Microsatellite markers developed for Utricularia reniformis (Lentibulariaceae)

? Premise of the study: Microsatellite markers were developed to study the genetic diversity and population structure of the carnivorous bladderwort Utricularia reniformis, which is endemic to the Atlantic Forest of southern and southeastern Brazil. Cross-species amplification was tested in U. gibba, U. neottioides, U. subulata, and Pinguicula benedicta. ? Methods and Results: The U. reniformis genome was sequenced in a 454 GS FLX sequencer, and eight primer sets were developed based on the microsatellites identified from the reads. All loci are polymorphic, showing 1.6 to 4.8 alleles per population. Preliminary results show that primer sets are suitable for population-level studies. Cross-species amplification was successful in three other Utricularia species and one Pinguicula species. ? Conclusions: Markers developed in this study provide tools for analyses of intra- and interpopulation genetic diversity in Utricularia and Pinguicula.     

Isolation and characterization of microsatellites in the lichen Buellia frigida (Physciaceae), an Antarctic endemic

? Premise of the study: Microsatellite markers were characterized for an Antarctic endemic, Buellia frigida, to investigate population structure and origin of Antarctic lichens. ? Methods and Results: Five primer sets were characterized. All loci were polymorphic with eight to 16 alleles per locus in a sample of 59 lichens. ? Conclusions: The microsatellite markers potentially provide insight into population structure and gene flow of B. frigida.     

Nuclear microsatellite loci for Arabidopsis halleri (Brassicaceae), a model species to study plant adaptation to heavy metals

? Premise of the study: Arabidopsis halleri is a model species to study the adaptation of plants to soils contaminated by zinc, cadmium, and lead. To provide a neutral genetic background with which adaptive genetic markers could be compared, we developed highly polymorphic neutral microsatellite markers. ? Methods and Results: Using a microsatellite-enriched library method, we identified 120 microsatellite loci for quantitative trait locus (QTL) mapping analysis, of which eight primer pairs were developed in a single multiplex for population genetic studies. Analyses were performed on 508 individuals from 26 populations. All loci were polymorphic with six to 23 alleles per locus. Genetic diversity varied between 0.56 and 0.76. ? Conclusions: Our results demonstrated the value of these eight microsatellite markers to investigate neutral population genetic structure in A. halleri. To increase the resolution of population genetic analyses, we suggest adding them to the 11 markers previously developed independently.     

New microsatellite markers in chicken optimized for automated fluorescent genotyping

We have isolated and developed 180 new polymorphic chicken microsatellite markers. In addition, primers have been developed for 91 microsatellites derived from the GenBank sequence database (isolated by the laboratory of Terry Burke, Leicester University), of which 89 were polymorphic, and six existing polymorphic markers (HUJ) have been modified. The primer sequences were designed to allow optimal performance of the markers, in sets containing multiple microsatellites, on ABI sequencers. The average number of alleles for the 275 polymorphic markers described was 4·0. Of these markers, 93% were polymorphic in the Wageningen resource population whereas 57% of the markers were polymorphic in the East Lansing reference population and only 44% could be mapped in the Compton reference population. The microsatellite markers described in this paper, in combination with the microsatellite markers published previously, are particularly well suited for performing a total genome scan for the detection of quantitative trait loci (QTL).     

Development of microsatellite markers in heterostylous Hedyotis chrysotricha (Rubiaceae)

? Premise of the study: Microsatellite primers were developed for a heterostylous herb, Hedyotis chrysotricha to investigate the effect of habitat fragmentation on its genetic diversity and population structure. ? Methods and Results: Twelve primer sets were developed and their polymorphisms were tested on 47 individuals from two island populations of H. chrysotricha in Thousand Island Lake of China. The number of alleles per locus ranged from five to 10, with an average of seven alleles. Expected heterozygosity per locus ranged from 0.284 to 0.821 and observed heterozygosity ranged from 0.191 to 0.851. ? Conclusions: We showed that all of the 12 microsatellite markers developed for H. chrysotricha are polymorphic within populations, which should provide a powerful tool for assessing population structure and genetic diversity across fragmented and continuous populations, and for studying the genetic effects of habitat fragmentation on this species.     

Development of microsatellite markers for the invasive weed Parthenium hysterophorus (Asteraceae)

? Premise of the study: Microsatellite primers were developed for Parthenium hysterophorus to investigate its genetic structure and genetic diversity. ? Methods and Results: Using the combined biotin capture method, 15 microsatellite primer sets were isolated and characterized. All markers showed polymorphism, and the number of alleles per locus ranged from two to nine across 60 individuals from two populations. The observed and expected heterozygosities ranged from 0.117 to 0.750 and from 0.182 to 0.835, respectively. ? Conclusions: These markers will be useful for investigating the invasion history of this weed globally and to help characterize its invasiveness.     

Sequence characterized amplified region markers for identifying biotypes of Bemisia tabaci (Hem., Aleyrodidae)

Abstract:  Bemisia tabaci (Gennadius) is an important pest of agriculture and horticulture crops that includes many morphologically indistinguishable biotypes. Molecular markers can provide a scientific method to rapidly identify biotypes. In this study, we attempted to develop specific primer sets for rapidly and stably identifying various biotypes of B. tabaci . We developed sequence characterized amplified region (SCAR) markers for each of six B. tabaci (Gennadius) biotypes (A, B, Q, Nauru, An and S). Two primer sets (BaAF/BaAR and BaQF/BaQR) were designed from random amplified polymorphic DNA-specific fragments for the A and Q biotypes. Four forward primers, BaBF, BaNaF, BaANF and BaSF, with a common reverse primer, L2-N-3014, for the other four biotypes of B, Nauru, An and S, respectively, were used based on their individual mitochondrial cytochrome oxidase I sequences. Six of these SCARs were useful in distinguishing each biotype from the others. Application of these SCARs will be helpful in rapidly identifying biotypes for quarantine pest control to prevent the invasion of the various biotypes.     

Isolation and characterization of microsatellite markers in an endangered species Dracaena cambodiana (Liliaceae)

?Premise of the study: The development of microsatellite primers in the endangered species Dracaena cambodiana will be the foundation for genetic and conservation studies of D. cambodiana and several Dracaena species. ?Methods and Results: A total of 26 microsatellite markers were developed in Chinese populations of D. cambodiana, using the Fast Isolation by AFLP of Sequences Containing Repeats (FIASCO) protocol. Among them, sixteen primer pairs generated polymorphic loci (fourteen of them successfully amplified in other four Dracaena species) and ten primer pairs produced monomorphic loci. ?Conclusions: These microsatellite markers could be used in the further investigation of population genetics of D. cambodiana and other Dracaena species.     

Development of nuclear microsatellite markers for the fonio, Digitaria exilis (Poaceae), an understudied West African cereal

? Premise of the study: We developed nuclear microsatellite primers to explore the genetic diversity, population genetic structure, and evolutionary history of the fonio (Digitaria exilis), an understudied cereal cultivated in West Africa. ? Methods and Results: We used a microsatellite-enriched library approach to isolate and characterize 38 nuclear primer pairs (31 di-, five tri-, and two tetranucleotide repeats), of which 21 were polymorphic and exhibited a clear pattern in 36 accessions from West Africa. The number of alleles per locus ranged from two to 22, with a mean of 4.71, and expected heterozygosity ranged from 0.03 to 0.93. ? Conclusions: The developed set of 21 polymorphic SSR markers will provide tools for population and evolutionary genetics studies of the cultivated fonio.     

Microsatellite markers for population studies of Phytophthora megakarya (Pythiaceae), a cacao pathogen in Africa

? Premise of the study: Phytophthora megakarya is the agent of black pod disease of cacao and is the main pathogen of this crop in Africa. Population genetic studies are required to investigate how this pathogen emerged. To this end, we developed 12 novel polymorphic microsatellite markers for P. megakarya. ? Methods and Results: Microsatellite sequences were obtained by pyrosequencing of multiplex-enriched libraries. Candidate loci with di- or trinucleotide motifs were selected, and primer pairs were tested with nine P. megakarya isolates. The 12 most polymorphic and unambiguous loci were selected to develop three multiplex PCR pools. The total number of alleles varied from two to nine, depending on loci, and higher than expected heterozygosity was observed. ? Conclusions: These markers were used for population genetic studies of P. megakarya in Cameroon and for comparison with reference strains from West Africa. This is the first time that microsatellite markers have been developed for P. megakarya.     

Microsatellite markers for the native Texas perennial grass, Panicum hallii (Poaceae)

? Premise of the study: We developed microsatellites for Panicum hallii for studies of gene flow, population structure, breeding experiments, and genetic mapping. ? Methods and Results: Next-generation (454) genomic sequence data were used to design markers. Eighteen robust markers were discovered, 15 of which were polymorphic across six accessions of P. hallii var. hallii. Fourteen of the markers cross-amplified in a P. capillare accession. For the 15 polymorphic markers, the total number of alleles per locus ranged from two to 26 (mean: 11.0) across six populations (11-19 individuals per population). Observed heterozygosity (mean: 0.031) was 13.7 times lower than the expected heterozygosity (mean: 0.426). ? Conclusions: The deficit of heterozygous individuals is consistent with P. hallii having a high rate of self-fertilization. These markers will be useful for studies in P. hallii and related species.     

Sixteen novel microsatellite markers developed for Dendrocalamus sinicus (Poaceae), the strongest woody bamboo in the world

? Premise of the study: Microsatellite primers were developed in the strongest and most economically important bamboo species, Dendrocalamus sinicus, to investigate its genetic variability. ? Methods and Results: Using the Fast Isolation by AFLP of Sequences COntaining Repeats (FIASCO) protocol, 16 polymorphic primer sets were identified within 24 individuals from two geographic locations. The number of alleles per locus ranged from two to four, with a mean of 2.6. The observed and expected heterozygosities varied from 0 to 1.000 and from 0.311 to 0.754, respectively. The cross-amplification of the 16 primer pairs was tested in four other Dendrocalamus species, and nine to 13 (56.3-81.3%) primer pairs were successfully amplified. ? Conclusions: These markers will be useful for studies on the genetic diversity of D. sinicus, which may facilitate conservation of this species.     

Microsatellite loci for Gossypium davidsonii (Malvaceae) and other D-genome, Sonoran Desert endemic cotton species

? Premise of the study: Microsatellite primers previously developed for domesticated cotton (Gossypium hirsutum; tetraploid) were screened for their utility in investigating genetic structure and gene flow within G. davidsonii and five other wild, Mexican, D-genome cotton species (all diploid). ? Methods and Results: We screened 50 microsatellite primer pairs from the Cotton Marker Database, identifying 10 loci as polymorphic within G. davidsonii. In genotyping approximately 200 individuals from four populations, we found that the number of alleles per locus ranged from seven to 17, and mean observed and expected heterozygosity ranged from 0.145 to 0.492 and from 0.436 to 0.734, respectively. We genotyped six to 20 individuals from each of the remaining species, finding these 10 loci to cross-amplify in all cases and in most cases to be polymorphic. ? Conclusions: These markers may be useful for further investigation of population genetics of G. davidsonii and other wild D-genome cotton species.     

Development of DNA microsatellite markers in the multiband butterflyfish (Chaetodon multicinctus)

Twelve polymorphic microsatellite loci were developed in the multiband (pebbled) butterflyfish Chaetodon multicinctus. The loci were scored in 45 individuals from Hawaii. There were five to 21 alleles per locus with observed heterozygosity ranging from 0.419 to 0.883. Four of the primer sets also reliably amplified polymorphic loci in Chaetodon quadrimaculatus. We expect these markers to be useful for studies of genetic population structure and kinship, for example to determine whether new recruits settling onto reefs are related.     

Development of microsatellite loci for Pinus koraiensis (Pinaceae)

? Premise of the study: Microsatellite markers were developed for Pinus koraiensis to characterize its genetic diversity and understand its population structure. ? Methods and Results: Using the Fast Isolation by AFLP of Sequences COntaining (FIASCO) Repeats protocol, 20 primer sets were developed in Chinese populations of P. koraiensis. Three of the markers showed polymorphism with two alleles per locus when assessed in a sample of two populations of P. koraiensis from the Changbai Mountain in the Jilin Province of China. Five and three loci were successfully amplified in P. taiwanensis and P. massoniana, respectively. The amplification size of these loci matches those in P. koraiensis. ? Conclusions: These markers may be useful for further investigation of population genetics of P. koraiensis.     

Microsatellite markers for Saussurea gnaphalodes (Asteraceae), a native Himalayan mountain species

? Premise of the study: Microsatellite markers were developed in Saussurea gnaphalodes to investigate its genetic variation and population structure. ? Methods and Results: Using the combined biotin capture method, 46 microsatellite primer sets were isolated and characterized across 48 S. gnaphalodes individuals from three Tibetan populations. Seventeen of these markers showed polymorphism, and the number of alleles ranged from two to 17. The observed and expected heterozygosities per population ranged from 0 to 0.938 and from 0 to 0.905, respectively. ? Conclusions: These markers provide a useful tool to investigate the genetic diversity and population structure of S. gnaphalodes, and to facilitate further studies on conservation and utilization.     

Development and characterization of microsatellite markers for the wild South American Passiflora cincinnata (Passifloraceae)

? Premise of the study: We developed the first set of microsatellite markers for the passion fruit, Passiflora cincinnata, to provide tools for further study of its genetic diversity and to support current conservation and genetic studies. ? Methods and Results: We used a microsatellite-enriched library approach to isolate and characterize 25 new molecular markers. Seven primer pairs successfully amplified polymorphic microsatellite regions, with an average of five alleles per locus. The mean values of expected and observed levels of heterozygosity were 0.516 and 0.525, respectively. ? Conclusions: The microsatellite markers identified in this study may be valuable tools for population genetic studies, and this set of markers also may be useful in the design of a genetic conservation strategy, mating system, and hybridization studies.     

Microsatellites for the mangrove tree Avicennia germinans (Acanthaceae): Tools for hybridization and mating system studies

? Premise of the study: We developed a new set of microsatellite markers for the black mangrove Avicennia germinans, to provide new informative tools for further studies of the mating system, interspecific hybridization, and population genetics. ? Methods and Results: We used the microsatellite-enriched library approach to isolate and characterize 25 new primer pairs. Sixteen of them are polymorphic, showing a variable degree of variation in A. germinans, while nine were monomorphic in the samples examined. Eight exhibited private alleles in A. schaueriana. ? Conclusions: These results indicate that these new microsatellite markers will be useful molecular tools for further studies of A. germinans and A. schaueriana population genetics, mating systems, and hybridization.     

Development of microsatellite markers in Lupinus luteus (Fabaceae) and cross-species amplification in other lupine species

? Premise of the study: Microsatellite primers were developed in Lupinus luteus L., an emerging temperate protein crop, to investigate genetic diversity, population structure, and to facilitate the generation of better yellow lupine varieties. ? Methods and Results: Thirteen polymorphic primer sets were evaluated in a European and Eastern European accession collection of L. luteus. The primers amplified di-, tri-, and tetranucleotide repeats with 2-4 alleles per locus. These revealed a moderate to low level of genetic variation, as indicated by an average observed heterozygosity of 0.0126. Select loci also amplified successfully in the closely related species L. hispanicus Boiss. & Reut. and in the New World species L. mutabilis Sweet. ? Conclusions: These results indicate the utility of primers for the study of genetic diversity across L. luteus populations and related lupine species. The use of these microsatellite markers will facilitate the implementation of several molecular breeding strategies in yellow lupine.     

Development of EST-PCR markers and monitoring their intrapopulational genetic variation in Picea abies (L.) Karst.

Fifteen cDNA sequences are reported for the European coniferous forest tree species Norway spruce [Picea abies (L.) Karst.], including the results of similarity searches in public electronic databases. The sequences were subsequently employed for the design of specific primer pairs and PCR-based amplification of genomic fragments. For seven primer pairs, polymorphic EST-PCR markers were identified among 18 trees. Their mode of inheritance was verified by analysing single-tree offspring and studying segregation among haploid endosperms in comparison to diploid tissue. Codominant inheritance was indicated for six markers, while one marker was apparently dominant. Variation of the six codominant EST markers was tested by genotyping 110 randomly selected trees in a Bavarian Norway spruce population. For comparison, the same trees were genotyped at 18 enzyme coding gene loci. There were 3.33 alleles per locus for EST markers and 3.00 for isoenzyme gene markers. In general, a trend to more even frequency distributions and larger intrapopulational variation, including observed heterozygosities, was indicated more for EST markers than for isoenzyme gene markers. The benefits of these newly developed EST-PCR markers are outlined with respect to population genetics and ecological genetics. Received: 29 April 2000 / Accepted: 25 August 2000