Ten novel microsatellite markers for the freshwater sleeper Micropercops swinhonis (Günther, 1873), with testing of cross‐species amplification in two other fishes from suborder Gobioidei

Ten novel polymorphic microsatellite loci were developed for the freshwater sleeper Micropercops swinhonis, a relatively abundant and broadly distributed lake‐dwelling fish in Asia, using next generation sequencing. These markers were tested in a population of 48 individuals, and characteristics suggest that they may be useful for population genetic studies. The average number of alleles per marker was 6.6 (range: 2–26), the average expected heterozygosity was 0.55 (range: 0.081–0.955), and the average polymorphic information content value per marker was 0.507 (range: 0.077–0.942). No marker showed significant deviation from Hardy‐Weinberg Equilibrium following Bonferroni correction of significance values, and no markers showed evidence of null alleles, large allele dropout, or scoring errors from stutter bands. Two markers had successful amplification in Odontobutis potamophila, and one marker had successful amplification in Rhinogobius giurinus. These novel markers may be useful for further research in population genetic studies involving M. swinhonis.     

Isolation and characterization of novel microsatellite markers for Cymodocea serrulata (Cymodoceaceae), a seagrass distributed widely in the Indo‐Pacific region

Cymodocea serrulata is a tropical seagrass species distributed widely in the Indo‐Pacific region. We developed 16 novel microsatellite (simple sequence repeat) markers for C. serrulata using next‐generation sequencing for use in genetic studies. The applicability of these markers was attested by genotyping of 40 individuals collected from a natural population in the Philippines. Of the 16 loci, 15 showed polymorphism. For the 15 polymorphic markers, the number of alleles per locus ranged from two to seven, and the observed and expected heterozygosities ranged from 0.131–1.000 and 0.124–0.788, respectively. These markers are useful tools for elucidating genetic diversity, connectivity, and structure in this foundational coastal species.     

Development and characterization of 24 microsatellite markers in Primula tosaensis,an endangered primrose,using MiSeq

Primula tosaensis (Primulaceae) is an endangered primrose endemic to Japan. In this study, 24 novel microsatellite markers were developed using Illumina MiSeq sequencing to facilitate conservation of this endangered species. The genetic diversity and polymorphisms of these novel markers were measured in 32 individuals from a wild P. tosaensis population. The number of alleles and expected heterozygosities ranged from 2 to 5 (mean = 2.8) and from 0.119 to 0.724 (mean = 0.395), respectively. All loci were in Hardy–Weinberg equilibrium. The markers developed in this study will provide a powerful and practical tool for investigating the population structure and genetic diversity of P. tosaensis.     

CONSTRUCTION OF A GENETIC LINKAGE MAP FOR PORPHYRA HAITANENSIS (BANGIALES,RHODOPHYTA) BASED ON SEQUENCE‐RELATED AMPLIFIED POLYMORPHISM AND SIMPLE SEQUENCE REPEAT MARKERS1

Molecular markers and molecular genetic maps are prerequisites for molecular breeding in any plant species. A comprehensive genetic linkage map for cultivated Porphyra haitanensis T. J. Chang et B. F. Zheng has not yet been developed. In this study, 157 double haploid (DH) lines [derived from a YSIII (wildtype) × RTPM (red‐type artificial pigmentation mutant) cross] were used as a mapping population in P. haitanensis. A total of 60 pairs of sequence‐related amplified polymorphism (SRAP) primers and 39 pairs of simple sequence repeat (SSR) primers were used to detect polymorphisms between the two parents. Fifteen SRAP and 16 SSR polymorphic primer pairs were selected to analyze the DH population. A linkage genetic map comprising 67 SRAP markers and 20 SSR markers in five linkage groups, with a total length of 830.6 cM and an average of 10.13 cM between markers, was constructed. The markers were distributed evenly in all linkage groups without clustering. The linkage groups comprised 12–23 markers ranging in length from 134.2 to 197.3 cM. The estimated genome length of P. haitanensis was 942.4 cM, with 88.1% coverage. This is the first report of a comprehensive genetic map in P. haitanensis. The map presented here will provide a basis for the development of high‐density genetic linkage maps and lay the foundation for molecular breeding work in P. haitanensis.     

Enzyme function and polymorphism: A test in two Anolis lizard species

Electrophoretic surveys of two lizard species were used to test hypotheses that relate levels of enzyme variability to enzyme function (single-substrate versus multiple-substrate enzymes, regulatory versus nonregulatory enzymes). Anolis roquet behaviorally regulates its body temperature, but its congener A. gundlachi is passive to variations in the thermal environment. As a result, populations of A. gundlachi probably experience the thermal environment as temporally coarse-grained, whereas populations of A. roquet do not. We therefore predicted that A. gundlachi would exhibit greater enzyme heterozygosity than A. roquet and that different enzyme classes would contribute disproportionately to this interspecific difference. The data show (1) that A. gundlachi does have a higher heterozygosity and (2) that this difference appears to result from high levels of heterozygosity at loci coding for multiple-substrate enzymes. The dichotomy between regulatory and nonregulatory enzymes offers no explanation for the variability in heterozygosity among enzyme loci in these species.E.Z. was supported by a grant from the Natural Sciences and Engineering Council of Canada. The study was accomplished while P.E.H. held a postdoctoral fellowship from the Killam Trust of Dalhousie University and a grant from the Research Development Fund of Dalhousie University. The collection of material was made possible by grants (to P.E.H.) from National Science Foundation (DEB 75-16334), the Explorers Club of New York, Sigma Xi, and the Richmond and Anderson Funds of Harvard University. We thank Dr. D. W. Foltz for his help with the calculations.     

The roles of allopatric divergence and natural selection in quantitative trait variation across a secondary contact zone in the lizard Anolis roquet

Populations of the Caribbean lizard, Anolis roquet, are thought to have experienced long periods of allopatry before recent secondary contact. To elucidate the effects of past allopatry on population divergence in A. roquet, we surveyed parallel transects across a secondary contact zone in northeastern Martinique. We used diagnostic molecular mitochondrial DNA markers to test fine‐scale association of mitochondrial DNA lineage and geological region, multivariate statistical techniques to explore quantitative trait pattern, and cline fitting techniques to model trait variation across the zone of secondary contact. We found that lineages were strongly associated with geological regions along both transects, but quantitative trait patterns were remarkably different. Patterns of morphological and mitochondrial DNA variation were consistent with a strong barrier to gene flow on the coast, whereas there were no indications of barriers to gene flow in the transitional forest. Hence, the coastal populations behaved as would be predicted by an allopatric model of divergence in this complex, while those in the transitional forest did not, despite the close proximity of the transects and their shared geological history. Patterns of geographical variation in this species complex, together with environmental data, suggest that on balance, selection regimes on either side of the secondary contact zone in the transitional forest may be more convergent, while those either side of the secondary contact zone on the coast are more divergent. Hence, the evolutionary consequences of allopatry may be strongly influenced by local natural selection regimes.     

Fifty‐two polymorphic microsatellite loci in the rust fungus,Cronartium quercuum f.sp. fusiforme

We report on 52 microsatellite markers for use in Cronartium quercuum f.sp. fusiforme. The markers were developed from di‐, tri‐, and tetranucleotide repeat‐enriched genomic libraries. In 46 isolates collected from two natural populations in the southeastern USA, the number of alleles per locus ranged from two to 20 (mean 6.94) with gene diversity values ranging from 0.043 to 0.933 (mean 0.537). The markers should prove highly useful for genetic ‘fingerprinting’ of single‐spore isolates commonly used in host–pathogen gene interaction studies, as marker loci for linkage mapping studies, and for examining fine‐scale population genetic structure in natural populations of the fungus.     

Identification of microsatellite loci from Epimedium koreanum and cross‐species amplification in four species of Epimedium (Berberidaceae)

Nineteen polymorphic microsatellite markers were isolated and characterized from a trinucleotide enriched partial genomic library of Epimedium koreanum. The average allele number of these microsatellites was 5.9 per locus, ranging from two to 11. The observed heterozygosity (H_O) and expected heterozygosity (H_E) at the population level were 0.00–0.90 and 0.12–0.90, respectively. In addition, the results of cross‐species amplification of this set of microsatellite markers in four closely related Epimedium species, E. brevicornum, E. sagittatum, E. pubescens and E. wushanense, revealed that these microsatellite markers were useful for population genetic structure evaluation and genotype analysis of major Epimedium species that have been used as traditional Chinese medicines.     

Isolation and characterization of polymorphic nuclear microsatellite loci in Pinus cembra L

We developed eight polymorphic nuclear microsatellite markers for the Swiss stone pine (Pinus cembra L.), of which seven may be amplified in a multiplex polymerase chain reaction. Allelic polymorphism across all loci and 40 individuals representing two populations in the Swiss Alps was high (mean = 7.6 alleles). No significant linkage disequlibrium was displayed between pairs of loci. Significant deviation from Hardy–Weinberg equilibrium was revealed at three loci in one population. Cross–amplification was achieved in two related species within the genus (P. sibirica and P. pumila). Thus, the markers may be useful for population genetic studies in these three pine species. They will be applied in ongoing projects on genetic diversity and patterns of gene flow in P. cembra.     

Isolation and characterization of novel tetranucleotide microsatellite DNA markers for the spotted salamander,Ambystoma maculatum

Fifteen tetranucleotide microsatellite loci were identified and characterized for spotted salamanders (Ambystoma maculatum) collected from three vernal pools in the south‐eastern USA. These markers revealed a high degree of genetic diversity (7–32 alleles per locus), heterozygosity (31.6–86.3%) and allelic heterogeneity (91% of comparisons were statistically significant). Considerable differentiation among populations was observed as genetic distances (chord) ranged between 0.50 and 0.65, and all F_ST values (0.08–0.14) were statistically significant. Moreover, genotypic assignment tests correctly classified all individuals to their respective collection. These markers should prove useful for investigating fine‐scale population structure and mating system.     

Development and characterization of microsatellite markers in Zanthoxylum ailanthoides (Rutaceae)

Nine microsatellite markers were developed for Zanthoxylum ailanthoides, a typical pioneer tree. Averaged over the nine loci, the number of alleles per locus was 5.1. The observed and expected heterozygosities ranged from 0.233 to 0.833 and from 0.314 to 0.823, with averages of 0.606 and 0.641, respectively. No loci showed significant departures from Hardy–Weinberg equilibrium or linkage equilibrium after Bonferroni correction (P > 0.05). These markers will be useful for parentage analyses and studies of population genetic structure in the species.     

Isolation and characterization of a set of microsatellite loci in the submerged macrophyte,Vallisneria spinulosa Yan (Hydrocharitaceae)

Twenty‐six microsatellites were isolated and characterized from an AAG‐enriched genomic library of Vallisneria spinulosa, a dominant submerged macrophyte in the middle‐lower reaches of the Yangtze River. Microsatellite polymorphism was evaluated in a random population and 11 microsatellite loci were found polymorphic with 2–18 alleles per locus and observed heterozygosity ranging from 0.281 to 1.000. These polymorphic markers were expected to be valuable for population genetic and demographic analyses of V. spinulosa.     

Novel tetranucleotide microsatellite DNA markers for the wood frog,Rana sylvatica

Fifteen tetranucleotide microsatellite loci were identified and characterized for wood frogs (Rana sylvatica) collected from three vernal pools in the southeastern US. These markers revealed a high degree of genetic diversity (nine to 34 alleles per locus), heterozygosity (30.6–92.3%) and allelic heterogeneity (69% of comparisons were statistically significant). Considerable differentiation among populations was observed as genetic distances (chord) ranged between 0.40 and 0.55 and all F_ST values (0.02–0.05) were statistically significant. Genotypic assignment tests correctly classified 103 of 113 individuals to their respective collection. These markers should prove useful for investigating fine‐scale population structure and metapopulation dynamics.     

Development of 28 EST‐SSR markers based on transcriptome sequences of Gobiobotia filifer and cross‐species amplification

Gobiobotia filifer is a small benthic fish distributed in Yangtze River Basin. The abundance of G. filifer increased after impoundment of Xiluodu Dam and Xiangjiaba Dam. The state of population structure and changes of genetic diversity before and after impoundment of Xiluodu Dam and Xiangjiaba Dam were interesting issues. However, efficient molecular markers were rare, which will limit us to solve above problems. Twenty‐eight expressed sequence tag SSRs (EST‐SSRs) were successfully identified and verified as stable amplification and polymorphic loci by polyacrylamide gel electrophoresis (PAGE) and capillary electrophoresis. The number of alleles at these EST‐SSR loci ranged from 3 to 14, the polymorphism information content values were 0.125–0.897, and the observed and expected heterozygosities were 0.0–0.857 and 0.132–0.928, respectively. Cross‐species amplification of the 28 loci developed in this study was examined in seven individuals of each of the 7 taxa. The amplification efficiency of 28 EST‐SSRs primer pairs is related to the distance of genetic relationship between cross‐species with G. filifer, and same subfamily species (Xenophysogobio boulengeri and Xenophysogobio nudicorpa) showed the highest (50%) amplification efficiency. These EST‐SSR markers could be used to analyse genetic diversity and population structure of G. filifer and related species.     

Molecular inferences about the genus Hypostomus Lacépède, 1803 (Siluriformes: Loricariidae): a review

This review compiles and discusses the use of genetic markers applied in the study of the fish genus Hypostomus Lacépède, 1803 (Siluriformes: Loricariidae). The database comprises 51 peer-review articles that were published in the last 52 years (1968–2020) and that approach analysis based on different classes of genetic markers. The use of cytogenetic and enzymatic markers was predominantly especially in population studies with the genus Hypostomus, while mitochondrial markers were the majority in phylogenetic studies. Although significant methodological advances have occurred for molecular evaluation, they are still modestly applied to the study of neotropical fish genera, in which Hypostomus is included. New perspectives, especially on integrative approaches, are needed to improve our knowledge of the genetic functionality of fishes.

     

Ten polymorphic microsatellites from freshwater pearl mussel,Hyriopsis cumingii

Freshwater pearl mussel, Hyriopsis cumingii, is the most economically important mussel species in China. Due to overexploitation and changes of water quality, H. cumingii is facing serious population decline and local extinction. We have isolated and characterized 10 polymorphic microsatellite loci from a genomic DNA library enriched for CA and AG repeats with the aim of developing a set of codominant DNA markers for analysing genetic diversity and population structure of this species. The average allele number of the 10 markers was 7.8 per locus, ranging from two to 16 in 24 unrelated individuals. Nine of 10 markers conformed to Hardy–Weinberg equilibrium and inherited independently, suggesting these microsatellites could be useful for studying population genetics, designing conservation strategies and developing breeding programs.     

Isolation and development of microsatellite markers for the Japanese dormouse, Glirulus japonicus

Eight microsatellite markers were developed for the Japanese dormouse (Glirulus japonicus), a natural monument and near‐threatened species in Japan. The markers amplify in individuals from all of the mitochondrial lineages detected in a previous study. Numerous polymorphisms were detected in specimens from a local population in central Honshu (11–21 alleles per locus; n = 31) and from the entire distribution range of the species (19–41 alleles per locus; n = 152). These microsatellites will be useful in conservation genetic studies of G. japonicus.     

Genotype‐by‐sequencing of the plant‐pathogenic fungi Pyrenophora teres and Sphaerulina musiva utilizing Ion Torrent sequence technology

Genetic and genomics tools to characterize host–pathogen interactions are disproportionately directed to the host because of the focus on resistance. However, understanding the genetics of pathogen virulence is equally important and has been limited by the high cost of de novo genotyping of species with limited marker data. Non‐resource‐prohibitive methods that overcome the limitation of genotyping are now available through genotype‐by‐sequencing (GBS). The use of a two‐enzyme restriction‐associated DNA (RAD)‐GBS method adapted for Ion Torrent sequencing technology provided robust and reproducible high‐density genotyping of several fungal species. A total of 5783 and 2373 unique loci, ‘sequence tags’, containing 16 441 and 9992 single nucleotide polymorphisms (SNPs) were identified and characterized from natural populations of Pyrenophora teres f. maculata and Sphaerulina musiva, respectively. The data generated from the P. teres f. maculata natural population were used in association mapping analysis to map the mating‐type gene to high resolution. To further validate the methodology, a biparental population of P. teres f. teres, previously used to develop a genetic map utilizing simple sequence repeat (SSR) and amplified fragment length polymorphism (AFLP) markers, was re‐analysed using the SNP markers generated from this protocol. A robust genetic map containing 1393 SNPs on 997 sequence tags spread across 15 linkage groups with anchored reference markers was generated from the P. teres f. teres biparental population. The robust high‐density markers generated using this protocol will allow positional cloning in biparental fungal populations, association mapping of natural fungal populations and population genetics studies.     

Identification and characterization of microsatellite markers derived from expressed sequence tags of the sea cucumber Stichopus japonicus

Eleven polymorphic microsatellite markers were identified in expressed sequence tags generated from Stichopus japonicus cDNA libraries. The numbers of alleles ranged from three to 10, and the expected and observed heterozygosities ranged from 0.378 to 0.870 and from 0.077 to 0.690, respectively. Significant deviations from Hardy–Weinberg expectations were observed at eight loci due to homozygote excess, suggesting the widespread occurrence of null alleles. The microsatellite markers will be useful for examining genetic population structure, parentage analysis and mapping studies of S. japonicus.