Development and characterization of polymorphic microsatellite loci in Ficus altissima

Abalone, genus Haliotis, are single shelled nocturnal herbivorous marine gastropods. There are nearly 56 currently described species of haliotids worldwide. About 10% of these are commercially important as wild fisheries or aquaculture species. Significant progress had been made in abalone aquaculture in general but genetic improvement and restocking programmes are still in their infancy. A single tropical species, Haliotis asinina of the Indo-Pacific, is extensively harvested in Southeast Asia. H. asinina, with a shorter generation time than the temperate species and predictable spawning cycle, is well suited for aquaculture and an ideal species to investigate the molecular mechanisms regulating reproduction and growth in abalone. Molecular genetics and associated technologies have facilitated the analysis and understanding of the underlying genetics of commercially important traits in plants and animals. The genetic markers, both protein and DNA markers, have been found to be efficient in addressing genetic issues associated with management of fisheries and aquaculture. Among the available genetic markers, microsatellites are increasingly being used to answer genetic questions of fisheries and aquaculture. Microsatellites are short tandem repeats of 2-6 base pairs and are abundant in eukaryotic genome. These markers are highly variable, selectively neutral and co-dominantly inherited. Polymorphic nature of microsatellites is helpful in identification of quantitative trait loci, parentage and pedigrees and genetic diversity between populations. In abalone aquaculture, marker assisted selection for growth has the potential to enhance industry development, by identifying individuals and broodstock with genetic productivity for faster growth. The focus of this study is to develop polymorphic microsatellite DNA markers and demonstrate their application in H. asinina fisheries and aquaculture. A partial genomic library of H. asinina, enriched for microsatellite clones, was screened for dinucleotide microsatellite DNA using biotinylated (AC)18 primer. Isolated microsatellite-containing DNA were cloned and sequenced. Out of 27 microsatellite sequences, 7 contained microsatellite motifs and priming sequences for detailed studies on the natural and cultured populations. These microsatellite loci and four other loci that were isolated earlier by Sandie Degnan, were characterised using H. asinina samples from Heron Island Reef. All 11 microsatellite loci were polymorphic with the number of alleles ranging from 2-30 per locus and the observed and expected heterozygosity between 0.25-0.97 and 0.31-0.95 respectively. These primers were also able to amplify homologous loci in other tropical abalone species, H. ovina and H. varia.Five of these microsatellite markers were used to investigate the genetic structuring of natural populations of H. asinina in Australia and Southeast Asia. The allelic diversity and average heterozygosities were high at these loci in all the populations. Significant genetic differentiation was observed among all the wild populations analysed. Genetic structuring of H. asinina populations appears to follow isolation-by-distance model. These microsatellite loci were used to test whether the hatchery-reared abalone at SEAFDEC, the Philippines, could be used to restock some of the Philippines populations. The hatchery population did not show significant differentiation with one of the populations but showed a significant differentiation with the other. Based on this observation, suggestions are made regarding the use of hatchery-bred abalone for reseeding the wild populations in the Philippines. The implications for future restocking programmes are also discussed. In aquaculture, microsatellite DNA markers are used to genotype parental broodstock, to assess fertilization success and to maintain pedigree information for selective breeding. In this study individual H. asinina larvae were genotyped by analysing a suit of polymorphic microsatellite loci. At least 10 loci can be analysed from a single abalone veliger larvae. Five polymorphic loci were assayed to identify the parents of individual larvae produced in three separate crosses. In all cases, the parents of an individual veliger could be determined from as few as 3 loci. The microsatellite analysis revealed a substantial inequality in the contribution of each broodstock to the new generation of veligers, as only a subset of broodstock males sired most of the offspring. These observations suggest that highly controlled breeding practices may be required to ensure that the genetic diversity of an abalone population produced for aquaculture. The individual larval genotyping technique was used to test for sperm competition in abalone crosses. The sperm age was the predominant factor affecting the rate of fertilization success. A difference in fertilization success was evident between two males which spawned at the same time, which might be due to natural variation in viability or genetic differences. The microsatellite loci isolated and characterised in this study are found to be efficient genetic markers for management of H. asinina fisheries and aquaculture. These markers have the potential to be used in population structure studies and are polymorphic enough to reveal small genetic differences between populations. These markers could be used to make informed decisions on translocation of animals for reseeding and aquaculture purposes. The polymorphism, exhibited at these microsatellite loci, demonstrates their potential to be used as genetic tags to monitor the success of reseeding programmes and to maintain pedigree information. These microsatellites markers along with development of AFLP markers could be used to develop linkage and genetic maps for H. asinina to identify and characterise quantitative trait loci.

Microsatellite markers and clonal genetic structure of the fungal pathogen Phialophora gregata

Human activities often lead to the disturbed and fragmented habitat and consequently threated species. Dipterocarpus dyeri (Dipterocarpaceae) is widely distributed in lowland rainforests in southeast Vietnam. Due to over- exploitation and habitat destruction in the 1980s and 1990s, the species is listed as threatened. Understanding the genetic variation and mating rate within D. dyeri population that occurs in forest patches is necessary to establish effectively conservation strategies for this species. To conserve the species in tropical forests, genetic diversity and mating rate were investigated on the basis of eight microsatellites (single sequence repeat, SSR). All of the eight loci were polymorphic. A total of 33 different alleles were observed across the screened loci. The SSR data indicated high genetic diversities (NA = 4.1; HO = 0.461 and HE = 0.543) and the inbreeding value was high (0.185). The mating system parameters were determined using the mixed mating model and indicated high outcrossing rates (tm = 0.884 and ts = 0.645). The differences of significant tm - ts value indicated that inbreeding contributed to selfing rate for this species in lowland tropical forests of Ma Da. This study also indicated the importance of conserving the genetic resources of Dipterocarpus dyeri species in Ma Da rainforests. Genetic conservation should derive from correlated outcrossing. The conservation strategy should be established an ex-situ conservation site with new big population for this species from all genetic groups, which might improve its fitness under different environmental stresses.

Isolation and characterization of fifteen polymorphic microsatellite loci in the threatened freshwater mussel Solenaia oleivora (Bivalvia: Unionidae)

BackgroundMicrosatellite markers have proven useful in genetic studies in many organisms, yet microsatellite-based studies of the dengue and yellow fever vector mosquito Aedes aegypti have been limited by the number of assayable and polymorphic loci available, despite multiple independent efforts to identify them. Here we present strategies for efficient identification and development of useful microsatellites with broad coverage across the Aedes aegypti genome, development of multiplex-ready PCR groups of microsatellite loci, and validation of their utility for population analysis with field collections from Haiti.ResultsFrom 79 putative microsatellite loci representing 31 motifs identified in 42 whole genome sequence supercontig assemblies in the Aedes aegypti genome, 33 microsatellites providing genome-wide coverage amplified as single copy sequences in four lab strains, with a range of 2-6 alleles per locus. The tri-nucleotide motifs represented the majority (51%) of the polymorphic single copy loci, and none of these was located within a putative open reading frame. Seven groups of 4-5 microsatellite loci each were developed for multiplex-ready PCR. Four multiplex-ready groups were used to investigate population genetics of Aedes aegypti populations sampled in Haiti. Of the 23 loci represented in these groups, 20 were polymorphic with a range of 3-24 alleles per locus (mean = 8.75). Allelic polymorphic information content varied from 0.171 to 0.867 (mean = 0.545). Most loci met Hardy-Weinberg expectations across populations and pairwise FST comparisons identified significant genetic differentiation between some populations. No evidence for genetic isolation by distance was observed.ConclusionDespite limited success in previous reports, we demonstrate that the Aedes aegypti genome is well-populated with single copy, polymorphic microsatellite loci that can be uncovered using the strategy developed here for rapid and efficient screening of genome supercontig assemblies. These loci are suitable for genetic and population studies using multiplex-PCR.

A total number of 76 randomly selected birds i.e. 20 of Hill fowl (HF), 14 of Rhode Island Red (RIR), 14 of Kadaknath (KN), 14 of White LeghWDDorn (WLH) and 14 of White Cornish (WC) were genotyped using 25 microsatellite markers in the present study. Out of 25 microsatellite loci, 17 (~70%) were found to be polymorphic among these breeds. Across the breeds, total number of alleles ranged from 2 to 3 at polymorphic locus and average number of alleles per locus was 2.41. The allele size ranged between 98 bp and 340 bp. Among all the polymorphic microsatellite loci, observed heterozygosity were 0.441, 0.415, 0.287, 0.296 and 0.376 and expected heterozygosity were 0.435, 0.443, 0.387, 0.384 and 0.442 in HF, RIR, KN, WLH and WC, respectively. Polymorphic Information Content (PIC) estimates, across all the polymorphic loci were 0.346, 0.342, 0.305, 0.297 and 0.330 in HF, RIR, KN, WLH and WC, respectively.

Landscape alterations have strong impacts on the distribution of genetic variation within and between populations, and understanding these effects can provide insights for design conservation strategies. We analyzed 20 georeferenced individuals of pudu deer (Pudu puda) of three populations from southern Chile to obtain genetic diversity and distance parameters based on five polymorphic microsatellite loci. The genetic population structure was assessed by using STRUCTURE 2.3.4 and the effective population sizes (Ne) with NeEstimator 2.0 software. Habitat fragmentation metrics were also analyzed to correlate these data with the genetic variation focused to assess the effect of native forest fragmentation on the genetic structure of pudu deer. The mean number of alleles ranges from 2.8 to 4.2, while the mean number of private alleles ranged from 0.4 to 1.2. The mean observed heterozygosity ranged from 0.423 to 0.547. All populations displayed significant deficits of heterozygotes (FIS = 0.091-0.189, P < 0.0001). The FST on the whole populations and loci was low (mean FST = 0.0942) and without statistical significance (P > 0.05), indicating no genetic differentiation among populations. The STRUCTURE analysis did not reveal a population structure, and the NeEstimator found a markedly low Ne in a population compared to other analyzed populations. Multiple regression analysis indicates that two fragmentation metrics, namely, the proportion of the landscape covered by forest (PLAND) and the patch density (PD), negatively and significantly affected the number of alleles per locus as well as the observed heterozygosity (PLAND, β = -0.8740, P < 0.05; PD, β = -0.6594, P < 0.05). This result suggests that a greater degree of habitat fragmentation contributes to a decrease in genetic variation of pudu deer. Therefore, maintaining the native forest continuity across the distribution range of pudu deer could contribute to preserving the genetic diversity, enhancing the long-term survival of this mammal.

Mating systems have important implications for understanding the population and evolutionary dynamics of species. However, most theoretical and empirical work on vertebrate mating systems has focused on bird and mammalian species, and the mating systems of amphibian remain poorly understood. To better understand the mating systems of amphibian, in this study we describe the isolation and characterization of ten polymorphic microsatellite loci in the Leishan moustache toad (Vibrissaphora leishanensis) from genomic DNA-enriched libraries. The number of alleles per polymorphic locus in 24 individuals ranged from 2 to 15, the average observed and expected heterozygosity per locus ranged from 0.333 to 0.875 and from 0.284 to 0.916, respectively. All polymorphic loci followed Hardy–Weinberg equilibrium. No evidence of significant linkage association between locus pairs was found. These polymorphic loci will be useful in studying mating systems of the Leishan moustache toad.

The isolation and development of 15 polymorphic dinucleotide microsatellite loci were described for Ophicephalus argus from the Huaihe River. All loci were polymorphic in the 30 individuals tested. The number of alleles per variable locus ranged from nine to seventeen, with a mean of 12.00. These novel microsatellite loci showed high level of polymorphism. Observed and expected heterozygosities ranged from 0.793 to 0.929 and from 0.841 to 0.952, respectively. Two loci were found deviated from HWE in the sampled population after Bonferroni correction. These microsatellite loci will be useful for revealing population structure, genetic diversity, and phylogeography of Ophicephalus argus.

Research objective To conduct a comparative analysis of the genetic diversity of the Nenets reindeer breed (Rangifer tarandus tarandus) in Russia using a new microsatellite panel. Material and methods used In total, 228 individuals from four populations of the Nenets (47 samples), Yamalo-Nenets (151), and Khant-Mansi (30) regions of the Russian Federation were studied. The new panel consists of 16 microsatellite loci: 10 dinucleotide loci - Rt6, BMS1788, Rt30, Rt1, Rt9, Rt7, Rt24, FCB193, BMS745, NVHRT16 and 6 tetranucleotide STR markers - OheQ, C217, C32, T40, C276, C143. GeneMapper ID-X 1.4 software (Applied Biosystems) used to analyze the obtained data. The statistical analysis was performed in the R package, 3.6.1 version (R Core Team, 2019). Research results For 16 microsatellite loci 132 alleles were found, from 2 alleles (locus C217) to 16 alleles (locus OheQ). The average number of alleles per locus for all samples was 8.25. The smallest number of alleles per sample was observed in the samples of reindeer from the Nen.NAO and Nen.KhMAO, 103 alleles, and the largest from the Nen.YaNAO, 119. Allelic richness (Ar) ranged from 5.85 to 6.25. For each sample, the distribution of individual inbreeding coefficients f was constructed. We can talk about the presence of inbreeding in all samples, since the high values of f indicate a decrease in the heterozygosity of individual genomes in the samples due to inbreeding. The Polymorphism Information Content (PIC) values were calculated for each pair of “locus-sample” which were estimated based on the number and frequency of alleles at the locus. The average PIC value turned out not to be very high: 0.613 ± 0.049. The highest levels of polymorphism were obtained for 3 loci: Rt9 (PIC = 0.906), OheQ (PIC = 0.913), and Rt1 (PIC = 0.920). It is proposed to investigate the polymorphism of microsatellite loci of all four Russian reindeer breeds. The following will be determined for each breed: genetic structure, its effective number, inbreeding coefficient; the degree of differentiation between the breeds has been established. As a result of a detailed analysis we will build a model for the demographic history of the reindeer.

Isolation and characterization of thirteen polymorphic microsatellite loci for Lithocarpus glaber

Stone flounder (Kareius bicoloratus) is a rare fish species in China. Here, we reported 11 polymorphic microsatellite loci isolated from a repeat-enriched genomic library of stone flounder. The number of alleles, observed and expected heterozygosity per locus in 32 individuals ranged from 3 to 6, from 0.1613 to 0.8667 and from 0.1549 to 0.7932, respectively. Two loci significantly deviated from Hardy–Weinberg equilibrium after Bonferroni correction and no significant linkage disequilibrium between pairs of loci was found. Cross-species amplification of these microsatellite loci in additional three fish species was performed. These polymorphic microsatellite loci would be useful for investigating genetic population structure and construction of genetic linkage map in Kareius bicoloratus.

Anopheles (Nyssorhynchus) triannulatus is a complex of 3 species. Thirteen polymorphic microsatellite loci were isolated and characterized in 20 to 25 individuals from Manaus (AM, Brazil). The number of alleles per locus varied from 3 to 10 (mean = 6.0). The observed heterozygosity ranged from 0.250 to 0.875 (mean = 0.680) and expected heterozygosity ranged from 0.376 to 0.844 (mean = 0.698). Two loci exhibited null alleles and all loci were in Hardy-Weinberg equilibrium. No linkage disequilibrium between loci was observed. These loci were used in 4 congeneric species and provide a useful tool for studying population genetics and other aspects of the biology of this and other Anopheles species.

Low-density species are species that occur with a low density of mature individuals. They are the most common species in tropical forests. Knowledge and protection of low-density species are key tobiodiversity conservation in tropical forests. The meanings of low-density species is broader than that of rare species. Low-density species also include species that have low density, wide distribution range and a huge number of mature individuals. Low survivorship of seeds and seedlings near the parent individual is an important mechanism of creation of low-density species. Low-density species probably arise from intraspecific density-dependent effects and limited population recruitment. The possible mechanisms of maintenance of low-density species lie in both external factors, such as capacities of insect pollinating, and inner factors, including flower number and flowering duration of tropical plants. Some tropical species are self-fertilized, compensating for the decline in opportunity for cross-breeding due to extreme low-density. Up to now, few studies have been carried out in this field. We should design more detailed research in combination with research on the properties of tropical forests, so that we can provide a theoretical basis and practical advice for biodiversity conservation in tropical forests of our country.

Logged forests cover four million square kilometers of the tropics, capturing carbon more rapidly than temperate forests and harboring rich biodiversity. Restoring these forests is essential to help avoid the worst impacts of climate change. Yet monitoring tropical forest recovery is challenging. We track the abundance of early-successional species in a forest restoration concession in Indonesia. If the species are carefully chosen, they can be used as an indicator of restoration progress. We present SLIC-UAV, a new pipeline for processing Unoccupied Aerial Vehicle (UAV) imagery using simple linear iterative clustering (SLIC)to map early-successional species in tropical forests. The pipeline comprises: (a) a field verified approach for manually labeling species; (b) automatic segmentation of imagery into “superpixels” and (c) machine learning classification of species based on both spectral and textural features. Creating superpixels massively reduces the dataset's dimensionality and enables the use of textural features, which improve classification accuracy. In addition, this approach is flexible with regards to the spatial distribution of training data. This allowed us to be flexible in the field and collect high-quality training data with the help of local experts. The accuracy ranged from 74.3% for a four-species classification task to 91.7% when focusing only on the key early-succesional species. We then extended these models across 100 hectares of forest, mapping species dominance and forest condition across the entire restoration project.