Development and characterization of microsatellite loci in the sub-canopy tree Chengiopanax sciadophylloides (Araliaceae) using next-generation sequencing technology

Chengiopanax sciadophylloides is a deciduous broad-leaved sub-canopy tree species that is endemic to Japan and widely distributed in its temperate forests. Recent analyses indicate that climate change will reduce its distributional range and cause habitat fragmentation, leading to a loss of genetic variation. Here we describe the development of 15 microsatellite markers for this species using next-generation sequencing techniques. A total of 44 individuals from two populations were genotyped at each marker. The number of alleles per locus ranged from two to 13 with a mean of 4.8, and the expected heterozygosity ranged from 0.215 to 0.895 with a mean of 0.560. None of the loci exhibited linkage disequilibrium or any deviation from Hardy–Weinberg equilibrium. Because these 15 microsatellite markers exhibit high degrees of genetic variation, they should be useful tools for studying the conservation genetics of C. sciadophylloides.     

Development and characterization of 15 polymorphic microsatellite loci in Sonneratia alba (Lythraceae) using next-generation sequencing

A set of 15 new microsatellite loci was developed and characterized for the widespread mangrove tree species Sonneratia alba (Lythraceae) by using next-generation sequencing. Forty-eight S. alba samples from seven populations in the Indo-West Pacific region were genotyped; all loci were polymorphic, with the number of alleles ranging from three to eight. The mean observed heterozygosity per locus was 0.21 for a population from Sabah, Malaysia. No null allele or significant linkage disequilibrium was detected, indicating the robustness of the markers. Only one locus (SA103) showed deviation from Hardy?CWeinberg equilibrium. As characterization of these microsatellite loci was done with samples covering most of the species?? distribution range, the markers can be applied to genetic diversity studies over the broad geographical range of the species.     

Development and characterization of 14 microsatellite loci in the beach wolf spider (Arctosa littoralis), using next-generation sequencing

The beach wolf spider, Arctosa littoralis, inhabits areas near water. A technique based on 454 sequencing was used for obtain microsatellite loci for the wolf spiders. Fourteen polymorphic microsatellites were designed and tested as specific markers for the species. All markers were genotyped on 36 individuals from Las Playitas on the Baja California Peninsula. We observed low to medium genetic variation across most loci (with a mean of seven alleles per locus). Expected heterozygosity across all markers was considered intermediate (mean He = 0.57, range 0.05–0.91). These loci are the first to be characterized explicit for A. littoralis and should be useful for investigation of population structure and gene flow in this species.     

Development of microsatellite markers for the Asian Stubtail Urosphena squameiceps by using next-generation sequencing technology

The Asian Stubtail Urosphena squameiceps winters in Southeast Asia which is facing a serious threat from deforestation and the species is categorized as a threatened species at some of the breeding populations. Monitoring population dynamics of the species in the field is difficult because they are secretive and prefer dense undergrowth. Therefore, developing genetic markers from the species is useful for detecting appropriate conservational management units and to understanding their unique breeding system. We isolated and characterized 13 microsatellite loci of the Asian Stubtail by using a next-generation sequencer. The number of alleles and the expected heterozygosity at each locus were 2–26 (mean 10.31) and 0.14–0.92 (mean 0.64), respectively. The combined first-parent non-exclusion probability was <0.005.     

Development and characterisation of 20 novel microsatellite markers for the little blue penguin (Eudyptula minor) using next-generation sequencing

Conservation Genetics Resources - The world’s smallest penguin, Eudyptula minor, exhibits substantial mtDNA differentiation among populations, suggesting the possibility of multiple taxa. We...     

Development of microsatellite markers for grasscutter (Thryonomys swinderianus, RODENTIA) using next-generation sequencing technology

The grasscutter (Thryonomys swinderianus) is a hystricomorph rodent that inhabits mainly Sub-Saharan Africa and its meat is highly relished by most people in this region. The animal is hunted in the wild using techniques that are sometimes detrimental to the environment. It is therefore being domesticated to make the meat readily available to forestall the negative consequences of hunting. There are currently no genetic markers available and we report here, 33 novel microsatellite markers developed using next-generation sequencing technology. The number of alleles ranged from 3 to 11 (mean 6.4) while the observed and expected heterozygosities ranged from 0.188 to 1.000 (mean 0.591) and 0.322?C0.873 (mean 0.713), respectively. Cumulative probability of identity (PI) for all loci was very low (PI?=?3.1?×?10^?33), indicating that these markers can be used for individual identification in the domestic setting. They will also be useful to study the genetic diversity of the grasscutter in the wild for population monitoring and conservation.     

New microsatellite loci isolated via next-generation sequencing for two endangered pronghorn from the Sonoran Desert

We isolated 16 novel microsatellite loci in two subspecies of endangered desert pronghorns (Antilocapra americana sonoriensis and Antilocapra americana peninsularis) using a shotgun pyrosequencing approach. All and 87.5 % of the loci were polymorphic within each subspecies, respectively. The mean number of alleles per locus was 4.86 (range 2–8) and 2.5 alleles per locus (range 1–4 alleles), and observed heterozygosity ranged from 0.13 to 0.78 (mean 0.48) and 0.00 to 0.61 (mean 0.31), respectively. We did not find significant linkage disequilibrium among loci pairs and only one locus deviated significantly from Hardy–Weinberg equilibrium in peninsularis.     

Development of 11 polymorphic microsatellite markers for Xylocarpus granatum (Meliaceae) using next-generation sequencing technology

Human impacts have seriously damaged mangroves, and conservation of mangroves will require information on local and regional population genetic structures. Here, we report the development and polymorphism of eleven novel microsatellite markers, developed using next-generation sequencing on 56 samples of widespread mangrove species Xylocarpus granatum (Meliaceae) from nine populations across the Indo-West Pacific region. All loci were found to be polymorphic, with the number of alleles per locus ranging from four to 19. In a population from Sabah (Malaysia), the mean observed and expected heterozygosity per locus was 0.59 and 0.58, respectively. No null allele, significant linkage disequilibrium or deviation from Hardy–Weinberg equilibrium was detected among all loci. The eleven markers developed can be valuable tools to conservation genetics of this species across its distributional range.     

Development of 38 microsatellite loci from the Arceye hawkfish, Paracirrhites arcatus, using next-generation sequencing and cross-amplification in other Cirrhitid species

The Arceye hawkfish, Paracirrhites arcatus, exhibits two distinct color morphotypes found sympatrically throughout the Indo-Pacific. To provide tools for addressing evolutionary and management questions, we used 454 sequencing to construct a microsatellite-enriched genomic library for both P. arcatus morphotypes. Thirty-eight polymorphic microsatellites were isolated and characterized. We found a mean of 10.47 and 10.94 alleles per locus and observed levels of heterozygosity from 0.208?C0.958 to 0.333?C1.000 for the pink-white-striped and melanistic morphotypes, respectively. Most loci conformed to Hardy?CWeinberg equilibrium expectations with no significant evidence of null alleles or linkage disequilibrium. Five multiplex PCR panels were designed and optimized. Most loci reliably amplified when cross-tested with Paracirrhites forsteri and Cirrhitus pinnulatus. We expect these markers to be useful tools for these species and potentially related taxa.     

Development of 31 polymorphic microsatellite markers for guinea fowl (Numida meleagris) using next-generation sequencing technology

Though efforts to domesticate guinea fowl (Numida meleagris) have been productive, both feral and wild populations continue to decline due to habitat fragmentation and heavy hunting, increasing the need to monitor and sustain this species. This study reports the first 31 original polymorphic microsatellite markers for guinea fowl by the next-generation sequencing technology. Observed (H _O ) and expected heterozygosities (H _E ) ranged from 0.033 to 1.000 (mean 0.396) and 0.033 to 0.799 (mean 0.419), respectively. Number of alleles ranged between 2 and 9 per locus (mean 3.387). Nine loci significantly deviated from Hardy–Weinberg Equilibrium (p < 0.05), after Bonferroni correction. Null allele frequency was less than 0.2 in all loci except locus Nmg22 (0.3). Results from this study can serve as baseline information for genetic diversity studies and also inform breeding strategies for the improvement and conservation in both domestic and wild populations of the species.     

Characterization of microsatellite loci for a threatened species, the King Rail, Rallus elegans, using a next-generation sequencing protocol

The King Rail Rallus elegans (Audubon) has experienced population declines of 4.6 % per year on average since the 1960s. Wetland loss, most severely affecting inland marshes, has significantly reduced this species’ distribution to the coastal margins of its historic range. Polymorphic microsatellite markers were generated by 454 pyrosequencing of genomic DNA from King Rails, and Clapper Rails R. longirostris from Louisiana after AFLP enrichment and barcoding of restriction fragment cut sites across individuals. Of 1,419 microsatellite-containing sequences, 20 hypervariable microsatellite loci with up to 20 different alleles were identified at the alignment stage. We characterized nine loci, tested variability in 45 Atlantic coast King Rail samples, and detected 4–19 alleles per locus. Cross-species amplification revealed variability in the Virginia Rail, R. limicola, and Sora, Porzana carolina. These loci will be useful for studying secretive marsh rails, many of which are threatened or endangered.     

Isolation and characterization of 21 polymorphic microsatellite loci in the iconic Australian lungfish, Neoceratodus forsteri, using the Ion Torrent next-generation sequencing platform

We isolated and characterized 21 microsatellite loci in the vulnerable and iconic Australian lungfish, Neoceratodus forsteri. Loci were screened across eight individuals from the Burnett River and 40 individuals from the Pine River. Genetic diversity was low with between one and six alleles per locus within populations and a maximum expected heterozygosity of 0.774. These loci will now be available to assess effective population sizes and genetic structure in N. forsteri across its natural range in South East Queensland, Australia.     

Development of microsatellite markers for the endangered grassland species Vincetoxicum pycnostelma (Apocynaceae) by using next-generation sequencing technology

Vincetoxicum pycnostelma (Apocynaceae) is distributed widely in East Asia; however, over the last several decades, the number of populations of the species in Japan has been rapidly decreasing because of the loss of its grassland habitat. Existing population sizes are extremely small. Here, we isolated and characterized 9 microsatellite loci of V. pycnostelma by using a next-generation sequencer. The number of alleles and the expected heterozygosity at each locus were 5?C22 (mean, 8.7) and 0.41?C0.92 (mean, 0.74), respectively. The markers described in this study will be useful for parentage analysis of V. pycnostelma (combined first-parent non-exclusion probability?<0.005) and conservation genetic studies such as evaluating the genetic diversity within and between the remaining populations.     

Characterization of sixteen polymorphic microsatellite DNA loci in the chum salmon (Oncorhynchus keta) isolated by next-generation sequencing

Conservation Genetics Resources - Chum salmon (Oncorhynchus keta) is an important fisheries resource around the North Pacific region. However, the genetic characteristics of this species mostly...     

Development of microsatellite markers for Astrotrichilia asterotricha (Meliaceae), an endemic tree in Madagascar, using next-generation sequencing technology

Astrotrichilia asterotricha is an endemic large-seeded tree in Madagascar, which depends on large-bodied frugivorous primates for seed dispersal. Because such primates are threatened by habitat destruction and hunting, seed dispersal and regeneration of A. asterotricha are vulnerable to human activities due to the loss of seed dispersers. We developed 19 microsatellite markers using next-generation sequencing technology. The number of alleles ranged from two to nine, and the ranges of observed and expected heterozygosities were 0.158–0.950 and 0.261–0.851, respectively. The cumulative probability of identity for all loci (excluding Ast01) was very low (8.6 × 10^?11), indicating that these markers can be used for individual discrimination. They will be useful for examining gene flow via seed dispersal by animals, as well as the regeneration dynamics of A. asterotricha, in Malagasy forests.     

High-throughput microsatellite marker development in Amur catfish (Silurus asotus) using next-generation sequencing

The Amur catfish, Silurus asotus, is an economically important fish species in East Asia, but few genetic studies have been conducted on this species, especially those based on nuclear markers. Here, we isolated and characterized 47 novel polymorphic microsatellite loci in the genome of S. asotus using 454 sequencing. We screened 70 primers and 48 of them generated amplification products. Forty-seven of the amplification products were polymorphic in a population of 40 collected from the upper Yangtze River. The number of alleles varied from 3 to 15, and the observed and expected heterozygosities varied from 0.300 to 0.800 and 0.305 to 0.866, respectively. The average polymorphic information content (PIC) of all loci was 0.682, indicating high levels of polymorphism. In addition, cross-species amplification in a congener species, Silurus meridionalis showed a high level of transferability (79.2 %), which confirmed that the microsatellite markers developed here could be used effectively for other related catfish species.     

The discovery of microsatellite markers for Hardenbergia violacea (Fabaceae), using next-generation sequencing

Hardenbergia violacea is a climbing to prostrate shrub endemic to woodlands in the south east of Australia. We used next generation sequencing to develop a species-specific primer set. We obtained 106,529 reads in FASTA format with an average read length of 548?bp. DNA was successfully amplified for 20 markers and eight markers were characterized. The degree of polymorphism of these loci was tested on 95 samples from five populations. The total number of alleles ranged from two to 11 and observed and expected heterozygosities ranged from 0.0 to 0.807 and 0.0 to 0.933, respectively. Inbreeding coefficients were highly variable among populations, suggesting a mixture of selfing and outcrossing in nature. The characterized loci will be useful for studies of population structure, gene flow, mating systems, and the conservation and selection of provenances for restoration projects in degraded habitats.     

Isolation and characterisation of ten microsatellite markers for the tetraploid Stypandra glauca R.Br. (Hemerocallidaceae) identified using next generation sequencing

Ten polymorphic microsatellite loci were developed for the tetraploid plant species Stypandra glauca which is common on granite outcrops in Western Australia. Amplification and genotyping trials were conducted on 48 individuals from two sampling localities. All 10 loci revealed multi-banding patterns with up to 4 bands visible in individuals consistent with tetraploidy. The number of alleles per locus ranged from 6 to 32 (mean = 12.5). The proportion of observed heterozygotes at each locus ranged from 0.32 to 0.98 (mean = 0.73). These polymorphic microsatellite markers will facilitate further analysis of population genetic structure and connectivity in Stypandra glauca.     

Development of 16 microsatellite markers in Eusideroxylon zwageri by next-generation sequencing

The Ulin, Eusideroxylon zwageri, is a useful tropical tree now threatened by excessive logging. We used next-generation sequencing to isolate and characterize 16 polymorphic and codominant microsatellite (simple sequence repeat) markers in 32 E. zwageri trees in east Kalimantan, Indonesia. The number of alleles for each locus ranged from 2 to 19 and observed and expected heterozygosities ranged from 0.13 to 0.91 and from 0.18 to 0.92, respectively. These microsatellites may be useful for genetic studies and conservation of E. zwageri.     

Development of microsatellite markers for the near threatened eagles Harpia harpyja and Morphnus guianensis using next-generation sequencing

We isolated and characterized 11 microsatellite loci for Harpia harpyja using the IonTorrent PGM. The loci were validated in 22 Harpia harpyja and 16 Morphnus guianensis individuals. Ten of the loci were variable in H. harpyja, ranging from two to six alleles per locus. Seven of the loci were variable in M. guianensis ranging from two to six alleles per locus. Genotypes did not deviate from Hardy–Weinberg equilibrium expectation, and no linkage disequilibrium was observed. These markers have a promise to provide an important resource for genetic population studies, conservation and monitoring of these two near threatened accipitrid species.