xrz69, linked to Pc71 are linked also in rice; however the genetic distance in oat is much less than the genetic distance in rice (6.2 cM and 20 cM, respectively).     

DNA sequence polymorphism at the human tumor necrosis factor (TNF) locus. Numerous TNF/lymphotoxin alleles tagged by two closely linked microsatellites in the upstream region of the lymphotoxin (TNF-beta) gene.

TNF-alpha and lymphotoxin (LT, TNF-beta) genes are tandemly arranged and map within the MHC centromeric to HLA-B and telomeric to the class III genes. Both cytokines encoded by these genes are potent immunomodulators. On the other hand, some MHC-linked autoimmune diseases are characterized by abnormal levels of their expression or inducibility. A search for the putative disease-associated TNF/LT alleles depends on the informative genetic markers at the TNF locus. Previously, a low degree of genetic polymorphism at the human TNF locus has been reported, mostly bi-allelic RFLP. To localize and define additional polymorphic markers, we probed the collection of genomic clones with synthetic tandemly repeated dinucleotides, corresponding to the sequences known as microsatellites. We mapped and characterized three (TC/GA) and one (AC/GT) repeats within cloned 40-kb DNA comprising the human TNF locus. Using a polymerase chain reaction-based technique, we analyzed three of these four microsatellites and observed their length of polymorphism. Using DNA samples from blood donors, two families, and three human cell lines, we detected 13 distinct alleles of the AC/GT microsatellite neighboring human TNF genes. The variability was further increased by simultaneous analysis of the second linked microsatellite. This linked TC/GA repeat showed at least five alleles, whereas the least polymorphic TC/GA repeat located in the first intron of LT (TNF-beta) gene had two alleles. TNF alleles defined by microsatellites were stably inherited and segregated in the Mendelian way. Therefore, we describe thus far the most informative level of DNA sequence polymorphism in this part of human MHC. We propose a nomenclature for microsatellite tagged LT/TNF alleles based on their size and variability, which could also be extended to include RFLP and other not yet identified polymorphic markers. Microsatellite tagged polymorphism described here can be used in systematic linkage studies of HLA-associated diseases.     

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     

Characterization of highly variable (GA/CT) n microsatellites in the bur oak,Quercus macrocarpa

The objective of this study was to ascertain the usefulness of polymerase chain reaction (PCR)-based microsatellite analysis for studying pollination and parentage in a wind-pollinated temperate tree. A small insert genomic library of the bur oak (Quercus macrocarpa) was constructed and screened for the presence of (CA/GT) _n and (GA/CT) _n repeats. The proportion of positive clones yielded estimates of 3×10⁵ such dinucleotide repeats per genome, roughly comparable to abundances reported in other eukaryotic genomes. Thirteen positive clones were sequenced. In contrast to mammalian genomes, the (GA/CT) _n motif was more abundant than the (CA/GT) _n motif in these clones. The (GA/CT) _n repeats also showed longer average repeat length (mean n=16.2 versus 7.3), suggesting that they are better candidates for yielding polymorphic genetic markers in oak genomes. Indeed, a survey of adult bur oaks and offspring in a small stand in northern Illinois at 3 of these (GA/CT) _n microsatellite loci revealed Mendelian inheritance and extremely high levels of polymorphism, with the number of alleles at each locus ranging from 11–20 and heterozygosity ranging from 0.66 to 0.75. These results, indicating that (GA/CT) _n microsatellites are both abundant and highly polymorphic in the bur oak genome, suggest that such genetic markers have tremendous potential for applications for studies of parentage, pollination and dispersal in temperate trees.     

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.     

Isolation and characterization of polymorphic microsatellite loci in Camellia nitidissima Chi (Theaceae)

? Premise of the study: Microsatellite markers were developed in Camellia nitidissima for further population genetic studies. ? Methods and Results: Eight microsatellite markers were newly developed from C. nitidissima and 7 were transferred from other Camellia species. Two to 13 alleles per locus were identified for these microsatellites. Observed and expected heterozygosities ranged from 0.040 to 0.909, and 0.184 to 0.916, respectively. Four loci showed a significant deviation from Hardy-Weinberg equilibrium and five locus pairs displayed linkage disequilibrium. ? Conclusions: These microsatellite markers will be useful to assess the genetic variation and genetic structure of C. nitidissima.     

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.     

Development of a set of highly polymorphic genomic microsatellites (gSSRs) in Sitka spruce (Picea sitchensis (Bong.) Carr.)

Robust, polymorphic microsatellite DNA markers (simple sequence repeats—SSRs) are valuable tools for a range of tree conservation and breeding applications. SSRs are routinely used in the study of population genetic structure and diversity, pedigree reconstruction and genetic linkage mapping. Their abundance in the genome, co-dominant inheritance and potential for cross-species amplification make microsatellites highly prized markers. This paper characterises 22 novel genomic polymorphic microsatellite loci for Sitka spruce (Picea sitchensis (Bong.) Carr.). Amplification of DNA from Sitka spruce material was carried out both with a set of unrelated trees to obtain diversity statistics for each locus, and with the progeny of a full-sib family to test simple Mendelian inheritance. Observed heterozygosity ranged from 0.38 to 0.91 and allele number per locus ranged from 6 to 21, with a mean of 12.2. In addition, the primer pairs were tested with DNA from Norway spruce (P. abies) and white spruce (P. glauca) to investigate their potential for cross-species amplification and ten loci amplified in all three species. The results from these genomic microsatellites are compared to data generated from microsatellites derived from Picea EST libraries. In summary, this novel, highly polymorphic markers represent a significant addition to the rapidly expanding Picea genomics tool-box. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Development of microsatellite markers and analysis of intraspecific genetic variability in chickpea (Cicer arietinum L.)

Paucity of polymorphic molecular markers in chickpea (Cicer arietinum L.) has been a major limitation in the improvement of this important legume. Hence, in an attempt to develop sequence-tagged microsatellite sites (STMS) markers from chickpea, a microsatellite enriched library from the C. arietinum cv. Pusa362 nuclear genome was constructed for the identification of (CA/GT) _n and (CT/GA) _n microsatellite motifs. A total of 92 new microsatellites were identified, of which 74 functional STMS primer pairs were developed. These markers were validated using 9 chickpea and one C. reticulatum accession. Of the STMS markers developed, 25 polymorphic markers were used to analyze the intraspecific genetic diversity within 36 geographically diverse chickpea accessions. The 25 primer pairs amplified single loci producing a minimum of 2 and maximum of 11 alleles. A total of 159 alleles were detected with an average of 6.4 alleles per locus. The observed and expected heterozygosity values averaged 0.32 (0.08–0.91) and 0.74 (0.23–0.89) respectively. The UPGMA based dendrogram was able to distinguish all the accessions except two accessions from Afghanistan establishing that microsatellites could successfully detect intraspecific genetic diversity in chickpea. Further, cloning and sequencing of size variant alleles at two microsatellite loci revealed that the variable numbers of AG repeats in different alleles were the major source of polymorphism. Point mutations were found to occur both within and immediately upstream of the long tracts of perfect repeats, thereby bringing about a conversion of perfect motifs into imperfect or compound motifs. Such events possibly occurred in order to limit the expansion of microsatellites and also lead to the birth of new microsatellites. The microsatellite markers developed in this study will be useful for genetic diversity analysis, linkage map construction as well as for depicting intraspecific microsatellite evolution.     

Development and mapping of microsatellite (SSR) markers in wheat

Microsatellite DNA markers are consistently found to be more informative than other classes of markers in hexaploid wheat. The objectives of this research were to develop new primers flanking wheat microsatellites and to position the associated loci on the wheat genome map by genetic linkage mapping in the ITMI W7984 × Opata85 recombinant inbred line (RIL) population and/or by physical mapping with cytogenetic stocks. We observed that the efficiency of marker development could be increased in wheat by creating libraries from sheared rather than enzyme-digested DNA fragments for microsatellite screening, by focusing on microsatellites with the [ATT/TAA]_n motif, and by adding an untemplated G-C clamp to the 5-end of primers. A total of 540 microsatellite-flanking primer pairs were developed, tested, and annotated from random genomic libraries. Primer pairs and associated loci were assigned identifiers prefixed with BARC (the acronym for the USDA-ARS Beltsville Agricultural Research Center) or Xbarc, respectively. A subset of 315 primer sets was used to map 347 loci. One hundred and twenty-five loci were localized by physical mapping alone. Of the 222 loci mapped with the ITMI population, 126 were also physically mapped. Considering all mapped loci, 126, 125, and 96 mapped to the A, B, and D genomes, respectively. Twenty-three of the new loci were positioned in gaps larger than 10 cM in the map based on pre-existing markers, and 14 mapped to the ends of chromosomes. The length of the linkage map was extended by 80.7 cM. Map positions were consistent for 111 of the 126 loci positioned by both genetic and physical mapping. The majority of the 15 discrepancies between genetic and physical mapping involved chromosome group 5.Electronic Supplementary Material Supplementary material is available for this article at     

Mining microsatellites in eukaryotic genomes

During recent decades, microsatellites have become the most popular source of genetic markers. More recently, the availability of enormous sequence data for a large number of eukaryotic genomes has accelerated research aimed at understanding the origin and functions of microsatellites and searching for new applications. This review presents recent developments of in silico mining of microsatellites to reveal various facets of the distribution and dynamics of microsatellites in eukaryotic genomes. Two aspects of microsatellite search strategies--using a suitable search tool and accessing a relevant microsatellite database--have been explored. Judicious microsatellite mining not only helps in addressing biological questions but also facilitates better exploitation of microsatellites for diverse applications.     

Instability of (GATA)<Subscript> n</Subscript> microsatellite loci in the parthenogenetic Caucasian rock lizard<Emphasis Type="Italic"> Darevskia unisexualis</Emphasis> (Lacertidae)

Mini- and microsatellites, comprising tandemly repeated short nucleotide sequences, are abundant dispersed repetitive elements that are ubiquitous in eukaryotic genomes. In humans and other bisexual species hypervariable mini- and microsatellite loci provide highly informative systems for monitoring of germline and somatic instability. However, little is known about the mechanisms by which these loci mutate in species that lack effective genetic recombination. Here, multilocus DNA fingerprinting was used to study M13 minisatellite and (GATA) _n microsatellite instability in the parthenogenetic Caucasian rock lizard Darevskia unisexualis (Lacertidae). DNA fingerprinting of 25 parthenogenetic families, from six isolated populations in Armenia (comprising a total of 84 siblings), using the oligonucleotide (GATA)₄ as a hybridization probe, revealed mutant fingerprinting phenotypes in 13 siblings that differed from their mothers in several restriction DNA fragments. In three families (8 siblings), the mutations were present in the germline. Moreover, the mutant fingerprint phenotypes detected in siblings were also present in population DNA samples. No intrafamily variations in DNA fingerprint patterns were observed with the M13 minisatellite probe. Estimates of the mutation rate for (GATA) _n microsatellite loci in D. unisexualis showed that it was as high as that seen in some bisexual species, reaching 15% per sibling or 0.95% per microsatellite band. Furthermore, in one case, a somatic (GATA) _n microsatellite mutation was observed in an adult lizard. These findings directly demonstrate that mutations in (GATA) _n microsatellite loci comprise an important source of genetic variation in parthenogenetic populations of D. unisexualis.Communicated by G. P. Georgiev     

A comparative map of wild rice (Zizania palustris L. 2n=2x=30)

We present the first genetic map of wild rice (Zizania palustris L., 2n=2x=30), a native aquatic grain of northern North America. This map is composed principally of previously mapped RFLP (restriction fragment length polymorphism) genetic markers from rice (Oryza sativa 2n=2x=24). The map is important as a foundation for genetic and crop improvement studies as well as a reference for genome organization comparisons among species of Gramineae. A comparative mapping approach with rice is especially useful because wild rice is grouped in the same subfamily, Oryzoideae, and no other mapping comparison has yet been made within the subfamily. As rice is the reference point for mapping and gene cloning in cereals, establishing a consensus map within the subfamily identifies conserved and unique regions. The genomes of wild rice and rice differ in total DNA content (wild rice has twice that of rice) and the number of chromosome pairs (wild rice=15 versus rice=12). The wild rice linkage map reported herein consists of 121 RFLP markers on 16 linkage groups spanning 1805 cM. Two linkage groups consist of only two markers. Colinear markers were found representing all rice linkage groups except #12. The majority of rice loci mapped to colinearly arranged arrays in wild rice (92 of 118). Features of the map include duplication of portions of three rice linkage groups and three possible translocations. The map gives basic information on the composition of the wild rice genome and provides tools to assist in the domestication of this important food source. Received: 25 August 1998 / Accepted: 20 February 1999     

Twelve novel microsatellite loci from an endangered marine fish species golden pompano Trachinotus blochii

The golden pompano (Trachinotus blochii) is a marine fish species in tropical regions. No information about genetic variation and population structure of wild populations is available. A first set of 12 polymorphic microsatellites isolated from this species were characterized. The number of alleles ranged from 5 to 14 with an average of 8.0 alleles per locus. All 12 markers conformed to HWE and were in linkage equilibrium. These 12 microsatellite markers supply the first set of co-dominant DNA markers for studying population genetics of the species T. blochii.     

Comparative genome analysis of mungbean (Vigna radiata L. Wilczek) and cowpea (V. unguiculata L. Walpers) using RFLP mapping data

Genome relationships between mungbean (Vigna tradiata) and cowpea (V. Unguiculata) based on the linkage arrangement of random genomic restriction fragment length polymorphism (RFLP) markers have been investigated. A common set of probes derived from cowpea, common bean (Phaseolus vulgaris), mungbean, and soybean (Glycine max) PstI genomic libraries were used to construct the genetic linkage maps. In both species, a single F₂ population from a cross between an improved cultivar and a putative wild progenitor species was used to follow the segregation of the RFLP markers. Approximately 90% of the probes hybridized to both mungbean and cowpea DNA, indicating a high degree of similarity in the nucleotide sequences among these species. A higher level of polymorphism was detected in the mungbean population (75.7%) than in the cowpea population (41.2%). Loci exhibiting duplications, null phenotypes, and distorted segregation ratios were detected in both populations. Random genomic DNA RFLP loci account for about 89% of the currently mapped markers with a few cDNA and RAPD markers added. The current mungbean map is comprised of 171 loci/loci clusters distributed in 14 linkage groups spanning a total of 1570cM. On the other hand, 97 markers covered 684 cM and defined 10 linkage groups in the current cowpea map. The mungbean and cowpea genomes were compared on the basis of the copy number and linkage arrangement of 53 markers mapped in common between the two species. Results indicate that nucleotide sequences are conserved, but variation in copy number were detected and several rearrangements in linkage orders appeared to have occurred since the divergence of the two species. Entire linkage groups were not conserved, but several large linkage blocks were maintained in both genomes.     

Development of 81 new polymorphic EST-derived microsatellite markers for the olive flounder,Paralichthys olivaceus

Expressed sequence tags (ESTs) can be used to identify microsatellite markers. We developed 81 polymorphic microsatellite markers from 4,940 ESTs of the olive flounder, Paralichthys olivaceus. Out of 100 EST-derived microsatellites for which PCR primers were designed, 81 loci were polymorphic in 30 individuals from a single natural population with 2–28 (mean 10.6) alleles per locus. The observed and expected heterozygosities of these loci were 0.033–1.000 and 0.033–0.965, respectively. Segregation analysis within a mapping family revealed non-amplifying null alleles at five loci. These new EST-derived microsatellite markers should be useful for population genetic analyses, pedigree tracing and constructing a linkage map for olive flounder.     

Isolation and characterization of polymorphic microsatellite loci from bluefin leatherjacket (Navodon septentrionalis Gunther, 1877)

The first set of 18 polymorphic microsatellite markers were developed from bluefin leatherjacket (Navodon septentrionalis Gunther, 1877). From a (GT)_n-enriched genomic library, we got 121 microsatellites, of which 60 were randomly selected for designing microsatellite primers. Eighteen of these loci were polymorphic in a test population of 32 individuals with alleles ranging from 2 to 9, and expected and observed heterozygosities from 0.1463 to 0.8517 and from 0.1562 to 1.0000, respectively. No significant linkage disequilibrium between pairs of loci was found, but three loci significantly deviated from Hardy–Weinberg equilibrium after Bonferroni correction. These polymorphic microsatellite loci should provide sufficient level of genetic diversity to investigate population structure in bluefin leatherjacket.