Enriched Genomic Library Research Articles

Polymorphic microsatellite markers in the brown seaweed Fucus vesiculosus.

BackgroundFucus vesiculosus is a brown seaweed dominant on temperate rocky shores of the northern hemisphere and, is typically distributed in the mid-upper intertidal zone. It is an external fertilizer that reproduces sexually, providing an excellent model to address conflicting theories related to mating systems and sexual selection. Microsatellite markers have been reported for several Fucus species, however the genomic libraries from where these markers have been isolated, have originated from two or more species pooled together (F. vesiculosus and F. serratus in one library; F. vesiculosus, F. serratus and Ascophyllum nodosum in a second library), or when the genomic DNA originated from only one species it was from Fucus spiralis. Although these markers cross-amplify F. vesiculosus individuals, the level of polymorphism has been low for relatedness studies.FindingsThe microsatellite markers described here were obtained from an enriched genomic library, followed by 454 pyrosequencing. A total of 9 microsatellite markers were tested across 44 individuals from the North of Portugal. The mean number of alleles across loci was 8.7 and the gene diversity 0.67.ConclusionsThe high variability displayed by these microsatellite loci should be useful for paternity analysis, assessing variance of reproductive success and in estimations of genetic variation within and between populations.

Isolation and screening of microsatellite markers from the pearl oyster, Pinctada fucata using FIASCO method.

Pearl oyster, Pinctada fucata is an important commercial species that is cultured for pearl in China (Meng et al. 1996). Pearls produced by this species are well-known worldwide and called ‘South China Sea Pearl’. In China, artificial reproduction of this species was successful in 1965. Since then, hundreds of hatcheries have been established (Meng et al. 1996), and currently the industry of pearl production relies mainly on hatchery produced spats. Each year, the hatcheries use cultured stocks as the brood stocks to produce the next generation for culture. This operation has been continued for more than 40 years which lead to some degeneration of growth traits, such as miniaturization of body size, reduction of resistance to pathogens and adverse environments, and decrease of cultured pearl quality. Thus, it is desirable to investigate the genetic variability of this species to provide background information for its genetic improvement. Microsatellite DNA is one of the most popular molecular markers, because of its high level of polymorphism and codominant nature it is increasingly being used for identity test, population genetics and gene mapping (Evans et al. 2006; Herbinger et al. 2006). Here, we report nine polymorphic microsatellite loci that would be useful in genetic studies in the pearl oyster. In a total of 32 individuals, samples for construction of enriched library were collected from Sanya Bay, Hainan Province, and samples for screening and characterization of polymorphic microsatellite loci were collected from Beibu Bay, Guangxi Province. Genomic DNA was extracted from the adductor muscle by proteinase K digestion, phenol– chloroform protocol. The enriched genomic library was constructed following the protocol of the fast isolation by AFLP of sequences containing repeats (FIASCO) as described by Zane et al. (2002). DNA was simultaneously digested with MseI and ligated to MseI-adaptor. The digestion ligation mixture was diluted (1:10) and directly amplified with

Development and characterization of microsatellite markers for Piptadenia gonoacantha (Fabaceae).

• Premise of the study: Microsatellite primers were designed for Piptadenia gonoacantha (Fabaceae) and characterized to estimate genetic diversity parameters. The species is a native tree from the Atlantic Forest biome commonly used in forest restoration; it has medicinal potential and the wood is economically useful.• Methods and Results: Twenty-eight microsatellite loci were identified from an enriched genomic library. Fifteen loci resulted in successful amplifications and were characterized in a natural population of 94 individuals. Twelve loci were polymorphic, with allele numbers ranging from three to 15 per locus, and expected and observed heterozygosities ranging from 0.2142 to 0.8325 and 0.190 to 0.769, respectively.• Conclusions: The developed markers will be used in further studies of population genetics of P. gonoacantha, aimed at conservation and management of the species in natural populations and in forest restoration projects.

Development of microsatellite markers for Hancornia speciosa Gomes (Apocynaceae).

Herein, we describe 34 microsatellite loci developed using an enrichment genomic library for the tree species Hancornia speciosa Gomes (Apocynaceae). Thirty-five individuals were genotyped using 34 primers to analyze the polymorphisms at each locus. The number of alleles per locus ranged from 4 to 20. The average number of alleles was 8.11, and the expected heterozygosity ranged from 0.62 to 0.94. These microsatellite primers will be useful in population genetics studies for this species.

Development and characterization of microsatellite loci for Cedrela fissilis Vell (Meliaceae), an endangered tropical tree species

Abstract The timber of the Neotropical tree Cedrela fissilis is used in construction, shipbuilding, carpentry and for medical purposes. In this study, polymorphic microsatellite (SSR) markers derived from an enriched genomic library were characterized using 120 adult trees from four different C. fissilis populations. No substantial genotypic linkage disequilibrium was detected among all possible pairs of SSR loci. The number of alleles per locus ranged from 2 to 20, the average allele number ranged from 8 to 9.7, depending on the population. The observed heterozygosity among the different SSR loci varied from 0.0 to 1.00 , the expected heterozygosity varied from 0.07 to 0.95 On the population level, the average observed and expected heterozygosities ranged from 0.50 to 0.63 and from 0.64 to 0.70, respectively. The average fixation index among populations ranged from 0.09 to 0.24. Thus, the SSR loci revealed high poly - morphism rates and can be used to study the genetic diversity,structure, mating system, and gene flow in C. fissilis.

Development of novel polymorphic microsatellite markers for four bird species exploited by the illegal wildlife trade in Brazil

Paroaria dominicana, Saltator similis, Sporophila frontalis and Cyanoloxia brissonii are bird species severely exploited by the illegal wildlife trade in Brazil. As part of an effort to characterize the genetic populations within each species to infer the most likely population of origin of seized animals, four microsatellite enriched genomic libraries were constructed and cross amplification of selected primers was tested for all four species. Eight polymorphic loci were selected for P. dominicana with mean observed and expected heterozigosities (mean Ho and He) of 0.750 and 0.768 respectively. Nine were selected for S. similis with mean Ho and He of 0.688 and 0.760. Seven for C. brissonii with mean Ho and He of 0.805 and 0.811. Three loci for S. frontalis with mean Ho and He of 0.851 and 0.903. These markers will be useful for population studies and for the development of paternity tests to be used by commercial breeders.

New advances in precision medicine for hepatocellular carcinoma recurrence prediction and treatment

New advances in precision medicine for hepatocellular carcinoma recurrence prediction and treatment

Development and characterization of microsatellite loci in Opisthopappus taihangensis (Compositae), a rare herb endemic to China

Using an enriched genomic library, we developed and characterized 10 polymorphic microsatellite primers for Opisthopappus taihangensis, a rare herb endemic to China. Number of alleles varied from 2 to 8 per locus, observed and expected heterozygosities ranged from 0.214 to 0.929 and 0.262 to 0.746 respectively. Significantly deviations from HWE were detected in two loci. These microsatellite loci would be useful for studies of genetic variation, genetic structure, and mating systems of O. taihangensis, which are important for the further conservation of this rare species.

Genetic analysis of Cheirostylis species based on microsatellite markers

Microsatellite markers specific to Cheirostylis yunnanensis Rolfe were developed using an enriched genomic DNA library technique. The library was constructed using (AG)20 and (CAG)20 oligonucleotide repeats. A total of 48 primer pairs were designed and tested with 48 C. yunnanensis Rolfe samples, resulting in 11 polymorphic loci. The number of alleles per locus ranged from 2 to 12, with an average of six alleles. The observed and expected heterozygosity ranged from 0.0426 to 0.8085 and 0.0421 to 0.9078, respectively. Of the 11 polymorphic loci, three showed a significant deviation from Hardy–Weinberg equilibrium and one exhibited linkage disequilibrium. Cross-species amplification was tested with five samples of Cheirostylis of unknown species resulting in eight loci that could be amplified, with the number of alleles ranging from one to two. The microsatellite markers developed in this study will be useful for the genetic analysis of C. yunnanensis in order to differentiate species as well as to establish a conservation plan for this species.

Isolation and characterization of microsatellite loci in the dog-faced water snake Cerberus rynchops (Colubridae)

The dog-faced water snake (Cerberus rynchops) is a viviparous colubrid snake. This species occurs in coastal South Asia, peninsular India, Sri Lanka, Bangladesh, Myanmar, the Andaman Islands, eastward to the coastal areas of the Indochinese and Malaysian Peninsulas, southwards into Indonesia and eastwards to Palau and Micronesia. It has been listed on Appendix III of CITES. In India, this species is protected under the Indian Wildlife Act, due to its historic trade as skin. Accordingly, we aimed to characterize new microsatellite markers, which could be helpful for us to get a better understanding of population structure and evolutionary history of the snake. Here, eight polymorphic microsatellite loci were isolated from an enriched genomic library of C. rynchops. One hundred and fifteen individuals were collected from Singapore. These markers revealed a high degree of genetic diversity (5–14 alleles per locus) and heterozygosity (HO ranged from 0.565 to 0.965, and HE ranged from 0.701 to 0.882). No locus exhibited significant deviations from Hardy–Weinberg equilibrium. There was no evidence of linkage disequilibrium between any pair of loci. These loci are first identified for C. rynchops and will be useful for future study of population structure, genetic diversity and conservation strategy design of this species.

GENOMIC AND EST MICROSATELLITE LOCI DEVELOPMENT AND USE IN OLIVE: MOLECULAR TOOLS FOR GENETIC MAPPING AND ASSOCIATION STUDIES

Identification of QTLs (quantitative trait loci) related to agronomic traits based on segregating population and/or on a collection of unrelated genotypes needs the use of molecular markers. Here, we aimed to develop and characterize genomic and expressed sequence tag (EST)-derived microsatellite markers in olive ( Olea europaea L.) and assess their Mendelian inheritance on the pseudo-F 1 population derived from the cross 'Oliviere' * 'Arbequina'. Based on an enriched genomic DNA library constructed on the French cultivar 'Oliviere', we designed 211 primer pairs among the total 584 identified SSR loci. Following 454 sequencing of cDNA fragments obtained on the Slovenian cultivar 'Istrska belica', we designed 113 primer pairs among 976 identified as EST-SSR loci. We screened these SSR markers for their informativeness for the saturation of the previously published linkage map based on the same cross, designed for bearing regularity study (in Montpellier, France). A set of 59 genomic and 40 expressed SSR loci were selected to genotype the 147 offspring. Twenty four (25.53%) SSRs show a Mendelian distortion segregation at P<0.05. From genotyped SSRs, 85 (90%), 64 (68%) and 94 (100%) SSR loci are now available for completing respectively 'Oliviere' and 'Arbequina', and the integrated maps published earlier.

Microsatellite primers for Camissoniopsis cheiranthifolia (Onagraceae) and cross-amplification in related species.

• Premise of the study: We developed 24 nuclear microsatellite primers from an enriched genomic library for the Pacific coastal dune endemic Camissoniopsis cheiranthifolia to study the consequences of mating system differentiation, the genetics of species’ range limits, and hybridization with its closest sister taxon, C. bistorta.• Methods and Results: Twenty-four primer pairs were developed and characterized in four populations of C. cheiranthifolia and one population of C. bistorta. We also tested eight additional taxa for cross-amplification. The average number of alleles per locus per species was 4.3 and 6.0, respectively. The number of loci that amplified and were variable within the eight related taxa ranged from six to 17.• Conclusions: These markers will be useful in studying mating system evolution, the genetic structure of species’ ranges, hybridization, and the provenance of material used for habitat restoration in C. cheiranthifolia, C. bistorta, and related species.

Development of microsatellite markers in the rice paddy snake Enhydris plumbea (Colubridae)

The rice paddy snake (also known as Boie’s mud snake) Enhydris plumbea is a viviparous colubrid snake. This is a widely distributed species found from the Andaman Islands (India) and Myanmar eastwards across the Southeast Asian Peninsula and southern China southwards into Indonesia; and it is present on most of the Indonesian islands and eastwards to Sulawesi and the Moluccas. The snake has been overexploited by local people for traditional medicine to treat skin diseases, and is listed on the “Lists of terrestrial wildlife under state protection, which are beneficial or of important economic or scientific value” promulgated by China’s State Forestry Administration in 2000. Knowledge of population structure and genetic diversity is crucial for effective wildlife preservation. Specific molecular markers such as mitochondrial DNA and microsatellites are useful tools for achieving this goal. Here, eight polymorphic microsatellite loci were isolated from an enriched genomic library of E. plumbea. One hundred and twenty individuals were collected from Wenzhou population in Zhejiang, China. These markers revealed a high degree of genetic diversity (4–8 alleles per locus) and heterozygosity (H O ranged from 0.148 to 0.789, and H E ranged from 0.347 to 0.766). No locus exhibited significant deviations from Hardy-Weinberg equilibrium. There was no evidence of linkage disequilibrium among pairs of loci. These loci will be useful for future study of population structure, genetic diversity and conservation strategy design of E. plumbea.

Isolation and characterization of twenty polymorphic microsatellites for Hemibarbus labeo (Cyprinidae)

Hemibarbus labeo is a valuable freshwater fish species distributed in the Heilongjiang River basin in China. Wild resources have declined sharply in recent years because of overfishing and pollution. Herein, twenty polymorphic microsatellites were isolated from an enriched genomic library in H. labeo for genetic conservation. These markers were characterized in 27 wild and 48 cultured individuals. The number of alleles per locus ranged from two to ten, with an average of 5.200. The observed heterozygosity and the expected heterozygosity varied from 0.086 to 0.870 (mean = 0.538) and from 0.116 to 0.861 (mean = 0.5441), respectively. Three and two loci significantly deviated from Hard–Weinberg equilibrium in wild and cultured individuals, respectively. Three pairwise comparisons of six loci showed significant genotypic linkage disequilibrium in cultured individuals. These polymorphic markers represent a powerful tool to study population genetic diversity, genetic conservation and breeding systems in H. labeo.

Development and characterization of polymorphic microsatellites markers in endangered Aquilaria malaccensis

Aquilaria malaccensis is a critically endangered species in Asia because of its overexploitation to obtain high valued agarwood, used in medicine, perfume, and incense production. Its genetic improvement faces serious problem due to lack of genomic resources. In this study, eighteen novel highly polymorphic microsatellite markers derived from enriched genomic libraries were characterized using 45 individuals of three populations, would be an asset to its non-existing marker recourse. Allelic variations per locus ranged from 2 to 6 with mean allele number was 3.28. The average observed and expected heterozygosity varied from 0.811 and 0.597, respectively. Reproducibility rate, polymorphic potential and ability to detect heterozygosity, novel microsatellites markers would contribute in evaluation of genetic diversity and population structure, and selection of elite germplasm for conservation of genetic resources and commercial utilization.

Isolation and characterization of the first microsatellite markers in the Neotropical freshwater fish piau-verdadeiro, Leporinus obtusidens (Valenciennes, 1837)

Ten microsatellite markers were isolated and characterized for piau-verdadeiro (Leporinus obtusidens), an important Characiform fish species in South American basins. The markers were obtained from an enriched microsatellite genomic library, and polymorphisms were evaluated in a sample of 44 individuals caught in the upper Sao Francisco River. The number of alleles varied from 4 to 39. The observed and expected heterozygosities ranged from 0.614 to 0.977 and from 0.541 to 0.977, respectively. All markers were in Hardy–Weinberg equilibrium and no null alleles were detected, thus making them suitable for assessing the diversity structure of the populations of this species.

Polymorphic microsatellite markers in Thrips hawaiiensis (Thysanoptera: Thripidae)

Thrips hawaiiensis (Morgan, 1913) is an important insect pest of fruits and vegetables. At present, it is primarily distributed in tropical and subtropical area. For a better understanding of the genetic makeup and migration patterns of T. hawaiiensis throughout the world, we isolated 11 novel polymorphic microsatellite loci from an enriched genomic library based on a biotin/streptavidin capture protocol. Genetic parameters were estimated on 80 individual thrips from two natural populations. The results showed that all 11 loci were highly polymorphic; the number of alleles ranged from six to 37, and nine loci demonstrated polymorphic information content (PIC) > 0.5. The observed (H O) and expected (H E) heterozygosities ranged from 0.350 to 0.925 and 0.307 to 0.952, respectively. Furthermore, only four locus/population combinations significantly deviated from Hardy–Weinberg equilibrium (HWE). These microsatellite markers have potential utility in population structure and gene flow studies of this species.

Isolation and characterization of microsatellite markers and analysis of genetic diversity in Chinese jujube (Ziziphus jujuba Mill.).

Chinese jujube (Ziziphus jujuba Mill, 2n = 2× = 24, Rhamnaceae) is an economically important Chinese native species. It has high nutritional value, and its medicinal properties have led to extensive use in traditional oriental medicine. The characterization of genotypes using molecular markers is important for genetic studies and plant breeding. However, few simple sequence repeat (SSR) markers are available for this species. In this study, 1,488 unique SSR clones were isolated from Z. jujuba ‘Dongzao’ using enriched genomic libraries coupled with a three-primer colony PCR screening strategy, yielding a high enrichment rate of 73.3%. Finally, 1,188 (80.87%) primer pairs were amplified successfully in the size expected for ‘Dongzao’. A total of 350 primer pairs were further selected and evaluated for their ability to detect polymorphisms across a panel of six diverse cultivars; among these, 301 primer pairs detected polymorphisms, and the polymorphism information content (PIC) value across all loci ranged from 0.15 to 0.82, with an average of 0.52. An analysis of 76 major cultivars employed in Chinese jujube production using 31 primer pairs revealed comparatively high genetic diversity among these cultivars. Within-population differences among individuals accounted for 98.2% of the observed genetic variation. Neighbor-joining clustering divided the cultivars into three main groups, none of which correspond to major geographic regions, suggesting that the genetics and geographical origin of modern Chinese jujube cultivars might not be linked. The current work firstly reports the large-scale development of Chinese jujube SSR markers. The development of these markers and their polymorphic information represent a significant improvement in the available Chinese jujube genomic resources and will facilitate both genetic and breeding applications, further accelerating the development of new cultivars.

Characterization of 10 new nuclear microsatellite markers in Acca sellowiana (Myrtaceae).

• Premise of the study: Microsatellite primers were identified and characterized in Acca sellowiana in order to expand the limited number of pre-existing polymorphic markers for use in population genetic studies for conservation, phylogeography, breeding, and domestication.• Methods and Results: A total of 10 polymorphic microsatellite primers were designed from clones obtained from a simple sequence repeat (SSR)–enriched genomic library. The primers amplified di- and trinucleotide repeats with four to 27 alleles per locus. In all tested populations, the observed heterozygosity ranged from 0.269 to 1.0.• Conclusions: These new polymorphic SSR markers will allow future genetic studies to be denser, either for genetic structure characterization of natural populations or for studies involving genetic breeding and domestication process in A. sellowiana.

Characterization of nineteen microsatellite markers and development of multiplex PCRs for the wedge clam Donax trunculus (Mollusca: Bivalvia).

The wedge clam Donax trunculus is an Atlantic-Mediterranean warm-temperate species found from Senegal to the northern coast of France, including the Mediterranean and Black Sea. It is commercially exploited in several European countries and constitutes an important fishing resource due to its high economical value. To contribute to its conservation and management, nineteen microsatellite markers were isolated from two enriched genomic libraries. These loci were characterized in 30 clams from a single population from northwest Spain. The number of alleles per locus ranged from 2 to 17 and observed and expected heterozygosity varied from zero to 0.714 and from 0.078 to 0.950, respectively. Linkage disequilibrium was not detected and nine loci were in agreement with Hardy-Weinberg equilibrium. Fifteen polymorphic markers were arranged into three multiplex PCR sets to reduce both time and cost of microsatellite genotyping. This is the first time that polymorphic microsatellite markers have been reported for D. trunculus. These new markers provide a valuable resource for future population genetics studies and management and culture of this species.