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.     

Interspecies and intergenus transferability of barley and wheat D-genome microsatellite markers

A selection of 147 wheat D-genome and 130 barley genomic simple sequence repeat (gSSR) markers were screened for their utility in Hordeum chilense, as an alien donor genome for cereal breeding. Fifty-eight wheat D-genome and 71 barley PCR primer pairs consistently amplified products from H. chilense. Nineteen wheat D-genome and 20 barley gSSR markers were polymorphic and allowed wide genome coverage of the H. chilense genome. Twenty-three of the wheat D-genome and 11 barley PCR primer pairs were suitable for studying the introgressions of H. chilense into wheat, amplifying H. chilense products of distinct size. In 88% of the markers tested, H. chilense products were maintained in the expected homeologous linkage group, as revealed by the analysis of wheat/H. chilense addition lines. Twenty-nine microsatellite markers (eight gSSRs and 21 expressed sequence tags-SSRs) uniformly distributed across the genome were tested for their utility in genetic diversity analysis within the species. Three genetic clusters are reported, in accordance with previous morphological and amplified fragment length polymorphism data. These results show that it is possible to discriminate the three previously established germplasm groups with microsatellite markers. The reported markers represent a valuable resource for the genetic characterisation of H. chilense, for the analysis of its genetic variability, and as a tool for wheat introgression. This is the first intraspecific study in a collection of H. chilense germplasm using microsatellite markers.     

Molecular diversity of species of the Elymus trachycaulus species complex and their relationships to non-North American taxa

Species of the E. trachycaulus complex species are known for their morphological variability, but little is known about their genetic basis. The delimitation of taxa within the complex has been controversial and difficult. E. trachycaulus is predominantly self-pollinating, and lacks clear morphological boundaries between it and E. alaskanus. Another controversial taxonomic issue of E. trachycaulus is the relationships of this complex species to non-North American E. caninus. The objectives of this study were to examine genetic diversity and the systematic relationships among the species of the E. trachycaulus complex and their relationships with E. caninus, E. alaskanus and E. mutabilis. Random amplified polymorphic DNA method was used to study 35 accessions of E. trachycaulus complex and other Elymus species. Higher genetic variation was detected within species of E. trachycaulus complex. Eurasian accessions are as variable as the North American ones. Both UPGMA and NJ analyses did not show clearly separation among species of the E. trachycaulus complex. No clear association between geographic origin and genetic grouping among these species was found. Eurasian E. trachycaulus probably originated from multiple North American populations.     

Molecular diversity and relationships of North American Elymus trachycaulus and the Eurasian E. caninus species

The morphological similarity of Elymus trachycaulus to the Eurasian E. caninus has often been noted. This has lead to controversial and contradicting taxonomic treatments. Nevertheless, there has been no systematic investigation on molecular genetic similarity between E. trachycaulus and E. caninus. In this study, random amplified polymorphic DNA (RAPD) analysis was used to study the similarity between the two species. RAPD analysis of 38 samples representing E. caninus and E. trachycaulus complex yielded 111 interpretable RAPD bands. The Jaccard’s similarity values for E. caninus ranged from 0.38 between accessions H10345 and H10353 to 0.97 between accessions H8745 and H10096, with an average of 0.67. The Jaccard’s similarity values for E. trachycaulus complex ranged from 0.09 between E. trachycaulus ssp. subsecundus (PI 537321) and E. trachycaulus ssp. violaceus (PI 272612) to 0.78 between accessions PI 315368 and PI 372644, with an average of 0.43. The results from different analyses (NJ and PCA) were similar but not identical. The molecular genetic separation between E. caninus and E. trachycaulus was consistent. The PCA analysis clearly separated all E. caninus accessions from E. trachycaulus and its subspecies. The NJ analysis also showed separation between most accessions of E. caninus and E. trachycaulus. Further analysis excluding E. trachycaulus ssp. subsecundus and ssp. violaceus revealed that E. caninus species and E. trachycaulus species were clearly separated into two distinct groups. The RAPD data thus support the treatment of E. caninus and E. trachycaulus as distinct species. The analyses further indicate that E. violaceus is nested within E. trachycaulus, and more related to E. trachycaulus complex rather than to E. caninus.     

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     

The development of sequence-tagged sites (STSs) in Lolium perenne L.: the application of primer sets derived from other genera

Genetic analysis, particularly the development of genetic linkage maps in forage grass species, lags well behind other members of the Poaceae. Comparative mapping within this family has revealed extensive conservation in gene and marker synteny among chromosomes of diverse genera. Recently, the ability to transfer mapped STS markers between barley and wheat has been demonstrated. The transfer of mapped STS markers between cereals and forage grasses could provide PCR-based markers for comparative mapping in these species providing they amplify homologous sequences. In this study, primers derived from three barley genes of defined function and a gene from Phalaris coerulescens were used to amplify homologous fragments in Lolium perenne. Primers derived from two barley and two oat cDNA clones were also tested along with eight barley and two Triticum tauchii STS markers. Twenty one primer pairs derived from 18 loci were tested. Eleven primer pairs (52%) amplified homologous sequences in L. perenne from ten (55%) of the loci targetted. Thirteen new STS markers were generated in L. perenne, of which ten have been mapped in barley or rye and amplify homologous sequences in L. perenne. Received: 20 October 2000 / Accepted: 13 January 2001     

Development and characterization of polymorphic microsatellite markers from Fragaria viridis,a wild diploid strawberry

To date, the development of microsatellite (SSR) markers in the genus Fragaria has focused on F. vesca. However, further species are thought to have contributed to the complex allo‐octoploid genome of the cultivated strawberry, F.×ananassa. Here, we present 22 new SSR markers developed from the diploid species F. viridis. Twenty‐one of the primer pairs amplified polymorphisms in six F. viridis accessions, with an average of 4.95 alleles per primer pair and an average expected heterozygosity of 0.68. Fourteen of these primer pairs, and a locus monomorphic in F. viridis, amplified polymorphic alleles in the parents of a F. vesca mapping population.     

Isolation and characterization of microsatellite loci in Penstemon rostriflorus (Plantaginaceae) and cross‐species amplification

Eight novel microsatellite primer pairs are presented for Penstemon rostriflorus, representing the first microsatellite markers available for this genus. Loci were characterized for 20 individuals from two populations in the Great Basin, USA. All loci are polymorphic within P. rostriflorus (seven to 13 alleles per locus; observed heterozygosity between 0.40 and 0.95), and therefore useful for population genetic studies within the species. Cross‐species transferability was tested on 40 additional species of Penstemon, and results indicate that these primers pairs will likely be useful for population genetic studies on many Penstemon species.     

Analysis of tetraploid Elymus species using wheat microsatellite markers and RAPD markers.

An analysis of Amplification fragment polymorphism of DNA from 27 accessions of 19 tetraploid Elymus species was carried out using 18 wheat microsatellite (WMS) primer pairs and 10 decamer primers. Ten WMS primer pairs produced multiple polymorphism on all accessions tested. Two independent phenograms, one based on WMS-PCR and one on RAPDs, separated the 19 tetraploid species into two main groups, viz., the SH genome species group and the SY genome species group. The results coincide with the genomic classification of these species and hence support previous studies showing that Elymus is not a monophyletic genus. The assays indicated that accessions within a species cluster together, which concurs with the morphological classification. Interspecific and intraspecific polymorphisms were detected by the WMS-PCR and RAPD analyses. Variation was observed among accessions of Elymus caninus. The WMS-PCR detected a much higher level of polymorphism than the RAPD analysis. WMSs seem to be more efficient markers than RAPD markers for studying the population diversity of Elymus species. The potential of cross-species amplification of microsatellite markers as an additional source for genetic analysis and applications in Elymus is discussed in the context of these results.     

Characterization and analysis of microsatellite loci in Elymus caninus (Triticeae: Poaceae)

 Microsatellites are highly variable DNA sequences that can be used as markers for the genetic analysis of plants. The potential of microsatellite markers for use in a genetic diversity study in Elymus species was evaluated. Genomic libraries of Elymus caninus were constructed. The libraries were screened with two dinucleotide, (GA)n and (GT)n, and two trinucleotide repeats, (TCT)n and (CAC)n. A total of 19 positive clones were found for the two dinucleotide repeats; no positive clone was found for the trinucleotide repeats. Positive clones were sequenced to confirm the presence of microsatellites and to generate polymerase chain reaction (PCR) primers based on the sequences flanking the microsatellite. All sequenced (GA)n clones have repeats of n>10; over half of the (GT)n microsatellites have n<10 repeats. Primer pairs were designed and evaluated for 8 selected microsatellites. PCR products were amplified from 15 Elymus caninus accessions. The number of alleles found for the eight loci varied from 1 for ECGA89 and ECGT35 to 13 for ECGA22, as determined by non-denaturing polyacrylamide electrophoresis. Six microsatellite loci were found to be polymorphic in E. caninus. The eight primer pairs were tested on three other species; seven were successful in amplifying DNA from Elymus alaskanus and E. mutabilis, and four amplified DNA from E. caucasicus. Based on these results, microsatellites appear to be useful markers in detecting variation in E. caninus. Received: 8 September 1997/Accepted: 6 October 1997     

Transferability and Level of Heterozygosity of Microsatellite Markers in <Emphasis Type="Italic">Citrus</Emphasis> Species

Microsatellite markers are a powerful tool for genetic studies, including germplasm conservation, cultivar identification, and integration of linkage maps. Several works have shown that primer pairs designed for one species can be used in related species to facilitate wider application because it reduces the costs for primer development. The objective of this study was to evaluate the transferability of microsatellite primers which was previously developed from the genomic library of Pêra sweet orange (Citrus sinensis L. Osbeck) and to determine the level of heterozygosity between citrus accessions and related genera. Twenty-four microsatellite loci were evaluated on 12 genotypes of Citrus, Poncirus, and an intergeneric hybrid. All analyzed markers were transferable across all genotypes. Seventeen loci were polymorphic, and the number of alleles per loci ranged from one to six. The lowest level of heterozygosity was observed for Poncirus trifoliata (L.) Raf. cultivars while the highest level was for Swingle citrumelo. In general, microsatellite markers showed wide genetic variation and demonstrated that they can be useful in citrus breeding programs.     

Utility of barley and wheat simple sequence repeat (SSR) markers for genetic analysis of Hordeum chilense and tritordeum

A selection of 36 wheat and 35 barley simple sequence repeat markers (SSRs) were studied for their utility in Hordeum chilense. Nineteen wheat and nineteen barley primer pairs amplified consistent H. chilense products. Nine wheat and two barley SSRs were polymorphic in a H. chilense mapping population, producing codominant markers that mapped to the expected homoeologous linkage groups in all but one case. Thirteen wheat and 10 barley primer pairs were suitable for studying the introgression of H. chilense into wheat because they amplified H. chilense products of distinct size. Analysis of wheat/H. chilense addition lines showed that the H. chilense products derived from the expected homoeologous linkage groups. The results showed that wheat and barley SSRs provide a valuable resource for the genetic characterization of H. chilense, tritordeums and derived introgression lines. Received: 20 November 2000 / Accepted: 12 April 2001     

Anonymous single‐copy nuclear DNA (scnDNA) markers for two endemic log‐dwelling beetles: Apasis puncticeps and Adelium calosomoides (Tenebrionidae: Lagriinae: Adeliini)

Three approaches — microsatellite library screening, consensus primer PCR (polymerase chain reaction) and sequencing with arbitrary primer pairs (SWAPP) — were used to develop single‐copy nuclear DNA (scnDNA) markers for log‐dwelling beetles Apasis puncticeps and Adelium calosomoides. We are unaware of other nuclear markers for Adeliini. We tested > 70 primer pairs per species, but despite exhaustive optimization, we obtained only five polymorphic markers. Nonetheless, the markers are valuable in detection of effects of habitat fragmentation.     

Development and validation of genomic simple sequence repeat markers in <Emphasis Type="Italic">Erianthus arundinaceus</Emphasis>

Erianthus arundinaceus, a member of the Saccharum complex, is of interest as a potential resource for sugarcane improvement and as a bioenergy crop. Genetic analyses of germplasm collections of E. arundinaceus are being used increasingly. To expand the genomic resources in E. arundinaceus, we aimed at developing simple sequence repeat markers. Using pyrosequencing on the 454 GS FLX system, we sequenced genomic DNA from “JW630” collected in Japan. A total of 1682 candidate loci were used to design the primers, and 1234 primer pairs amplified fragments of the expected size in the primer screening with three wild E. arundinaceus accessions (JW630, “JW4,” and “IJ76-349”). The efficiency of genotyping was validated with a subset of 174 primer pairs and 8 E. arundinaceus accessions. Of these primer pairs, 171 amplified fragments in all accessions tested and 162 detected polymorphic loci. The average values of genetic parameters were estimated as 0.30 (range, 0.09–0.49) for polymorphic information content, 1.65 (0.00–5.87) for marker index, and 2.78 (0.00–8.75) for resolving power. Using these parameters, we selected 61 primer pairs with large discriminatory power for the analyzed loci. Of the 174 primer pairs, 45 (25.9%) were also applicable to Saccharum and 33 (19.0%) to Miscanthus species. These markers would provide a valuable tool for estimating genetic diversity and constructing linkage maps in E. arundinaceus, which would be useful for genetic study and breeding.     

Onion microsatellites for germplasm analysis and their use in assessing intra- and interspecific relatedness within the subgenus Rhizirideum

We have identified a set of informative STMS markers in onion (Allium cepa L.) and report on their application for genotyping and for determining genetic relationships. The markers have been developed from a genomic library enriched for microsatellites. Integrity of the microsatellite polymorphism was confirmed by amplicon sequencing. The microsatellite genotypes of 83 onion accessions and landraces from living onion collections were compared. As few as four primer pairs were sufficient to assign unique microsatellite patterns to the 83 accessions. Some of the microsatellite markers can be used for interspecific taxonomic analyses among close relatives of Allium cepa. Generally, our data support and extend results obtained from recently performed analyses using ITS, RAPD and morphology. Received: 8 October 1999 / Accepted: 3 November 1999     

Microsatellite variation in <Emphasis Type="Italic">Avena sterilis</Emphasis> oat germplasm

The Avena sterilis L. collection in the Plant Gene Resources of Canada (PGRC) consists of 11,235 accessions originating from 27 countries and is an invaluable source of genetic variation for genetic improvement of oats, but it has been inadequately characterized, particularly using molecular techniques. More than 35 accessions have been identified with genes for resistance to oat crown and stem rusts, but little is known about their comparative genetic diversity. This study attempted to characterize a structured sample of 369 accessions representing 26 countries and two specific groups with Puccinia coronata avenae (Pc) and Puccinia graminis avenae (Pg) resistance genes using microsatellite (SSR) markers. Screening of 230 SSR primer pairs developed from other major crop species yielded 26 informative primer pairs for this characterization. These 26 primer pairs were applied to screen all the samples and 125 detected alleles were scored for each accession. Analyses of the SSR data showed the effectiveness of the stratified sampling applied in capturing country-wise SSR variation. The frequencies of polymorphic alleles ranged from 0.01 to 0.99 and averaged 0.28. More than 90% of the SSR variation resided within accessions of a country. Accessions from Greece, Liberia, and Italy were genetically most diverse, while accessions from Egypt, Georgia, Ethiopia, Gibraltar, and Kenya were most distinct. Seven major clusters were identified, each consisting of accessions from multiple countries and specific groups, and these clusters were not well congruent with geographic origins. Accessions with Pc and Pg genes had similar levels of SSR variation, did not appear to cluster together, and were not associated with the other representative accessions. These SSR patterns are significant for understanding the progenitor species of cultivated oat, managing A. sterilis germplasm, and exploring new sources of genes for oat improvement.     

Polymorphic Microsatellite Markers Transferable Across <Emphasis Type="Italic">Capsicum</Emphasis> Species

Pepper (Capsicum annuum L.) is one of the most important crops in the family Solanaceae. However, the number of polymorphic molecular loci detected in this important crop is far behind that of other cultivated plant species. In the present study, a total of 45 microsatellite primer pairs were developed using Capsicum expressed sequence tags databases. Microsatellite primer pairs were tested using several species of Capsicum and several genera in the family Solanaceae including tomato, potato, eggplant, and tobacco. Results indicated that microsatellite primer pairs amplified genomic targets of C. annuum L., Capsicum baccatum L., Capsicum chacoense L., Capsicum chinense L., Capsicum frutescens L., and Capsicum pubescens Ruiz et Pavon, indicating species transferability within Capsicum. Further analyses revealed that amplicons of these primer pairs segregated 1:2:1 or 3:1 Mendelian fashions in 38 F₂ individuals of pepper. It was also noted that markers derived from sequences containing dinucleotide repeats were generally more polymorphic at the intraspecific level than sequences containing trinucleotide repeats. All the microsatellite primer pairs developed in this study will be useful for marker-assisted selection and mapping studies in pepper.     

Characterisation of microsatellites from Actinidia chinensis

We have identified a set of informative microsatellite markers for genome analysis in kiwifruit and related Actinidia species. A small-insert genomic library was constructed from Actinidia chinensis DNA, and screened for microsatellites. About 1.2% of the total colonies hybridised to a (GA)₈ probe, 0.4% to (GT)₈, and 0.1% to a mixture of three different trinucleotide repeat probes, (CAA)₅, (GAA)₅ and (CTA)₅. From the DNA sequences of 35 hybridising clones, 18 primer pairs were designed, and used to amplify genomic DNA from 38 individual plants, representing 30 different accessions of ten Actinidia species. The banding patterns for most of the dinucleotide repeats showed a high degree of polymorphism in the diploid and tetraploid A. chinensis, and in the hexaploid A. deliciosa (kiwifruit). Heterozygosity levels of up to 100% were found among eight diploid accessions of A. chinensis examined, and the number of different-sized bands among all the species varied from 3 to 36 for each microsatellite. One simple CT microsatellite gave 21 bands with sizes suggesting that the number of repeats ranged from 9 to 37. The highest number of bands (36) and the largest size variation (>100 bp) were observed with a complex microsatellite harbouring four different repeat motifs. The majority of primer pairs amplified bands from most of the ten Actinidia species tested. The most polymorphic primer pairs were used successfully to fingerprint a range of closely related varieties of kiwifruit (A. deliciosa).Abbreviations PCR polymerase chain reaction - RFLP restriction fragment length polymorphism - VNTR variable number of tandem repeats     

Novel polymorphic microsatellite markers in section Caulorrhizae (Arachis,Fabaceae)

This work reports the characterization of 11 polymorphic microsatellite loci in section Caulorrhizae. The primer pairs were designed from Arachis pintoi and showed full transferability to Arachis repens species. These new markers were used to evaluate the genetic diversity in germplasm (accessions and cultivars) of section Caulorrhizae. This new set of markers detected greater gene diversity than morphological and molecular markers such as AFLP (amplified fragment length polymorphism) and RAPD (rapid analysis of polymorphic DNA) previously used in this germplasm.     

Sequence characterization of microsatellites in diploid and polyploid Ipomoea

 The objectives of the present study were to evaluate the inheritance and nucleotide sequence profiles of microsatellite genetic markers in hexaploid sweetpotato [Ipomoea batatas (L.) Lam.] and its putative tetraploid and diploid ancestors, and to test possible microsatellite mutation mechanisms in polyploids by direct sequencing of alleles. Sixty three microsatellite loci were isolated from genomic libraries of I. batatas and sequenced. PCR primers were designed and used to characterize microsatellite loci in two hexaploid I. batatas populations, a tetraploid Ipomoea trifida population, and a diploid I. trifida population. Nine out of the sixty three primer pairs tested yielded a clearly discernible, heritable banding pattern; five showed Mendelian segregation. All other primer pairs produced either smeared banding patterns, which could not be scored, or no bands at all in I. batatas. All of the primers which produced discernible banding patterns from I. batatas also amplified products of similar size in tetraploid and diploid I. trifida accessions. The sequence analysis of several alleles in the three species showed differences due to mutations in the repeat regions consistent with small differences in the repeat number. However, in some cases insertions/deletions and base substitutions in the microsatellite flanking regions were responsible for polymorphisms in both polyploid and diploid species. These results provide strong empirical evidence that complex genetic mechanisms are responsible for SSR allelic variation in Ipomoea. Four I. batatas microsatellite loci showed polysomic segregation fitting tetraploid segregation ratios. To our knowledge this is the first report of segregation ratios for microsatellites markers in polyploids. Received: 4 January 1999 / Accepted: 4 January 1999