Genetic variation and phylogenetic relationship between two species of yellow catfish, <Emphasis Type="Italic">Horabagrus brachysoma</Emphasis> and <Emphasis Type="Italic">H. nigricollaris</Emphasis> (Teleostei: Horabagridae) based on RAPD and microsatellite markers

The two species of yellow catfish, Horabagrus brachysoma and H. nigricollaris are categorized as ‘endangered’ and ‘critically endangered’ respectively in their wild habitat. Proper knowledge of genetic structure and variability of these endangered species are highly essential for the management, conservation and improvement of fish stocks. Therefore, genetic variation and phylogenetic relationships between these species of yellow catfish sampled from Chalakkudy River in the hot spot of biodiversity-Western Ghats region, Kerala, India were analyzed by using Random amplified polymorphic DNA (RAPD) and microsatellite markers. 85 RAPD and five microsatellites loci were detected to analyze the genetic variation and phylogenetic relationships among these species. Out of 85 RAPD loci produced only 52.94% were polymorphic whereas in microsatellite, all 5 loci were polymorphic (100%). Species-specific RAPD bands were found in both species studied. In microsatellite, the number of alleles across the five loci ranged from 1 to 8. The observed heterozygosities in H. brachysoma and H. nigricollaris were 0.463 and 0.443, respectively. Here, both RAPD and microsatellite methods reported a low degree of gene diversity and lack of genetic heterogeneity in both species of Horabagrus which strongly emphasize the need of fishery management, conservation and rehabilitation of these species.     

Genetic variation and population structure of endemic yellow catfish, <Emphasis Type="Italic">Horabagrus</Emphasis><Emphasis Type="Italic">brachysoma</Emphasis> (Bagridae) among three populations of Western Ghat region using RAPD and microsatellite markers

Random amplified polymorphic DNA (RAPD) and microsatellite markers were applied to evaluate the genetic variation in endemic and endangered yellow catfish, Horabagrus brachysoma sampled from three geographic locations of Western Ghat, South India river systems. In RAPD, of 32 10-mer RAPD primers screened initially, 10 were chosen and used in a comparative analysis of H. brachysoma collected from Meenachil, Chalakkudy and Nethravathi River systems. Of the 124 total RAPD fragments amplified, 49 (39.51%) were found to be shared by individuals of all 3 populations. The remaining 75 fragments were found to be polymorphic (60.48%). In microsatellites, six polymorphic microsatellite loci were identified by using primers developed for Pangasius hypophthalmus, Clarias macrocephalus and Clarias gariepinus. The identified loci were confirmed as microsatellite by sequencing after making a clone. The nucleotide sequences of 6 loci were published in NCBI genbank. The number of alleles across the six loci ranged from 4 to 7 and heterozygosities ranged from 0.07 to 0.93. The mean number of alleles and effective number of alleles per locus were 5.00 and 3.314, respectively. The average heterozygosity across all investigated samples was 0.72, indicating a significant deficiency of heterozygotes in this species. RAPD and microsatellite methods reported a high degree of gene diversity and genetic distances depicted by UPGMA dendrograms among the populations of H. brachysoma.     

Cross-species amplification of 44 microsatellite loci developed for Chaetodon trifascialis, C. lunulatus and C. vagabundus in 22 related butterflyfish species

Forty‐four microsatellite primers developed for three species of butterflyfish were cross‐tested against 22 related confamilial species. Amplification success and cross‐species transferability of these markers were moderately high. Between 24 and 37 loci were amplified successfully in each species, with a mean success rate per species of 71.7% (± 1.8 SE). Rates of amplification success were comparable among primers designed for the three source species, ranging from a mean success rate per species of 16.9 loci (± 0.8 SE) for Chaetodon trifascialis source loci to 13.7 loci (± 1.5 SE) for C. vagabundus source loci. Polymorphism rates were high (76.1%± 3.1 SE of all successfully amplified loci), and 10 loci were polymorphic in all successfully amplified species (Tri14, B11, C5, D3, D113, D6, D117, D120, D111, D118). The number of alleles per polymorphic locus ranged from 2 to 8, and the average number of alleles across all polymorphic loci and all species was 3.6 (± 0.07 SE). Polymorphism rates were higher overall in primers designed for C. vagabundus (89.9%± 3.9 SE). Overall cross‐testing success was lowest for Heniochus chrysostomus, the most phylogenetically divergent species. The significant cross‐testing reported here provides a valuable resource that will enable population genetics studies to be undertaken on a range of butterflyfishes without the need for expensive and time‐consuming de novo microsatellite development.     

Isolation and characterization of microsatellite loci in the Pacific pleasure oyster,Crassostrea corteziensis,and their cross‐species amplification in four other oyster species

Microsatellite loci were isolated in Crassostrea corteziensis using (GT)_n, (CT)_n and (CTGT)_n‐enriched genomic libraries. Within each of 45 sequenced clones, an average of three microsatellite regions (156 total) were observed. Thirty‐three primers were designed, from which 11 microsatellite loci amplified. Ten of those were polymorphic, with a range of two to 30 alleles. Three loci were not in Hardy–Weinberg equilibrium, and linkage disequilibrium was found for six pairs of loci. These microsatellite loci will be further tested for segregation distortions and null alleles to establish a set for population genetic studies of the species in the Northwest coasts of Mexico, and for optimization of aquaculture development. Seven of the microsatellite loci cross‐amplified in Crassostrea palmula, a sympatric species, and will be useful in further genetic studies.     

Development of polymorphic microsatellite from RAPD bands of black sea bream Acanthopagrus schlegeli

Eight pairs of simple sequence repeat markers were developed from random amplified polymorphic DNA product in black sea bream Acanthopagrus schlegeli. Twenty microsatellites were selected for designing microsatellite primers, of which eight gave working primer pairs. They had between three and seven alleles. Observed and expected heterozygosities varied from 0.65 to 0.90, and from 0.58 to 0.82, respectively. Eight additional fish species assessed for cross‐species amplification revealed between two and five positive amplifications and between zero and three polymorphic loci per species.     

Identification of polymorphic microsatellite markers from RAPD product in turbot (Scophthalmus maximus) and a test of cross‐species amplification

Five polymorphic microsatellite loci have been isolated and characterized from random amplified polymorphic DNA product in turbot, Scophthalmus maximus. Twelve microsatellites were selected for designing microsatellite primers, of which five gave working primer pairs. They had between four and nine alleles. Observed and expected heterozygosities varied from 0.76 to 0.90 and from 0.63 to 0.83, respectively. Five additional fish species assessed for cross‐species amplification revealed between one and three positive amplifications and between zero and two polymorphic loci per species.     

Characterization of nuclear DNA microsatellite markers in the ant Cataglyphis cursor

The ant Cataglyphis cursor is exceptional in that unmated workers are potentially able to lay both male and female eggs. We characterized eight pairs of primers for microsatellite loci, developed from genomic DNA for this species. Variability was tested with DNA from 19 workers and all eight loci were highly polymorphic, displaying 5–10 alleles and a high level of heterozygosity. Cross‐species amplifications indicate that these microsatellites might be useful in genetic studies of other species belonging to the genus Cataglyphis.     

Cross-priming of microsatellite loci in subfamily cyprininae (family Cyprinidae): their utility in finding markers for population genetic analysis in three Indian major carps

This study is aimed to identify polymorphic microsatellite markers and establish their potential for population genetics studies in three carp (family cyprinidae; subfamily cyprininae) species, Labeo rohita, Catla catla and Cirrhinus mrigala through use of cyprinid primers. These species have high commercial value and knowledge of genetic variation is important for management of farmed and wild populations. We tested 108 microsatellite primers from 11 species belonging to three different cyprinid subfamilies, Cyprininae, Barbinae and Leuciscinae out of which 63 primers (58.33 %) successfully amplified orthologous loci in three focal species. Forty-two loci generated from 29 primers were polymorphic in these three carp species. Sequencing of amplified product confirmed the presence of SSRs in these 42 loci and orthologous nature of the loci. To validate potential of these 42 polymorphic loci in determining the genetic variation, we analyzed 486 samples of three focal species collected from Indus, Ganges and Brahmaputra river systems. Results indicated significant genetic variation, with mean number of alleles per locus ranging from 6.80 to 14.40 and observed heterozygosity ranging from 0.50 to 0.74 in the three focal species. Highly significant (P < 0.00001) allelic homogeneity values revealed that the identified loci can be efficiently used in population genetics analysis of these carp species. Further, thirty-two loci from 19 primers were useful for genotyping in more than one species. The data from the present study was compiled with cross-species amplification data from previous results on eight species of subfamily cyprininae to compare cross-transferability of microsatellite loci. It was revealed that out of 226 heterologous loci amplified, 152 loci that originated from 77 loci exhibited polymorphism and 45 primers were of multispecies utility, common for 2–7 species.     

Isolation and characterization of polymorphic microsatellite loci from an EST‐library of red sea bream (Chrysophrys major) and cross‐species amplification

Microsatellite markers have been developed from a complementary DNA (cDNA) library of red sea bream, Chrysophrys major. Twenty‐eight microsatellites were selected for designing microsatellite primers, of which 11 gave working primer pairs. Observed and expected heterozygosities varied from 0.33 to 1.00 and from 0.38 to 0.83, respectively. Five additional fish species assessed for cross‐species amplification revealed between one and six positive amplifications and between 0 and 6 polymorphic loci per species.     

Isolation and characterization of microsatellite markers in Labiotermes labralis (Isoptera,Termitidae, Nasutitermitinae)

We report here on the development of six polymorphic microsatellite loci for Labiotermes labralis. This soil‐feeding species is restricted to the Neotropical rainforest and then could represent a major candidate for being a bio‐indicator of anthropic disturbance resulting in the forest fragmentation. The microsatellite loci were isolated and characterized using a microsatellite‐enriched genomic library. The primers were tested on a French Guyanese population of L. labralis, represented by the sampling of one soldier in 22 nests. The primers were also tested against nine species of the same Nasutitermitinae subfamily and were successfully amplified at five loci in Armitermes minutus.     

Microsatellite loci for studies on population differentiation and connectivity of the red‐bellied yellow tail fusilier,Caesio cuning (Caesionidae)

Ten polymorphic microsatellites were isolated from the red‐bellied yellow tail fusilier Caesio cuning, a reef‐associated fish which occurs widely in the Indo‐Pacific region. The species is exploited by both small‐scale and commercial fisheries. Fifty individuals from six populations were genotyped using primers that reliably amplified 10 polymorphic loci. The number of alleles per locus ranged from three to 11. Observed heterozygosity (H_O) and expected heterozygosity (H_E) ranged between 0.400 to 0.880 and 0.337 to 0.843, respectively. These microsatellite loci may be used to study population structure, genetic diversity and connectivity of C. cuning in the range of its distribution.     

Isolation and characterization of polymorphic microsatellite DNA markers in the brown sole,Pleuronectes herzensteini

New microsatellite DNA markers from brown sole were developed and characterized. Fourteen primer sets were designed from 40 microsatellite regions. Eight of 14 loci exhibited variations comprising 8–31 alleles. Observed and expected heterozygosities ranged from 0.611 to 0.833 and from 0.647 to 0.968 among 36 individuals, respectively. Phz3, Phz8 and Phz12 significantly deviated from Hardy–Weinberg equilibrium, and there was a significant linkage disequilibrium between Phz2 and Phz12. Seven of eight loci conformed to the Mendelian manner of inheritance in a full‐sib family. Seven to four loci of three related species were cross‐amplified by primers for brown sole.     

Isolation and characterization of eight microsatellite loci from the endangered plant species Hypochaeris salzmanniana (Asteraceae)

We report the isolation and characterization of eight microsatellite loci for Hypochaeris salzmanniana, an endangered species endemic to the southwestern coast of Spain and the Atlantic coast of Morocco. A total of 32 alleles were detected across a sample of 45 individuals, with an average number of 4.0 alleles per locus. The average polymorphic information content (PIC) was 0.533 and the observed (H _O) and expected (H _E) heterozygosity values varied from 0.022 to 0.978 and from 0.434 to 0.759, respectively. Five loci exhibited significant deviation from Hardy–Weinberg equilibrium (P ≤ 0.001) and three pairs of loci showed significant linkage disequilibrium (P ≤ 0.01). The eight loci were tested for transferability in three others species (H. arachnoidea, H. glabra, and H. radicata) belonging to the same section of H. salzmanniana. With the exception of locus Hsalz7, all loci successfully amplified in the three species. These preliminary data confirm the usefulness of microsatellite markers for assessing the ecology and genetic structure of H. salzmanniana and to understand the evolution of species within the section Hypochaeris.     

Identification of microsatellite loci in Collinsia verna (Veronicaceae)

We developed eight polymorphic microsatellite loci for Collinsia verna (Veronicaceae). In a sample of 18–35 individuals from a single population, we found two to 15 alleles per locus (mean 8.3). We also tested these loci for cross‐amplification in all 22 species in the tribe Collinseae. Overall, more than half the species in the tribe amplified one microsatellite while three species most closely related to C. verna (Collinsia violacea, Collinsia parviflora and Collinsia grandiflora) amplified multiple microsatellite loci. These microsatellite loci will be used in future studies of mating system in this tribe and other quantitative genetic and population genetic studies.     

Improved genetic markers for monitoring recruitment dynamics in the endangered Mary River cod (Maccullochella mariensis)

The Mary River cod (Maccullochella mariensis) is a large predatory freshwater fish identified as a potential flagship for freshwater ecosystem conservation in Australia. The species is endemic to the Mary River catchment in eastern Australia, and is listed as Endangered. Previous conservation genetic assessment of Mary River cod was based on a small set of microsatellite loci developed for congeneric Maccullochella species. Here we develop a novel set of 15 microsatellite loci specific to M. mariensis, and demonstrate that these markers exhibit higher variability than those used previously. Mean number of alleles per locus was 4 and mean expected heterozygosity was 0.57. We genotyped 35 Mary River cod larvae belonging to a single cohort using the 15 novel loci and eight previously used loci, and found 10 full‐sib family groups along with clear genetic differentiation between individuals collected from the two sub‐catchments – Tinana Creek and Mary River. Microsatellites presented here will be useful for cost‐effective monitoring of genetic diversity and recruitment dynamics in this endangered fish species.     

The development of eight microsatellite loci in the wild daffodil Narcissus triandrus (Amaryllidaceae)

We report microsatellite primer pairs for the wild tristylous daffodil, Narcissus triandrus (Amaryllidaceae). From enriched libraries, we identified 58 unique microsatellite loci. We designed primer pairs for 27 of these loci and screened genomic DNA from 38 to 40 adults from a single population. For eight polymorphic loci, the number of alleles per locus ranged from five to 17. As six primers also amplified loci in three other Narcissus species, including two horticultural varieties, we expect that some of these markers will be transferable to other Narcissus species.     

Development and characterization of the first microsatellite markers in the mangrove species Pelliciera rhizophorae Triana & Planchon

Pelliciera rhizophorae is a unique Neotropical mangrove species belonging to Pelliciera genus. We isolated eight microsatellite loci from this species. All loci were polymorphic and showed three to nine alleles per locus in Colombian Pacific and Caribbean populations. Polymorphic information content ranged from 0.46 to 0.69. Two loci (PeRh‐14 and PeRh‐19) showed null alleles on the Caribbean coast, which suggest genetic differentiation between Pacific and Caribbean populations of P. rhizophorae. Development of these microsatellite loci constitutes a new molecular tool to carry out studies in the genome of the species and to evaluate its population dynamics.     

Characterization of microsatellites in the fungal plant pathogen,Sclerotinia sclerotiorum

Twenty‐five primers produced unambiguous amplification products of 23 microsatellite‐containing loci and two microsatellite‐like polymorphic loci, with 2–10 alleles at each locus in the plant pathogenic fungus, Sclerotinia sclerotiorum. Haplotypes are polymorphic among individuals sharing the same DNA fingerprint and DNA sequence haplotype, facilitating epidemiological monitoring worldwide. Fourteen of these primers also successfully amplified the closely related S. trifoliorum and S. minor.     

Isolation of polymorphic microsatellite loci in the genus Clusia (Clusiaceae)

Microsatellite flanking region sequences may provide phylogenetically useful information. We isolated 13 polymorphic microsatellite loci from two species, Clusia minor (five loci) and Clusia nemorosa (eight loci), to aid in the determination of phylogenetic relationships within the genus Clusia. Eleven loci amplified across all 17 Clusia species tested, while two loci amplified in 10 out of 17 species. The extensive cross‐species amplification suggests that these loci may be useful for an examination of phylogenetic relationships in this genus.     

Isolation and characterization of microsatellite markers for the clam Ruditapes philippinarum and cross-species amplification with the clam Ruditapes variegate

Ruditapes philippinarum and R. variegate are commercially important shellfish in Korea. In order to understand the processes and organization of genetic diversity and genetic resources for the sustainable management of fisheries resources we developed 13 primer pairs for microsatellite loci in R. philippinarum and tested their cross-amplification in R. variegate. Twelve primers amplified in both species. Nine loci were polymorphic in R. philippinarum and eight in R. variegate, each with nine to 26 alleles per locus. Unbiased expected heterozygosity levels varied from 0.73 to 0.94 in R. philippinarum. All polymorphic loci possessed species-specific alleles. No linkage disequilibrium was found. Results indicated that these microsatellite were highly polymorphic and will be useful for conservation genetics of both species.