Identification,development, and application of 12 polymorphic EST-SSR markers for an endemic Chinese walnut (Juglans cathayensis L.) using next-generation sequencing technology

The Chinese walnut (Juglans cathayensis L.), valued for both its nut and wood, is an ecologically important tree species endemic temperate southern China. Investigation of the genetic diversity of Chinese walnut has been limited to natural population genetics and genetic germplasm resources. Here, we describe the development of 12 polymorphic microsatellite markers using next-generation sequencing to screen 96 Chinese walnut individuals collected from 11 natural populations. The number of alleles per locus ranged from 5 to 12. The observed heterozygosity (0.288–0.748) overlapped well with the expected heterozygosity (0.337–0.751). This species has high genetic diversity and gene flow among different populations (F_ST = 0.075, Nm = 3.088). These markers will be useful for future studies on population genetic structure, evolutionary ecology, and genetic breeding of this walnut tree or other Juglans species.     

Genetic diversity and population structure in the narrow endemic Chinese walnut <Emphasis Type="Italic">Juglans hopeiensis</Emphasis> Hu: implications for conservation

The conservation of narrow endemic species relies on accurate information regarding their population structure. Juglans hopeiensis Hu (Ma walnut), found only in Hebei province, Beijing, and Tianjin, China, is a threatened tree species valued commercially for its nut and wood. Sequences of two maternally inherited mitochondrial markers and two maternally inherited chloroplast intergenic spacers, three nuclear DNA sequences, and allele sizes from 11 microsatellites were obtained from 108 individuals of J. hopeiensis, Juglans regia, and Juglans mandshurica. Haplotype networks were constructed using NETWORK. Genetic diversity, population differentiation, and analysis of molecular variance (AMOVA) were used to determine genetic structure. MEGA was used to construct phylogenetic trees. Genetic diversity of J. hopeiensis was moderate based on nuclear DNA, but low based on uniparentally inherited mitochondrial DNA and chloroplast DNA. Haplotype networks showed that J. hopeiensis haplotypes were different than haplotypes found in J. regia and J. mandshurica. Allelic variants in nuclear genes that were shared among J. hopeiensis populations were not found in J. regia or J. mandshurica. Sampled populations of J. hopeiensis showed clear genetic structure. The maximum parsimony (MP) tree showed J. hopeiensis to be distinct from J. mandshurica but threatened by hybridization with J. regia and J. mandshurica. J. hopeiensis populations are strongly differentiated from sympatric Juglans species, but they are threatened by small population sizes and hybridization.     

Development of microsatellite markers for <Emphasis Type="Italic">Manilkara maxima</Emphasis> T.D. Penn. (Sapotaceae) and their use in conservation genetics

Manilkara maxima is an endemic tree species of the Atlantic Forest in southern Bahia, Brazil. It is considered important for forest conservation due to its mutualistic interactions with endemic and endangered animals. Our aim was to develop microsatellite markers to estimate genetic diversity in order to provide information for effectiveness of future conservation programs. We used next generation sequencing technology to develop the first specific microsatellite markers for M. maxima. Seventeen new microsatellite loci were applied in 72 individuals sampled in three natural populations. On average, the number of alleles per loci was 8.8. The expected heterozygosity varied between 0.72 and 0.77, indicating that the developed set of molecular markers is useful for genetic diversity studies. Additionally, the estimated value for the combined probability of exclusion (Q) was greater than 0.999, which indicates the powerful of these molecular tools for paternity and kinship analysis. Our results demonstrate that the set of microsatellites developed in this work is a powerful tool for population genetics, molecular ecology and conservation biology purposes.     

Isolation and characterization of eight polymorphic microsatellite loci for the endangered,endemic Chilean tree Gomortega keule (Gomortegaceae)

Queule (Gomortega keule) is an endangered, Chilean tree in the endemic, monotypic family Gomortegaceae. Eight polymorphic microsatellite loci were developed and characterized for this species. The number of alleles per locus ranged from four to 12 and the observed heterozygosity ranged from 0.2 to 1.0. These microsatellite markers will be valuable tools for population genetics studies and conservation planning.     

Isolation and characterization of species-specific microsatellite markers for blue and black wildebeest (<Emphasis Type="BoldItalic">Connochaetes taurinus</Emphasis> and <Emphasis Type="BoldItalic">C. gnou</Emphasis>)

The blue wildebeest (Connochaetes taurinus) is distributed throughout southern and east Africa while the black wildebeest (Connochaetes gnou) is endemic to South Africa and was driven to near extinction in the early 1900s due to hunting pressure and disease outbreaks. Extensive translocation of both species throughout South Africa is threatening the genetic integrity of blue and black wildebeest. To effectively manage these species, genetic tools that can be used to detect hybrid individuals, identify genetically unique subpopulations and determine the levels of genetic diversity are required. In this study, 11 microsatellite markers were developed for wildebeest through next-generation sequencing. The microsatellite loci displayed 2.00–4.14 alleles, unbiased heterozygosity values ranged from 0.32 to 0.60 and observed heterozygosity values ranged from 0.26 to 0.52. The comparatively high level of polymorphism observed in the microsatellite markers indicates that these markers can contribute significantly to our knowledge of population genetic structure, relatedness, genetic diversity and hybridization in these species.     

Biotic and abiotic factors affecting the genetic structure and diversity of butternut in the southern Appalachian Mountains,USA

The abundance of butternut (Juglans cinerea L.) trees has severely declined rangewide over the past 50 years. An important factor in the decline is butternut canker, a disease caused by the fungus Ophiognomonia clavigigenti-juglandacearum, which has left the remaining butternuts isolated and sparsely distributed. To manage the remaining populations effectively, information regarding how butternut’s population genetic structure is affected by environmental and historical factors is needed. In this study, we assessed genetic structure and diversity of 161 butternut trees from 19 adjacent watersheds in the southern portion of butternut’s range using 12 microsatellite markers. We assessed the genetic diversity and genetic differentiation among trees grouped at various spatial scales. Our goal was to use historical abundance and land use data for these watersheds, which are now all a part of the Great Smoky Mountains National Park (GSMNP), to understand the ecological and evolutionary forces that challenge the conservation and management of butternut. In general, butternuts within the 19 neighboring watersheds were all part of one continuous population, with gene flow throughout. Significant genetic differentiation was detected between some groups of trees, but the differentiation was quite small and may not represent an ecologically significant distinction. The mean heterozygosity in all watersheds remained high, despite extensive mortality. Overall, genetic diversity and rare alleles were evenly distributed across all watersheds, with some variability in subpopulations containing butternut-Japanese walnut hybrids (Juglans x bixbyi or buarts). These results indicate that management of this species should focus on protection from future hybridization with Japanese walnut, promotion of regeneration, and persistence of all remaining butternut trees, which still retain high levels of genetic diversity.     

Isolation and characterization of microsatellite loci for the analysis of genetic diversity in Whitmania pigra

The objective of this study was to isolate microsatellite loci to analyze the genetic diversity of Whitmania pigra. Four new microsatellite markers of W. pigra were developed from an enriched library and ten from a modified SAMPL assay. A total of 127 alleles were detected, with an average of 9.1 alleles per locus. The expected heterozygosity (He) of each microsatellite locus varied from 0.451 to 0.857, with an average of 0.688. The polymorphism information content (PIC) of each microsatellite locus ranged from 0.361 to 0.838, with an average of 0.640. Analysis of molecular variance showed that the main variation component existed within the populations (81.64%) rather than among the populations (18.36%). Phylogenetic tree for 15 populations of Hirudo using the NJ method by MEGA 5.1 software were divided into two major clusters. These microsatellite markers will contribute to research on the individual identification, genetic diversity, population structure, genome mapping and conservation biology of Hirudo.     

Development of microsatellite markers using next-generation sequencing for the columnar cactus <Emphasis Type="Italic">Echinopsis chiloensis</Emphasis> (Cactaceae)

The aim of this study was to develop microsatellite markers as a tool to study population structure, genetic diversity and effective population size of Echinopsis chiloensis, an endemic cactus from arid and semiarid regions of Central Chile. We developed 12 polymorphic microsatellite markers for E. chiloensis using next-generation sequencing and tested them in 60 individuals from six sites, covering all the latitudinal range of this species. The number of alleles per locus ranged from 3 to 8, while the observed (Ho) and expected (He) heterozygosity ranged from 0.0 to 0.80 and from 0.10 to 0.76, respectively. We also detected significant differences between sites, with F_ST values ranging from 0.05 to 0.29. Microsatellite markers will enable us to estimate genetic diversity and population structure of E. chiloensis in future ecological and phylogeographic studies.     

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.     

Isolation and characterization of microsatellite markers in pangolins (Mammalia,Pholidota, Manis spp.)

Thirty‐four polymorphic dinucleotide microsatellite loci were developed in the Malayan pangolin Manis javanica. Of the 34 markers, 32 and 18 were also amplified, respectively, in the Chinese pangolin (Manis pentadactyla) and the African tree pangolin (Manis tricuspis). Analysis of 24 Malayan, 12 Chinese and 2 African tree pangolins showed high levels of variability (heterozygosity ranging from 0.321 to 0.708). These are the first available microsatellite markers in Pholidota and will be an invaluable tool for evolutionary and conservation genetic studies in pangolins.     

Development and characterisation of nine polymorphic microsatellite markers for Tephrosia calophylla Bedd. (Fabaceae)

Tephrosia calophylla Bedd. (Fabaceae) is an endangered tropical plant endemic to southwestern Ghats, India. The objective of this study was to contribute to the characterisation of the diversity of this rare species, which is necessary for its future conservation. Accordingly, microsatellite markers were designed, and their ability to detect polymorphisms was determined. Nine microsatellite markers were developed using genomic libraries, and all of the markers were successfully amplified in 42 individuals. Three to nine alleles per locus were observed, and the heterozygosity of the loci ranged from 0.381 to 0.905. The nine newly developed polymorphic markers recognise a sufficient number of varying loci to perform further studies on the conservation and breeding of this medicinal cultivar.     

Isolation and characterization of microsatellite loci in Eurycorymbus cavaleriei,a dioecious endemic tree species in China

Eleven dinucleotide microsatellites were developed and characterized for Eurycorymbus cavaleriei, a dioecious, rare and endemic tree species in China. A genomic DNA enrichment protocol was used to isolate microsatellite loci and polymorphism was explored using 30 trees from two natural populations. The observed heterozygosity (H_O) ranged from 0.379 to 0.931, with an average of 0.625. These markers provide powerful tools for the ongoing population and conservation genetics studies of E. cavaleriei.     

Development of microsatellite markers for <Emphasis Type="Italic">Myracrodruon urundeuva</Emphasis> (F.F. &amp; M.F. Allemão), a highly endangered species from tropical forest based on next-generation sequencing

Myracrodruon urundeuva is a tree species of high economic importance due the strength and durability of its wood. Threatened of extinction in Brazil, it is present only in a few forest remnants, mostly in conservation units. Currently, there is little information on the genetic diversity of natural populations in Brazil and even less information about the genome of this species. Here, new species-specific microsatellite loci were developed based on next-generation sequencing (Illumina). More than 100,000 loci were identified in the run, with di- to hexanucleotides motifs. Of these, 20 loci were selected for validation in 30 individuals, with 15 successfully polymorphic loci detected. The number of alleles ranged among loci from 3 to 16, with an average of 7.73, expected (H _e ) and observed (H _o ) heterozygosity ranged from 0.246 to 0.902 and from 0.103 to 0.867, respectively. These results point out that these new set of markers has a great potential for use in population genetic studies for genetic conservation of the species.     

Isolation and characterization of microsatellite markers in a highland fish, <Emphasis Type="BoldItalic">Pareuchiloglanis sinensis</Emphasis> (Siluriformes: Sisoridae) by next-generation sequencing

Pareuchiloglanis sinensis (Siluriformes: Sisoridae) is an endemic and highland fish species which occurs only in some rivers of south-west China. In this study, the isolation and characterization of polymorphic microsatellite loci of this fish species by next-generation sequencing is described. A total of 9471 simple-sequence repeats (SSRs) were observed from RNA-seq data. One hundred and twenty primer pairs were chosen randomly and validated across 48 P. sinensis individuals collected from the Dadu river (a branch of the Yangtze river) of which 28 polymorphic microsatellite loci were detected. The number of alleles ranged from 2 to 14, with an average of seven alleles per locus. Twenty loci exhibited high polymorphism with the polymorphism information content (PIC) larger than 0.5. The mean observed and expected heterozygosity varied from 0.104 to 0.958 and 0.157 to 0.844, with an average of 0.583 and 0.613, respectively. The microsatellite markers characterized in the current study serve as a useful tool for the conversation genetic studies and population evaluation of P. sinensis.     

Isolation and characterization of 24 microsatellite loci in Paphiopedilum rothschildianum, an endangered slipper orchid

This paper describes the characterization of 24 polymorphic microsatellite loci in Paphiopedilum rothschildianum, an endangered slipper orchid endemic to Sabah, Malaysia. A total of 36 clones containing microsatellite repeat motifs yielded 41 dinucleotide, 20 tetranucleotide and 30 cryptic simple repeat sequences. Thirty microsatellite loci were randomly selected to characterize the population, of which 24 were found to be polymorphic. The expected heterozygosity ranged from 0.0813 to 0.7279 (mean 0.4533) and the observed heterozygosity ranged from 0 to 1 (mean 0.3800). The results indicated that there was limited migration between the three subpopulations and a significant degree of genetic differentiation (F_ST = 0.5098) between populations implying that gene flow has been disrupted as a result of habitat fragmentation. The molecular markers have a potential application in population management, CITES enforcement and the forensic detection of specimens in international trade.     

Identification, characterization and utilization of unigene derived microsatellite markers in tea (Camellia sinensis L.)

Background

Despite great advances in genomic technology observed in several crop species, the availability of molecular tools such as microsatellite markers has been limited in tea (Camellia sinensis L.). The development of microsatellite markers will have a major impact on genetic analysis, gene mapping and marker assisted breeding. Unigene derived microsatellite (UGMS) markers identified from publicly available sequence database have the advantage of assaying variation in the expressed component of the genome with unique identity and position. Therefore, they can serve as efficient and cost effective alternative markers in such species.

Results

Considering the multiple advantages of UGMS markers, 1,223 unigenes were predicted from 2,181 expressed sequence tags (ESTs) of tea (Camellia sinensis L.). A total of 109 (8.9%) unigenes containing 120 SSRs were identified. SSR abundance was one in every 3.55 kb of EST sequences. The microsatellites mainly comprised of di (50.8%), tri (30.8%), tetra (6.6%), penta (7.5%) and few hexa (4.1%) nucleotide repeats. Among the dinucleotide repeats, (GA)n.(TC)n were most abundant (83.6%). Ninety six primer pairs could be designed form 83.5% of SSR containing unigenes. Of these, 61 (63.5%) primer pairs were experimentally validated and used to investigate the genetic diversity among the 34 accessions of different Camellia spp. Fifty one primer pairs (83.6%) were successfully cross transferred to the related species at various levels. Functional annotation of the unigenes containing SSRs was done through gene ontology (GO) characterization. Thirty six (60%) of them revealed significant sequence similarity with the known/putative proteins of Arabidopsis thaliana. Polymorphism information content (PIC) ranged from 0.018 to 0.972 with a mean value of 0.497. The average heterozygosity expected (H _E ) and observed (H _o ) obtained was 0.654 and 0.413 respectively, thereby suggesting highly heterogeneous nature of tea. Further, test for IAM and SMM models for the UGMS loci showed excess heterozygosity and did not show any bottleneck operating in the tea population.

Conclusion

UGMS markers identified and characterized in this study provided insight about the abundance and distribution of SSR in the expressed genome of C. sinensis. The identification and validation of 61 new UGMS markers will not only help in intra and inter specific genetic diversity assessment but also be enriching limited microsatellite markers resource in tea. Further, the use of these markers would reduce the cost and facilitate the gene mapping and marker-aided selection in tea. Since, 36 of these UGMS markers correspond to the Arabidopsis protein sequence data with known functions will offer the opportunity to investigate the consequences of SSR polymorphism on gene functions.     

Population Structure of Geosmithia morbida,the Causal Agent of Thousand Cankers Disease of Walnut Trees in the United States

The ascomycete Geosmithia morbida and the walnut twig beetle Pityophthorus juglandis are associated with thousand cankers disease of Juglans (walnut) and Pterocarya (wingnut). The disease was first reported in the western United States (USA) on several Juglans species, but has been found more recently in the eastern USA in the native range of the highly susceptible Juglans nigra. We performed a comprehensive population genetic study of 209 G. morbida isolates collected from Juglans and Pterocarya from 17 geographic regions distributed across 12 U.S. states. The study was based on sequence typing of 27 single nucleotide polymorphisms from three genomic regions and genotyping with ten microsatellite primer pairs. Using multilocus sequence-typing data, 197 G. morbida isolates were placed into one of 57 haplotypes. In some instances, multiple haplotypes were recovered from isolates collected on the same tree. Twenty-four of the haplotypes (42%) were recovered from more than one isolate; the two most frequently occurring haplotypes (H02 and H03) represented 36% of all isolates. These two haplotypes were abundant in California, but were not recovered from Arizona or New Mexico. G. morbida population structure was best explained by four genetically distinct groups that clustered into three geographic regions. Most of the haplotypes isolated from the native range of J. major (Arizona and New Mexico) were found in those states only or present in distinct genetic clusters. There was no evidence of sexual reproduction or genetic recombination in any population. The scattered distribution of the genetic clusters indicated that G. morbida was likely disseminated to different regions at several times and from several sources. The large number of haplotypes observed and the genetic complexity of G. morbida indicate that it evolved in association with at least one Juglans spp. and the walnut twig beetle long before the first reports of the disease.     

Fine-scale spatial genetic structure of Dalbergia nigra (Fabaceae), a threatened and endemic tree of the Brazilian Atlantic Forest

The Atlantic Forest is one of the most diverse ecosystems in the world and considered a hotspot of biodiversity conservation. Dalbergia nigra (Fabaceae) is a tree endemic to the Brazilian Atlantic Forest, and has become threatened due to overexploitation of its valuable timber. In the present study, we analyzed the genetic diversity and fine-scale spatial genetic structure of D. nigra in an area of primary forest of a large reserve. All adult individuals (N = 112) were sampled in a 9.3 ha plot, and genotyped for microsatellite loci. Our results indicated high diversity with a mean of 8.6 alleles per locus, and expected heterozygosity equal to 0.74. The co-ancestry coefficients were significant for distances among trees up to 80 m. The Sp value was equal to 0.017 and indirect estimates of gene dispersal distances ranged from 89 to 144 m. No strong evidence of bottleneck or effects of human-disturbance was found. This study highlights that long-term efforts to protect a large area of Atlantic Forest have been effective towards maintaining the genetic diversity of D. nigra. The results of this study are important towards providing a guide for seed collection for ex-situ conservation and reforestation programmes of this threatened species.     

Isolation and characterization of nine polymorphic microsatellite markers of the marbled rockfish Sebastiscus marmoratus (Scorpaeniformes, Scorpaenidae)

Sebastiscus marmoratus is a typical sedentary fish and usually lives at the bottom of the coastal zone from Japan to the East China Sea. Next-generation sequencing platform was used to obtain microsatellite loci sequences of S. marmoratus, among which forty-eight perfect microsatellite repeats were suitable for primer design. Nine polymorphic microsatellite markers were exploited and characterized for it. The allele number of all polymorphic microsatellite markers ranged from 4 to 12. Expected and observed heterozygosity varied from 0.302 to 0.909 and from 0.190 to 0.625, respectively. In further studies, the genetic variation and population structure of this species would be investigated using these microsatellite loci.