Interspecific Amplification Research Articles

Microsatellite DNA markers for the commercially important fish Hypophthalmus donascimientoi (Pisces: Siluriformes: Pimelodidae) in the Amazon basin: isolation, characterization and amplification in congeneric species.

The genus Hypophthalmus comprises six species (H. edentatus, H. marginatus, H. fimbriatus, and H. oremaculatus), and the recently described: H. donascimientoi and H. celiae. The popular name for Hypophthalmus spp. in Brazil is mapará, this name refers to the six species. This group of fish has commercial importance for the states of Amazonas and Pará and, for this reason, requires studies to identify fish stocks. One approach is to use molecular markers, which have been very useful in studies with identification and population analysis of fish. Microsatellite molecular markers (SSRs) are one of the most informative markers for this purpose. There is little populations study of Hypophthalmus using SSRs, and there are less than six loci for the species Hypophthalmus marginatus available in the literature. With the construction of a genomic library of H. donascimientoi, we aimed to isolate and characterize SSRS markers and evaluate the extent of interspecific amplification. A genomic library was constructed with regions enriched of microsatellite for Hypophthalmus donascimientoi. A total of 126 contigs with 42 SSRs were used to design flanking primers for 39 microsatellites. Fifteen loci were characterized in three locations of the Solimões/Amazonas Rivers. The number of alleles ranged from one to 17 with a total of 126 alleles. The mean observed heterozygosity (HO) and expected heterozygosity (HE) were 0.721-0.692, respectively (S.d. HO 0.061 and HE 0.060). Two loci showed significant deviation in the HWE. The PIC ranged from 0.375 to 0.908. Such identified, 12 highly informative loci, and two moderately informative loci. Among the fifteen loci characterized, seven were successfully amplified in four other species of the genus. The microsatellite showed promise for estimating the genetic variability of H. donascimientoi and can be used as an efficient tool in population analyses of this species and in congeneric species analyzed.

Development of Microsatellite Markers for the Hermatypic Coral <i>Porites lutea</i>

By mining the EST and genomic microsatellite sequence resources of Porites species in GenBank, and using interspecific amplification and resequencing, we developed 20 microsatellite markers for the stony coral Porites lutea , meanwhile, validated and excluded 43 microsatellite markers of its symbiotic zooxanthella. These markers of P. lutea were genotyped in one wild population (n=16) from the southwest of Hainan Island fringing reefs. Nineteen of the 20 markers showed polymorphism. The number of alleles was 2~7 (mean 3.5±1.8), and the observed heterozygosity ( H o ) and expected heterozygosity ( H e ) were 0.06~0.94 (mean 0.34±0.29) and 0.18~0.83 (mean 0.57±0.20), respectively. Four markers remained deviated from the Hardy-Weinberg Equilibrium (HWE) after multiple comparison correction for Bonferroin ( P HWE < 0.05). This is the first set of microsatellite markers of P. lutea . They should be useful for the research on the genetic diversity and connectivity of this species and the conservation of endangered coral reef ecosystems and reef-building coral communities in the Indo-Pacific region.

Isolation and characterization of novel microsatellite markers for Bletilla striata and inter-specific amplification in 2 congeneric species

Bletilla striata (Orchidaceae) is an endangered plant species with high ornamental and medicinal value. It is important to acquaint the molecular proofs to protect this endangered species. To evaluate the genetic diversity and population structure of B. striata, 10 polymorphic microsatellites were developed from microsatellite-enriched libraries. Microsatellite loci polymorphism was tested across a total of 31 individuals. The number of alleles per locus both ranged from 4 to 11, with an average of 7.5 per locus. The observed and expected heterozygosities ranged from 0.3077 to 0.6923 and from 0.4908 to 0.8153, respectively. Cross-species amplification of all loci was successful among two congeneric species. The 10 microsatellite loci identified in this study can be utilized as novel markers for further investigation of conservation genetics of B. striata and its congeneric species.

Development of 12 polymorphic microsatellite loci in the high alpine perennial Primula halleri (Primulaceae).

• Premise of the study: Primula halleri is a high alpine endemic species with a patchy distribution in Central and Eastern European mountains. Little is known about the structure of genetic variation and mating system in populations of this species.• Methods and Results: We report on the development of 12 novel, polymorphic microsatellite loci for P. halleri. Tests for amplification and polymorphism were performed on 45 individuals sampled from three populations in the Swiss Alps. Interspecific amplification of the loci was evaluated using samples of the closely related species P. farinosa, P. frondosa, and P. scotica.• Conclusions: The results show that the new microsatellite loci will be useful to study the structure of genetic diversity and mating system of P. halleri, and possibly its closely related species.

Microsatellite primers for the endangered beech tree, Fagus hayatae (Fagaceae)

Microsatellite primers were developed for the endangered species Fagus hayatae (Fagaceae) to investigate the genetic diversity of the population and to investigate species delimitation within Fagus. Ten polymorphic simple sequence repeat markers were developed for F. hayatae using a magnetic bead enrichment method. The primers amplified trinucleotides, hexanucleotides, and complex repeats with six to 16 alleles per locus. The observed and expected heterozygosities across loci varied, with a range of 0.05-0.71 and 0.63-0.91, respectively. Most of the primers also amplified DNA from F. crenata, F. grandifolia, F. japonica, F. longipetiolata, F. lucida, F. orientalis, and F. sylvatica. These results indicate that these polymorphic loci of F. hayatae will be potentially useful for future studies of the population genetic diversity within the species. In addition, the interspecific amplification indicates that these transferable microsatellite markers will also be useful for future phylogeographic and speciation studies among close Fagus species.

Characterization of microsatellite loci from Litsea hypophaea (Lauraceae), a tree endemic to Taiwan

Microsatellite primers were developed for the endemic tree Litsea hypophaea (Lauraceae) in Taiwan to investigate its genetic diversity and population genetic structure and to investigate species delimitation within Litsea. Fifteen new simple sequence repeat markers were developed from L. hypophaea with a magnetic bead enrichment method. Most loci were also amplified from three closely related species, L. coreana, L. lii, and L. acutivena. The number of alleles and observed and expected heterozygosities across loci varied with a range of 1-25, 0.000-1.000, and 0.000-0.956, respectively. The application of these microsatellite markers of L. hypophaea provides a tool for understanding genetic diversity and population differentiation. In addition, interspecific amplification suggests that these markers will also be useful for species identification of related taxa within Litsea in Taiwan.

Characterization of 24 transferable microsatellite loci in four skullcaps (Scutellaria , Labiatae)

Transferable polymorphic microsatellite loci for four skullcaps, Scutellaria indica, S. taiwanensis, S. austrotaiwanensis, and S. playfairii, were developed for future studies of the mating system and population structure of these species. Interspecific amplification was also tested in various Scutellaria species. Twelve novel polymorphic microsatellite loci were isolated from four S. taiwanensis specimens, and seven are interspecifically transferable. Microsatellite loci developed from S. austrotaiwanensis in a previous study were also analyzed in the other three species, and 12 loci were found to be transferable. Allele numbers of the total 24 loci for S. indica, S. taiwanensis, S. playfairii, and S. austrotaiwanensis are two to four, two, two to five, and two to three, respectively, with an expected heterozygosity ranging from 0.114-0.661, 0.062-0.499, 0.280-0.730, and 0.268-0.662, respectively. The interspecies transferability of these 24 loci was further tested in another 10 Scutellaria species, including three species native to Taiwan. Seventeen loci were found to be interspecifically amplifiable, especially among the Taiwan native species. These highly polymorphic and transferable loci will be useful for future studies of the mating system of closely related Scutellaria species.

Isolation and characterization of 25 microsatellite DNA loci for Anopheles albitarsis sensu lato and inter-specific amplification in 5 congeneric species

The Anopheles albitasis complex includes 6 species, and 3 are considered as malaria vectors in Brazil. Twenty-five polymorphic microsatellite DNA loci were isolated and characterized in 24-36 individuals from the neighborhood of Puraquequara, Manaus, Amazonas State, Brazil. The number of estimated alleles ranged from 2 to 10, the observed heterozygosity ranged from 0.182 to 0.897, and the expected heterozygosity ranged from 0.260 to 0.854. Eleven loci showed significant deviation from Hardy-Weinberg equilibrium. Eleven loci were cross-amplified successfully in 5 Anopheles species. These microsatellite loci will be useful in studies investigating population structure and evolutionary genetics in A. albitarsis sensu lato and other A. albitarsis complex species.

Microsatellite cross-species amplification in the genus &lt;i&gt;Centaurea&lt;/i&gt; (Compositae)

Microsatellite cross-species amplification in the genus Centaurea (Compositae).- Microsatellites are widely used for population genetic studies although the development of these species-specific markers is costly and time-consuming. One strategy for saving time and money is the use of markers developed for one species (source species) in a different species (target species). This is known as cross-amplification. In the present work, two sets of microsatellites are used to test their transferability to six narrow endemic Centaurea species: i) 16 nuclear loci previously published for three congeneric species and ii) 10 universal chloroplast markers designed from Nicotiana tabacum sequences. Seventeen of the 26 markers tested were transferable and 14 of them were also polymorphic and therefore useful for future works. Nuclear markers were more variable and thus more informative than chloroplast markers. Interspecific amplification performed better for the nuclear loci developed for different Centaurea species than for the universal chloroplast markers developed for Nicotiana tabacum. Likewise, transferability was more successful for the species from sect. Phalolepis than for sect. Lepteranthus. Therefore, our results support the idea that the success of the cross-amplification is influenced by the evolutionary distance between the target and the source species.

Polymorphic chloroplast microsatellite markers in the octoploid Lepidium meyenii (Brassicaceae) and cross‐species amplification in Lepidium

As a crop and medicinal plant, the octoploid Andean endemic Lepidium meyenii suffers from taxonomic uncertainty. Few molecular markers are available to genotype individuals or track gene flow in wild and cultivated material. • Using available sequence data, eight cpSSR primer pairs were developed for L. meyenii. Levels of polymorphism checked in 56 individual L. meyenii, including cultivated and wild material, revealed that the number of alleles per locus ranged from three to five, and intrapopulation allele frequencies ranged from 0.071 to 1.0. Polymerase-chain-reaction screens using our cpSSR primers in 27 other Lepidium species and three Coronopus species suggested a high degree of interspecific amplification. • These polymorphic cpSSR markers should prove useful in characterizing genetic variation among cultivated and wild L. meyenii. Additionally, interspecific amplifications suggest that these markers will be useful for the study of related taxa.

Cross‐species amplification of Eucalyptus microsatellite loci

This study examined the interspecific amplification of nuclear microsatellite loci developed mainly for eucalypts in the subgenus Symphyomyrtus across five species within the second most speciose subgenus, subgenus Eucalyptus. A set of eight to 10 loci, depending on taxon, have been identified that are highly variable and easily scored. The successful transfer of microsatellite loci to these eucalypt species sidesteps the expensive and time-consuming development of species-specific microsatellite libraries. This primer set will enable the examination and cross-species comparison of the genetic resources of commercially and ecologically important members of the subgenus Eucalyptus.

Isolation and characterization of microsatellite markers from Hibiscus rosa-sinensis (Malvaceae) and cross-species amplifications

We report on the development of 10 microsatellite markers in Hibiscus rosa-sinensis (Hrs). Three markers were obtained from sequences available in GenBank and seven were isolated using a two-step ‘primer extension’ procedure, based on the microsatellite-AFLP (M-AFLP) technique. Polymorphism was explored in 21 Hrs genotypes representing the genetic variation within commercial varieties. Inter-specific amplification was assessed on 12 Hibiscus wild species. A total of 45 and 56 alleles (ranging from 1 to 10 for each locus) was amplified respectively from the 21 Hrs varieties and among the full Hibiscus spp. genotype set. Primers and conditions for polymerase chain reaction (PCR) amplification of the detected loci are reported.

Development of microsatellite loci for Pelophylax plancyi and cross-amplification in other ranid species

Thirteen polymorphic microsatellite loci were obtained from the gold-striped pond frog, Pelophylax plancyi. The number of alleles per locus ranged from 5 to 13 with an average of 8.38. The observed and expected heterozygosities (HO and HE) ranged from 0.533 to 0.890 and from 0.620 to 0.897, with averages of 0.686 and 0.751, respectively. In order to assess interspecific amplification, all primer pairs were tested with the same PCR conditions on three other Pelophylax species: Pelophylax hubeiensis, Pelophylax fukienensis, Pelophylax nigromaculata and two species from the genus Hylarana and Hoplobatrachus, respectively. All of these loci can be amplified successfully in the Pelophylax species, with eight loci amplified in all species tested.

Development and characterization of microsatellite markers in Abies firma and interspecific amplification in other Japanese Abies species

Abstract Abies firma is a dominant coniferous tree species endemic to Japan. We isolated eight microsatellite loci from needles of this tree species and tested their polymorphism among 26 A. firma individuals. Six of them showed polymorphism, with two to 16 alleles per locus. Their expected heterozygosities ranged from 0.075 to 0.922. Moreover, interspecific amplification among Abies sachalinensis, Abies mariesii and Abies veitchii was successful in majority of the isolated loci, suggesting that these loci may be useful for characterization of other fir species.

Interspecific amplification of tea tree (Melaleuca alternifolia-Myrtaceae) microsatellite loci-potential implications for conservation studies

This study investigated the interspecific amplification of 35 microsatellite loci developed for M. alternifolia across seven other species within the Myrtaceae. All the primers used gave successful amplification of loci in at least one of the species tested. The level of success varied between species; 88.6% of primers gave amplification products for Melaleuca spp., 74.3% for Callistemon salignus, 45.7% for Eucalyptus spp. and 25.7% for Backhousia citriodora. Sequencing of a number of amplification products confirmed the presence of microsatellites in those loci. This study shows that the development of species-specific microsatellite libraries might not always be necessary. Cross-species amplification could enable the application of microsatellite technology to studies with limited resources, a feature characteristic of conservation projects.