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     

The use of microsatellites for detecting DNA polymorphism, genotype identification and genetic diversity in wheat

A set of 20 wheat microsatellite markers was used with 55 elite wheat genotypes to examine their utility (1) in detecting DNA polymorphism, (2)in the identifying genotypes and (3) in estimating genetic diversity among wheat genotypes. The 55 elite genotypes of wheat used in this study originated in 29 countries representing six continents. A total of 155 alleles were detected at 21 loci using the above microsatellite primer pairs (only 1 primer amplified 2 loci; all other primers amplified 1 locus each). Of the 20 primers amplifying 21 loci, 17 primers and their corresponding 18 loci were assigned to 13 different chromosomes (6 chromosomes of the A genome, 5 chromosomes of the B genome and 2 chromosomes of the D genome). The number of alleles per locus ranged from 1 to 13, with an average of 7.4 alleles per locus. The values of average polymorphic information content (PIC) and the marker index (MI) for these markers were estimated to be 0.71 and 0.70, respectively. The (GT)_n microsatellites were found to be the most polymorphic. The genetic similarity (GS) coefficient for all possible 1485 pairs of genotypes ranged from 0.05 to 0.88 with an average of 0.23. The dendrogram, prepared on the basis of similarity matrix using the UPGMA algorithm, delineated the above genotypes into two major clusters (I and II), each with two subclusters (Ia, Ib and IIa, IIb). One of these subclusters (Ib) consisted of a solitary genotype (E3111) from Portugal, so that it was unique and diverse with respect to all other genotypes belonging to cluster I and placed in subcluster Ia. Using a set of only 12 primer pairs, we were able to distinguish a maximum of 48 of the above 55 wheat genotypes. The results demonstrate the utility of microsatellite markers for detecting polymorphism leading to genotype identification and for estimating genetic diversity. Received: 15 May 1999 / Accepted: 27 July 1999     

Characterization of microsatellites and development of chromosome specific STMS markers in bread wheat

Microsatellites, or simple sequence repeats (SSRs), have become the markers of choice for genetic studies with many crop species including wheat. Currently an international effort is underway to enrich the repertoire of available sequence tagged microsatellite site (STMS) markers in wheat. As a part of this effort, we have sequenced 43 clones obtained from a microsatellite-enriched wheat genomic library; 34 clones contained 41 different microsatellites. These microsatellites (mono-, di-, tri- nucleotide repeats) were classified as 19 simple perfect, 18 simple imperfect and 4 compound imperfect types. Dinucleotide repeats were the most abundant (70%). Primer pairs for only 16 microsatellites could be designed, since the flanking sequences of the others were either too short or were otherwise not suitable for designing the microsatellite specific primers. Microsatellite loci of the expected size and polymorphism were successfully amplified from 15 of these 16 primer pairs using three wheat varieties. 14 loci detected by 12 out of the 15 functional primer pairs were assigned to 11 specific chromosomes. An erratum to this article is available at .     

Development and genetic mapping of 127 new microsatellite markers in barley

To enhance the marker density of existing genetic maps of barley (Hordeum vulgare L.), a new set of microsatellite markers containing dinucleotide motifs was developed from genomic clones. Out of 254 primer pairs tested, a total of 167 primer pairs were classifed as functional in a panel of six barley cultivars and three H. spontaneum accessions, and of those, 127 primer pairs resulting in 133 loci were either mapped or located onto chromosomes. The polymorphism information content (PIC) ranged from 0.05 to 0.94 with an average of 0.60. The number of alleles per locus varied from 1 to 9. On average, 3.9 alleles per primer pair were observed. The RFLP frameworks of two previously published linkage maps were used to locate a total of 115 new microsatellite loci on at least one mapping population. The chromosomal assignment of 48 mapped loci was corroborated on a set of wheat-barley chromosome addition lines; 18 additional loci which were not polymorphic in the mapping populations were assigned to chromosomes by this method. The microsatellites were located on all seven linkage groups with four significant clusters in the centromeric regions of 2H, 3H, 6H and 7H. These newly developed microsatellites improve the density of existing barley microsatellite maps and can be used in genetic studies and breeding research.Communicated by G. Wenzel     

GenBank数据库中鳜鱼微卫星位点的筛选及特征分析

微卫星(Microsatellite)是一类由2-6个核苷酸经多次单位串联组成的高度变异重复DNA序列(Schlotterer and Tautz,1992)。它具有按照孟德尔方式分离、突变快、多态信息含量丰富、呈共显性遗传等特点,其核心序列在同一物种中具有保守性,因此,可以根据微卫星的侧翼序列设计合适的引     

Isolation, characterization, and cross-species utility of microsatellites in yellow cedar (Chamaecyparis nootkatensis).

Chamaecyparis nootkatensis is an ecologically and economically important conifer of the north Pacific coastal forests. To aid in studies of clonal structure and genetic differentiation of this and related species, we isolated and characterized microsatellites from C. nootkatensis. A microsatellite-enriched library yielded 75 repeat-containing sequences for which primer pairs were designed. Only five showed reliable amplification and polymorphism, with an average of 13.7 alleles/locus and a mean expected heterozygosity of 0.592. In progeny tests with four families, few null alleles were directly detected and loci segregated according to Mendelian expectations. However, in one primer pair, high heterozygote deficiency was observed, suggesting the presence of a null allele. The ability of primer pairs to cross amplify was tested on 18 species of the Cupressaceae sensu lato; three primer pairs yielded polymorphic loci in Cupressus and Juniperus species, but not in other Chamaecyparis species. This also supports recent findings of a closer affinity of C. nootkatensis with Cupressus over other Chamaecyparis species.     

Characteristics of single- and multi-copy microsatellites from Pinus radiata

 Dinucleotide microsatellites were isolated from Pinus radiata using both a standard genomic library and libraries enriched for microsatellites. Locus-specific primers were designed to amplify 43 unique microsatellites. Thirty two of these loci had interpretable PCR patterns, 11 of which were polymorphic in a screen of 19 P. radiata individuals; all 11 polymorphic loci contained at least 17 repeats in the sequenced plasmid. Six of the eleven primer pairs amplified multiple fragments per individual (3–8), suggesting that these loci were present in multiple copies in the genome. Genotyping a 48-tree P. radiata production population with seven of the most polymorphic microsatellites revealed an average of 17 bands per locus (the multi-copy microsatellites were treated as one locus). When tested on known pedigrees, both single and multi-copy microsatellites exhibited co-dominant inheritance and Mendelian segregation. Two loci had null alleles and one locus had a high frequency of non-parental alleles, suggesting a high mutation rate. Eight of these microsatellites, including five multi-copy loci, were placed on a partially constructed P. radiata genetic map. Four of the five multi-copy microsatellites had two or more sets of alleles that mapped to the same locus, and the fifth mapped to two unlinked loci. All seven tested primer pairs amplified PCR products from other species of hard pine, three amplified products from soft-pine species, and one amplified bands in other conifers. Received: 10 November 1997 / Accepted: 5 January 1998     

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.     

EST-SSR DNA polymorphism in durum wheat (Triticum durum L.) collections

SSRs derived from EST were molecular markers belonging to the transcribed region of the genome. Therefore, any polymorphism detected using EST-SSRs might reflect the better relationship among species or varieties. Using wheat EST-SSR markers, 60 durum wheat (Triticum durum L.) accessions from seven countries were investigated. Twenty-five primer pairs could amplify successfully in the 60 durum wheat accessions, of which tri-nucleotide repeats were the dominant type, and revealed 26 loci on all seven wheat homologous chromosome groups. A total of 87 eSSR alleles were detected, and the number of alleles detected by a single pair of primers ranged from 1 to 11, with an average of 3.3 alleles per locus. Higher numbers of alleles and PIC were identified on the B genome than those on the A genome.     

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.     

Integration of trinucleotide microsatellites into a linkage map of Citrus

 We report the successful assignment of the first seven microsatellite markers to the Citrus RFLP and isozyme map. A total of 14 microsatellite primer pairs were developed and tested for amplification and product-length polymorphism within a population of plants previously used for linkage-map construction. In each case, the successfully assigned microsatellite mapped to the termini of a different linkage group indicating a widespread distribution throughout the genome. Analysis of allele segregation revealed that two of nine microsatellites displayed a significant deviation from expected ratios (P>0.5). This was compared with other marker types within Citrus and a similar proportion of skewed loci was also found to be present. The analysis of two markers was complicated by the non-amplification of an inherited null allele within the mapping population. The successful integration of microsatellites into the genetic map of Citrus demonstrates the utility of this marker type for genetic analysis within wide intergeneric plant crosses. Received: 16 September 1996 / Accepted: 18 October 1996     

Isolation and characterization of 20 polynucleotide microsatellite markers in a vulnerable Korean snail, <Emphasis Type="Italic">Ellobium chinense,</Emphasis> using 454 pyrosequencing

A small and air-breathing snail, Ellobium chinense (Ellobiidae), is a vulnerable species by International Union for Conservation of Nature (IUCN). To protect and manage habitat and population of E. chinense, microsatellite markers were developed using 454 pyrosequencing and 20 polymorphic microsatellite markers were identified. A total of 146,704 sequences containing a minimum of four repeat motifs (mean, 631 base pairs) were identified from 499,505 reads. Among 80 loci containing more than nine repeat units, 34 primer sets (42.5 %) produced strong PCR products, of which 20 were polymorphic among 48 samples of E. chinense. All loci exhibited high genetic variability, with an average of 18.9 alleles per locus, and the mean observed and expected heterozygosities were 0.65 and 0.90, respectively. In addition, cross-amplification was tested for all 20 loci in the same family species, Melampus sincaporensis. None of the primer pairs resulted in effective amplification, which might be due to their high mutation rates. Our work demonstrated the utility of next-generation 454 sequencing as a method for the rapid and cost-effective identification of microsatellites. The high degree of polymorphism exhibited by the 20 newly developed microsatellites will be useful in future conservation genetic studies of this species.     

Genetic differentiation of hexaploid wheat inferred from analysis of microsatellite loci

Landraces of wheat can serve as important potential sources for extending the genetic basis of selection cultivars. Analysis of microsatellites and typing of polymorphism in a representative sample of 347 genotypes, including landraces and selection cultivars, was performed using a set of 38 selected oligonucleotide primer pairs. Each genotype had a unique allele combination at 39 microsatellite loci examined. Classification of genotypes with respect to the level of their similarity was performed using cluster analysis. The data obtained pointed to genetic differentiation of hexaploid wheat. The groups of cultivars, the formation of which was thought to be associated with the main old areas of wheat cultivation in Europe and Asia, were identified. The basis of each of the groups was formed by landraces of common wheat. The differences between the groups identified were associated with multiple changes in the wheat genome and were expressed as quantitative differences in the allele frequencies of microsatellite loci. The results of the study are of interest in terms of understanding the structure of wheat genetic diversity and revealing the pathways of evolution of this culture.     

Transferability and utility of white oat (Avena sativa) microsatellite markers for genetic studies in black oat (Avena strigosa)

Preservation and use of wild oat species germplasm are essential for further improvement of cultivated oats. We analyzed the transferability and utility of cultivated (white) oat Avena sativa (AACCDD genome) microsatellite markers for genetic studies of black oat A. strigosa (A(s)A(s) genome) genotypes. The DNA of each black oat genotype was extracted from young leaves and amplified by PCR using 24 microsatellite primers developed from white oat. The PCR products were separated on 3% agarose gel. Eighteen microsatellite primer pairs amplified consistent products and 15 of these were polymorphic in A. strigosa, demonstrating a high degree of transferability. Microsatellite primer pairs AM3, AM4, AM21, AM23, AM30, and AM35 consistently amplified alleles only in A. sativa, which indicates that they are putative loci for either the C or D genomes of Avena. Using the data generated by the 15 polymorphic primer pairs, it was possible to separate 40 genotypes of the 44 that we studied. The four genotypes that could not be separated are probably replicates. We conclude that A. sativa microsatellites have a high transferability index and are a valuable resource for genetic studies and characterization of A. strigosa genotypes.     

Fifty new microsatellite loci for the wheat genetic map

 Hexaploid bread wheat (Triticum aestivum) has low levels of RFLP. Simple sequence repeats, however, show high levels of polymorphism and are therefore especially useful in intervarietal breeding applications. We present 53 newly mapped microsatellite loci for the wheat genetic map, 41 primary loci and 12 additional loci from these same primer pairs. Markers have been accredited with a quality score on a scale of 1–5 which describes the complexity of the amplification product profile from each primer pair. Received: 29 June 1997 / Accepted: 4 February 1998     

Assessment of genetic diversity among Syrian durum (Triticum turgidum ssp. durum) and bread wheat (Triticum aestivum L.) using SSR markers

Genetic diversity among 49 wheat varieties (37 durum and 12 bread wheat) was assayed using 32 microsatellites representing 34 loci covering almost the whole wheat genome. The polymorphic information content (PIC) across the tested loci ranged from 0 to 0.88 with average values of 0.57 and 0.65 for durum and bread wheat respectively. B genome had the highest mean number of alleles (10.91) followed by A genome (8.3) whereas D genome had the lowest number (4.73). The correlation between PIC and allele number was significant in all genome groups accounting for 0.87, 074 and 0.84 for A, B and D genomes respectively, and over all genomes, the correlation was higher in tetraploid (0.8) than in hexaploid wheat varieties (0.5). The cluster analysis discriminated all varieties and clearly divided the two ploidy levels into two separate clusters that reflect the differences in genetic diversity within each cluster. This study demonstrates that microsatellites markers have unique advantages compared to other molecular and biochemical fingerprinting techniques in revealing the genetic diversity in Syrian wheat varieties that is crucial for wheat improvement.     

Transferability of wheat microsatellites to diploid Triticeae species carrying the A, B and D genomes

Hexaploid wheat (Triticum aestivum L em Thell) is derived from a complex hybridization procedure involving three diploid species carrying the A, B and D genomes. In this study, we evaluated the ability of microsatellite sequences from T. aestivum to be revealed on different ancestral diploid species more or less closely related, i.e. to test for their transferability. Fifty five primer pairs, evenly distributed all over the genome, were investigated. Forty three of them mapped to single loci on the hexaploid wheat genetic map although only 20 (46%) gave single PCR products; the 23 others (54%) gave more than one band with either only one being polymorphic, the others remaining monomorphic, or with several co-segregating polymorphic bands. The other 12 detected two (9) or three (3) different loci. From the 20 primer pairs which gave one amplification pro- duct on hexaploid wheat, nine (45%) also amplified products on only one of the diploid species, and seven (35%) on more than one. Four microsatellites (20%) which mapped to chromosomes from the B genome of wheat, did not give any amplification signal on any of the diploid species. This suggests that some regions of the B genome have evolved more rapidly compared to the A or D genomes since the emergence of polyploidy, or else that the donor(s) of this B genome has(have) not yet been identified. Our results confirm that Triticum monococcum ssp. urartu and Triticum tauschii were the main donors of the A and D genomes respectively, and that Aegilops speltoides is related to the ancestor(s) of the wheat polyploid B genome. Received: 21 June 2000 / Accepted: 15 November 2000     

Assessment of genetic diversity among Syrian durum (<Emphasis Type="Italic">Triticum</Emphasis> ssp. <Emphasis Type="Italic">durum</Emphasis>) and bread wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) using SSR markers

Genetic diversity among 49 wheat varieties (37 durum and 12 bread wheat) was assayed using 32 microsatellites representing 34 loci covering almost the whole wheat genome. The polymorphic information content (PIC) across the tested loci ranged from 0 to 0.88 with average values of 0.57 and 0.65 for durum and bread wheat respectively. B-genome had the highest mean number of alleles (10.91) followed by A genome (8.3) whereas D genome had the lowest number (4.73). The correlation between PIC and allele number was significant in all genome groups accounting for 0.87, 074 and 0.84 for A, B and D genomes respectively, and over all genomes, the correlation was higher in tetraploid (0.8) than in hexaploid wheat varieties (0.5). The cluster analysis discriminated all varieties and clearly divided the two ploidy levels into two separate clusters that reflect the differences in genetic diversity within each cluster. This study demonstrates that microsatellites markers have unique advantages compared to other molecular and biochemical fingerprinting techniques in revealing the genetic diversity in Syrian wheat varieties that is crucial for wheat improvement.     

Development of microsatellite markers specific for the short arm of rye (Secale cereale L.) chromosome 1

We developed 74 microsatellite marker primer pairs yielding 76 polymorphic loci, specific for the short arm of rye chromosome 1R (1RS) in wheat background. Four libraries enriched for microsatellite motifs AG, AAG, AC and AAC were constructed from DNA of flow-sorted 1RS chromosomes and 1,290 clones were sequenced. Additionally, 2,778 BAC-end-sequences from a 1RS specific BAC library were used for microsatellite screening and marker development. From 724 designed primer pairs, 119 produced 1RS specific bands and 74 of them showed polymorphism in a set of ten rye genotypes. We show that this high attrition rate was due to the highly repetitive nature of the rye genome consisting of a large number of transposable elements. We mapped the 76 polymorphic loci physically into three regions (bins) on 1RS; 29, 30 and 17 loci were assigned to the distal, intercalary and proximal regions of the 1RS arm, respectively. The average polymorphism information content increases with distance from the centromere, which could be due to an increased recombination rate along the chromosome arm toward’s the telomere. Additionally, we demonstrate, using the data of the whole rice genome, that the intra-genomic length variation of microsatellites correlates (r = 0.87) with microsatellite polymorphism. Based on these results we suggest that an analysis of the microsatellite length variation is conducted for each species prior to microsatellite development, provided that sufficient sequence information is available. This will allow to selectively design microsatellite markers for motifs likely to yield a high level of polymorphism. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

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