Reverse ascertainment bias in microsatellite allelic diversity in owls (Aves, Strigiformes)

A rich source of markers may be overlooked by screening for polymorphism in the source species only. We screened 129 microsatellite loci isolated from the powerful owl (Ninox strenua) against two closely related species; Ninox connivens and Ninox novaeseelandiae. From the screening effort 20 polymorphic markers were isolated, including six loci which were originally discarded as they were monomorphic in the source species. Further cross-species amplification of all 20 loci across species from two families, Strigidae and Tytonidae, revealed unusually high levels of polymorphism within closely related species, and limited success within phylogenetically distant species. Routine screening of multiple species during the marker development phase can yield a wider range of polymorphic markers which can subsequently enhance cross-species amplification attempts.     

Identification of microsatellite DNA markers for population structure analysis in Indian major carp, Cirrhinus mrigala (Hamilton-Buchanan, 1882)

Forty‐four primer sequences available for four cyprinid fishes were tested to amplify microsatellite loci in Indian major carp, Cirrhinus mrigala. A total of 12 loci were successfully amplified with clear scorable patterns and five thereof were polymorphic. Suitability of the identified polymorphic loci in population structure analysis of C. mrigala was assessed. Genetic variation was examined in 76 specimens collected from five different rivers. The mean observed heterozygosity ranged from 0.247 to 0.333. Significant heterogeneity in allele frequencies was detected, indicating that the samples analysed did not belong to homogenous populations. The identified microsatellite markers are promising for the analysis of intraspecific divergence in C. mrigala across its distribution range.     

Isolation and characterization of microsatellite markers for Pinellia ternata and cross-species amplification

In this study, we isolated and characterized 12 microsatellite loci for Pinellia ternata. Polymorphism of these 12 loci was assessed in 46 individuals collected from two wild populations. All the loci were polymorphic with four to 13 alleles per locus and the observed and expected heterozygosities ranged from 0.312 to 0.680 and from 0.506 to 0.734, respectively. None of the loci showed significant deviation from Hardy–Weinberg equilibrium (P < 0.05). No significant linkage disequilibrium was observed between pairs of studied loci. In addition, most markers amplified successfully in three closely related taxa that are Pinellia cordata, P. peltata and P. pedatisecta. These microsatellite markers could provide a useful tool for genetic structure studies of the Pinellia species.     

Development and bin mapping of strawberry genic-SSRs in diploid <Emphasis Type="Italic">Fragaria</Emphasis> and their transferability across the Rosoideae subfamily

Cultivated strawberry (Fragaria × ananassa) together with other economically important genera such as Rosa (roses) and Rubus (raspberry and blackberry) belongs to the subfamily Rosoideae. There is increasing interest in the development of transferable markers to allow genome comparisons within the Rosaceae family. In this report, 122 new genic microsatellite (SSR) markers have been developed from cultivated strawberry and its diploid ancestor Fragaria vesca. More than 77% of the sequences from which the markers were developed show significant homology to known or predicted proteins and more than 92% were polymorphic among strawberry cultivars, representing valuable markers in transcribed regions of the genome. Sixty-three SSRs were polymorphic in the diploid Fragaria reference population and were bin-mapped together with another five previously reported but unmapped markers. In total, 72 loci were distributed across the seven linkage groups. In addition, the transferability of 174 Fragaria SSRs to the related Rosa and Rubus genera was investigated, ranging from 28.7% for genic-SSRs in rose to 16.1% for genomic-SSRs in raspberry. Among these markers, 33 and 16 were both localized in the diploid Fragaria reference map and cross-amplified in rose and raspberry, respectively. These results indicate that transferability of SSRs across the Rosoideae subfamily is limited. However, we have identified a set of Fragaria markers, polymorphic in the diploid reference population, which cross-amplified in both Rosa and Rubus, which represents a valuable tool for comparative mapping and genetic diversity analyses within the Rosoideae subfamily.     

Tetranucleotide microsatellite loci from the striped dolphin (Stenella coeruleoalba Meyen, 1833)

Five tetranucleotide microsatellite loci were isolated from the striped dolphin (Stenella coeruleoalba). Characterization of these loci revealed between three and 11 alleles at each locus and observed heterozygosity ranged from 0.192 to 0.775. All sets of primers amplified homologous loci when tested on four related species and showed polymorphisms in most locus–species combinations. A sixth microsatellite, which appeared to be monomorphic in the striped dolphin, was also shown to be polymorphic in three related species.     

Characterization of 23 Tri‐ and tetranucleotide Microsatellite Loci in the Brown Tiger Prawn,Penaeus esculentus

Twenty‐three unique tri and tetra‐nucleotide microsatellites were isolated and characterized from the genome of the brown tiger prawn Penaeus esculentus. Nine of the 23 microsatellite loci were found to be polymorphic and were tested across a sample of wild caught Penaeus esculentus. Good fits to Hardy–Weinberg expectations were observed for six of the markers when tested in a population of at least 180 individuals. The total number of alleles for these six markers ranged from eight to 19, with an average total of 13. These values will make them useful for future stock identification and population genetics studies.     

Construction of an oilseed rape (Brassica napus L.) genetic map with SSR markers

We constructed a Brassica napus genetic map with 240 simple sequence repeats (SSR) primer pairs from private and public origins. SSR, or microsatellites, are highly polymorphic and efficient markers for the analysis of plant genomes. Our selection of primer pairs corresponded to 305 genetic loci that we were able to map. In addition, we also used 52 sequence-characterized amplified region primer pairs corresponding to 58 loci that were developed in our lab. Genotyping was performed on six F2 populations, corresponding to a total of 574 F2 individual plants, obtained according to an unbalanced diallel cross design involving six parental lines. The resulting consensus map presented 19 linkage groups ranging from 46.2 to 276.5 cM, which we were able to name after the B. napus map available at , thus enabling the identification of the A genome linkage groups originating from the B. rapa ancestor and the C genome linkage groups originating from the B. oleracea ancestor in the amphidiploid genome of B. napus. Some homoeologous regions were identified between the A and the C genomes. This map could be used to identify more markers, which would eventually be linked to genes controlling important agronomic characters in rapeseed. Furthermore, considering the good genome coverage we obtained, together with an observed homogenous distribution of the loci across the genome, this map is a powerful tool to be used in marker-assisted breeding. Electronic Supplementary Material Supplementary material is available for this article at     

Isolation and characterization of polymorphic microsatellites in Iris ensata (Iridaceae)

• Premise of the study: Microsatellite primers were developed in Iris ensata (Iridaceae) to provide polymorphic markers for further studies into population genetics. • Methods and Results: Thirteen polymorphic microsatellite loci were isolated from I. ensata. These loci were successfully amplified in two natural populations of I. ensata from eastern China (Longwangshan, Zhejiang Province) and northeastern China (Jinchuan, Jilin Province). There was no significant linkage disequilibrium found for any pair of loci. These loci contained between two and 12 alleles per locus across all 48 individuals of I. ensata. The number of alleles per locus varied from two to 10 at the population level and the observed and expected heterozygosities ranged from 0.167 to 0.958 and from 0.284 to 0.853, respectively. • Conclusions: These loci showed high levels of polymorphism and could be used to study the population genetic structure, genetic relationships, and phylogeography of I. ensata.     

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     

Isolation and characterization of 21 microsatellite loci in Lycium chinense and cross-amplification in Lycium barbarum

The present study reports isolation and characterization of 21 polymorphic microsatellite markers developed from Lycium chinense Mill. These markers produced a total of 86 alleles across 30 L. chinense accessions, with an average of 4.1 alleles per locus. Values for observed heterozygosity and polymorphism information content ranged from 0.03 to 0.81 (mean = 0.35) and from 0.03 to 0.78 (mean = 0.31), respectively. At the significance threshold (P < 0.05), 12 loci deviated from Hardy–Weinberg equilibrium, whereas significant linkage disequilibrium values were observed between 52 pairs of loci. All loci were successfully amplified for all L. barbarum accessions. These newly developed polymorphic microsatellite markers will be very useful for programming in the genetic conservation and classification of L. chinense and L. barbarum.     

Development and interspecific transferability of genic microsatellite markers in Spartina spp. with different genome size

Flow cytometry analysis showed variation of nuclear DNA content among different species of Spartina. Spartina alterniflora had the biggest genome (1763.9 Mbp) and S. cynosuroides had the smallest genome (756.35 Mbp), whereas the genomes of S. patens (969.36 Mbp) and S. spartinae (979.78 Mbp) were comparable. Mining simple sequence repeats (SSR) from 1227 expressed sequence tags (EST) generated from salt stressed S. alterniflora showed an abundance of di- and tri-nucleotide repeats. Of 100 ESSR (EST-derived SSR) loci with five or more repeats, 81 loci were successfully amplified in eight S. alterniflora genotypes and 15 (22.2%) ESSR markers were polymorphic. Eleven of the 15 polymorphic ESSRs showed amplification across six different species of Spartina while 100% cross transferability was observed with at least one species of Spartina. The average number of alleles per marker was 3.9 and 5.8 within S. alterniflora and among Spartina species, respectively. The ESSR markers discriminated different members within and between species of Spartina genus.     

Isolation and characterization of 13 microsatellite loci in Rhinolophus pusillus (least horseshoe bat) with cross-amplification in five related species

We used the enriched genomic library method to isolate and characterize dinucleotide microsatellite loci in the least horseshoe bat, Rhinolophus pusillus. Seventeen loci were obtained and tested on 31 individuals sampled from Guangxi Province in southern China. Thirteen of these markers were polymorphic with expected heterozygosity ranging from 0.821 to 0.909. A total of 164 alleles were detected and the number of alleles per locus ranged from 9 to 16 (mean 12.6). These polymorphic markers will be used to assess population structure in R. pusillus. In addition, successful cross-amplification in five congeneric bat species suggests most of these markers will also be useful for studying related species.     

Isolation of microsatellite loci from a rainforest tree,Elaeocarpus grandis (Elaeocarpaceae), and amplification across closely related taxa

Elaeocarpus grandis (Blue Quandong) is a common rainforest tree with a widespread distribution across northeastern Australia, while the related species E. sp. Rocky Creek and E. williamsianus are more restricted in their distribution. Highly polymorphic markers are required to provide genetic information on these species, which will aid in developing effective conservation and management strategies. Eight microsatellite loci were isolated and characterized in E. grandis, and tested in the rare species and other species of the Elaeocarpaceae. Cross‐species transferability was most successful within Elaeocarpus, with six and eight loci transferring to E. sp. Rocky Creek and E. williamsianus, respectively.     

Genomic and cDNA microsatellites from apricot (Prunus armeniaca L.)

We developed primers for the amplification of 24 polymorphic nuclear microsatellites in apricot (Prunus armeniaca L.). Thirteen loci originated from three genomic libraries enriched for TC, TG and AAG motifs. Eight loci were developed from three fruit EST (Expressed‐Sequence‐Tag) libraries and three from a leaf cDNA microsatellite‐enriched library. There were up to nine alleles per polymorphic locus in 12 different cultivars. No difference in allele numbers were shown between cDNA and genomic‐source loci. Mean expected heterozygosity was 0.65 (range: 0.15–0.87). Mendelian segregation was confirmed for all loci. These markers should be helpful for diversity studies, genome mapping and cultivar identification in apricot and related species.     

Microsatellite Loci from Lanyu Scops Owl (Otus elegans botelensis) and their Cross-Species Application in Four Species of Strigidae

We developed six new microsatellite markers containing tetranucleotide repeat motifs (GATA/CTAT) for Lanyu scops owl (Otus elegans botelensis) from an enriched partial library. All these loci are polymorphic and conform to Hardy–Weinberg equilibrium. We cross-species tested these and 12 other microsatellite primer pairs previously developed from O. elegans on four other species of owls (O. lettia, O. spilocephalus, O. scops, and Ninox scutulata). Results showed that the degree of polymorphism decreased with increasing phylogenetic distance to O. elegans. Most loci (66.7, 83.3, and 100%) were polymorphic in the three Otus owls but only five (27.8%) were polymorphic in N. scutulata. These microsatellites should be very useful genetic markers in studying the mating system, population genetics, and conservation of other little studied Old World Otus owls.     

Characterization of microsatellite loci developed for <Emphasis Type="Italic">Amaranthus hypochondriacus</Emphasis> and their cross-amplifications in wild species

The present study reports isolation and characterization of 12 polymorphic microsatellite markers for Amaranthus hypochondriacus. A total of 92 alleles were detected across the 20 accessions, with an average of 7.7 alleles per locus. The observed (H _O ) and expected (H _E ) heterozygosity values ranged from 0 to 0.95 and from 0.49 to 0.92, respectively. At significance threshold (P < 0.05), nine loci deviated from Hardy-Weinberg equilibrium (HWE), whereas significant linkage disequilibria (LD) were observed between five pairs of loci. The 12 loci were successfully amplified in 18 other amaranth species representing cultivated grain and vegetable species, their putative progenitors and wild species. These results demonstrate wide potential applicability of these markers for the study of intra- and inter-specific genetic diversity as well as evolutionary relationships among cultivated and wild amaranths.     

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.     

Development of microsatellite markers in the marine phytoplankton Karenia mikimotoi (Dinophyceae)

The marine phytoplankton, Karenia mikimotoi, causes severe red tides which are associated with mass mortality of marine fish, and have expanded their distributions in the coastal waters of western Japan. To assess the dispersal mechanism, a population genetic study using highly polymorphic genetic markers is one of the crucial approaches. Here we developed 12 polymorphic microsatellite markers from K. mikimotoi. These loci provide a class of highly variable genetic markers, as the number of alleles ranged from 5 to 23, and the estimate of gene diversity was from 0.551 to 0.933 across the 12 microsatellites. We consider these loci potentially useful for detailing the genetic structure and gene flow among K. mikimotoi populations.     

Microsatellite loci for great white herons and great blue herons (Aves,Ardeidae, Ardea herodias)

We used an enrichment technique to isolate 60 microsatellite loci in Ardea herodias. We developed primers for 17 loci, screened for variation in A. herodias and attempted to amplify these loci in three closely related species (A. alba, A. cinerea and A. cocoi). Fifteen loci were polymorphic in A. herodias. Observed heterozygosities ranged from 0.10 to 0.81. Two loci appeared to be monomorphic in A. herodias, but exhibited variation in product size among species within the genus. Our ability to amplify polymorphic products in closely related species suggests that these markers may be useful in other herons.     

An integrated molecular linkage map of diploid wheat based on a <Emphasis Type="Italic">Triticum boeoticum</Emphasis> × <Emphasis Type="Italic">T. monococcum</Emphasis> RIL population

Diploid A genome species of wheat harbour immense variability for biotic stresses and productivity traits, and these could be transferred efficiently to hexaploid wheat through marker assisted selection, provided the target genes are tagged at diploid level first. Here we report an integrated molecular linkage map of A genome diploid wheat based on 93 recombinant inbred lines (RILs) derived from Triticum boeoticum × Triticum monococcum inter sub-specific cross. The parental lines were analysed with 306 simple sequence repeat (SSR) and 194 RFLP markers, including 66 bin mapped ESTs. Out of 306 SSRs tested for polymorphism, 74 (24.2%) did not show amplification (null) in both the parents. Overall, 171 (73.7%) of the 232 remaining SSR and 98 (50.5%) of the 194 RFLP markers were polymorphic. Both A and D genome specific SSR markers showed similar transferability to A genome of diploid wheat species. The 176 polymorphic markers, that were assayed on a set of 93 RILs, yielded 188 polymorphic loci and 177 of these as well as two additional morphological traits mapped on seven linkage groups with a total map length of 1,262 cM, which is longer than most of the available A genome linkage maps in diploid and hexaploid wheat. About 58 loci showed distorted segregation with majority of these mapping on chromosome 2A^m. With a few exceptions, the position and order of the markers was similar to the ones in other maps of the wheat A genome. Chromosome 1A^m of T. monococcum and T. boeoticum showed a small paracentric inversion relative to the A genome of hexaploid wheat. The described linkage map could be useful for gene tagging, marker assisted gene introgression from diploid into hexaploid wheat as well as for map based cloning of genes from diploid A genome species and orthologous genes from hexaploid wheat.