Enriched Genomic Library Research Articles

Microsatellite markers for Opisthorchis felineus to understand its genetic diversity and transmission patterns of opisthorchiosis.

Opisthorchis felineus is a food-borne trematode which causes opisthorchiosis and affects mainly the liver and bile ducts of the liver with a possible risk of bile duct carcinogenesis resulting in cholangiocarcinoma. In Russia, O. felineus is mainly endemic in Western Siberia (Ob and Irtysh river basins) and occurs throughout the Volga, Kama, Don, and Dnepr river basins. The prevalence, intensity, and clinical significance of human infections and the incidence of cholangiocarcinoma vary geographically in endemic regions. Currently, there is substantial evidence on genetic variation of O. felineus, but information on the population genetic structure is so far very scarce. Because microsatellite DNA of this parasite is not available, we for the first time isolated sufficient microsatellite loci to examine the genetic diversity and population structure of O. felineus, using multiple nuclear loci approach. A total of ten highly polymorphic microsatellite loci from a constructed enriched genomic DNA library were characterized, using 29 samples representing huge O. felineus metapopulation extended in latitude over 5000km from Middle Europe to Western Siberia. At least three populations can be discerned as result of analysis of the microsatellite loci genetic diversity. Based on the results for the first time, a hypothesis was put forward about the formation of a modern habitat of O. felineus.

Molecular insight for baru Dipteryx alata (Fabaceae) populations based on novel SSRs

Baru tree (Dipteryx alata) is an arboreal, fruitful plant native to the Cerrado biome with an important socioeconomic impact. Populations of this species are a good model to study anthropogenic disturbances on the biome through the genetic information. In this study, we developed seven new polymorphic microsatellite markers for D. alata, using an enriched genomic library. We performed loci characterization in three populations, obtaining a total of 49 alleles, with an average of 5 to 5.57 alleles per locus. A significant content of polymorphic information was obtained, as indicated by the average expected heterozygosity (uHE), with a total average of 0.58 to 0.65 per locus. The average value of the observed heterozygosity (Ho) was also high, with a total average of 0.73 to 0.85 per locus. Some of the loci are in linkage disequilibrium, such as DalatG6 with DalatB3, DalatH3 and DalatB4. The estimate of the combined loci for the probability of paternity exclusion obtained an average value of 1.00 for all loci, and the average combined probability of identity, the values were (1.210^-5) to (4.410^-6). All markers are informative and suitable for studies on genetic diversity and population structure, aiming at the conservation and management of the species.

Development of highly polymorphic microsatellite markers for Campanula glomerata L. (Campanulaceae).

Campanula glomerata L. (Campanulaceae) is a self-incompatible, insect-pollinated herb occurring in calcareous grasslands, and is declining and (critically) endangered in many parts of its European distribution range. It often exists as small and isolated populations. A recovery plan of C. glomerata has been implemented in southern Belgium, involving plant translocations. We developed microsatellite markers using an enriched genomic library and characterized 16 loci in 111 individuals from eight populations. These 16 loci were highly polymorphic, with 11 to 31 alleles per locus for a total of 329 alleles, and expected heterozygosity (He) ranging from 0.470 to 0.938. These highly polymorphic loci constitute a promising tool for detailed genetic analyses: assigning individuals to distinct multilocus genotypes will allow quantifying pollen dispersal, clonal propagation and sexual recruitment and identifying admixed seed progeny and their pollen donors. Evaluating the genetic status of existing populations and a genetic monitoring of the translocated populations will contribute to optimize success in restoring viable and evolutionary resilient populations.

Development of microsatellite markers for Calyptranthes clusiifolia (Myrtaceae)

The endemic tree species Calyptranthes clusiifolia (Myrtaceae) plays a relevant ecological role in the forest fragments where it has a common occurrence. In this study, we reported the development of microsatellite markers for C. clusiifolia what will allow a better understanding of the relationship between the forest fragmentation process and the genetic structure and diversity of tree populations. Seven microsatellite markers were developed using an enriched genomic library and characterized in 30 individuals (from three populations). These seven loci were polymorphic and resulted in a total of 23 alleles. The expected heterozygosity (HE) varied from 0.14 (Caly 06) to 0.73 (Caly 22). Linkage disequilibrium between the loci (p > 0.0007) pairs was not detected. The parentage exclusion power of the first (Pe-1) and the second (Pe-2) parents were 0.6099 and 0.8548, respectively. The microsatellite markers developed are indicated for future studies of the genetic diversity in natural populations of C. clusiifolia.

Isolation and characterization of microsatellite markers in two species of the neotropical Epicharis (Hymenoptera, Apidae, Centridini) genus and cross-amplification in related species.

Genetics can provide invaluable tools for management and conservation of bee populations, which are declining worldwide. Among these tools, microsatellite are very useful molecular markers for population analyses. The aim of this study was to isolate and characterize microsatellites for Epicharis (Anepicharis) dejeanii and Epicharis (Epicharis) nigrita, two Neotropical species of solitary bees, both exhibiting the habit of nesting in aggregations. Microsatellite loci were identified from two enriched genomic libraries. The characterization and analysis of loci were carried out using 35 females of E. dejeanii and 34 of E. nigrita. In total, we report the development of 12 microsatellite loci for E. dejeanii and 13 for E. nigrita. For E. dejeanii, all loci were polymorphic, the number of alleles per locus ranged from 2 to 12, averaging 8.7 and, observed and expected heterozygosity were 0.485 (range 0.229-0.857) and 0.633 (range 0.288-0.843), respectively. For E. nigrita, only nine out of 13 loci amplified were polymorphic, the number of alleles per locus ranged from 2 to 12, averaging 5.5. For this species, the observed and expected heterozygosity were 0.440 (range 0.118-0.676) and 0.545 (range 0.167-0.814), respectively. Cross-amplification of primers was successful in other Centridini species. The two sets of loci described for E. dejeanii and E. nigrita species are polymorphic and informative and show promising applicability for both population genetic approaches and relatedness on these and other Centridini species.

Development of tri-nucleotide microsatellite markers from Crassostrea hongkongensis using enriched genomic libraries and cross-species amplification in two closely related species

An enrichment protocol was used to isolate and characterize tri-nucleotide microsatellite markers in Crassostrea hongkongensis, an economically important and extensively cultured oyster species along the southern coastal areas of China. Thirty tri-nucleotide microsatellite loci were assessed in 32 individuals from a wild population. The number of alleles ranged from 2 to 12, averaging 7.63 alleles per locus. The observed and expected heterozygosity values ranged from 0.031 to 0.844 (averaging 0.579) and 0.062 to 0.881 (averaging 0.665), respectively. Six loci significantly deviated from Hardy-Weinberg equilibrium after Bonferroni correction. BLAST analysis showed that the tri-nucleotide microsatellite markers randomly developed from the genome of C.hongkongensis have a higher correlation with functional genes than di-nucleotide markers. Cross-amplification in C. ariakensis and C. gigas was performed and 21 loci showed reasonable polymorphism between C. hongkongensis and C. ariakensis, while 15 loci showed reasonable polymorphism between C. hongkongensis and C. gigas. The tri-nucleotide microsatellite markers identified in this study have greater potential for future use in gene mapping, molecular-marker assisted selection and comparative linkage mapping than previously identified di-nucleotide markers. Additionally, we found two microsatellite loci that can effectively identify the purebred and hybrid offspring of these three oyster species.

Research Article Development and transferability of microsatellite loci for <i>Spondias</i> <i>tuberosa</i> (Anacardiaceae: Sapindales), a species endemic to the Brazilian semi-arid region

The umbu tree (Spondias tuberosa) is one of the best known plants of the Brazilian semi-arid region; it has great potential for the fruit market due to excellent consumer acceptance. This tree is not presently cultivated; fruit commercialization is based on extrativism. Consequently, there has been little research on the genetics of this species. Our objective was to develop, evaluate and transfer single sequence repeat (SSR) loci to S. tuberosa to support work on genetic resources and agronomic improvement of this species. SSR loci for the umbu tree were developed from a new enriched genomic library and evaluated by PCR. Fourteen SSR loci developed for S. radlkoferi were evaluated for use in S. tuberosa, as well as 18 SSR loci previously identified for this species. DNA was extracted from leaf tissue of eight umbu trees available that are part of a germplasm collection located in Petrolina, PE, Brazil. Of the 14 pairs of primers that were tested, six yielded amplicons, and two showed polymorphism in the genotyped samples. All SSR loci of S. radlkoferi transferred to the umbu tree species, yielding amplicons; however, only four were polymorphic in this sample. Among the eighteen available species-specific SSR loci, six were polymorphic for these eight trees. Among the 38 loci that we evaluated, 12 were polymorphic in the sample, including two new loci for S. tuberosa. The number of alleles ranged from 2 to 12, and 10 of them were moderately to highly informative (PIC>0.50), while nine had heterozygosity greater than 0.50. The six new SSR loci and the 14 SSR loci transferred from S. radlkoferi increase the number of available loci for population studies, germplasm collection and resource management for the development of new umbu tree cultivars.

Development and characterization of microsatellite markers for Antennaria corymbosa (Asteraceae) and close relatives.

PremiseThe genus Antennaria has a complex evolutionary history due to dioecism, excessive polyploidy, and the evolution of polyploid agamic complexes. We developed microsatellite markers from A. corymbosa to investigate genetic diversity and population genetic structure in Antennaria species.Methods and ResultsTwenty‐four novel microsatellite markers (16 nuclear and eight chloroplast) were developed from A. corymbosa using an enriched genomic library. Ten polymorphic nuclear markers were used to characterize genetic variation in five populations of A. corymbosa. One to four alleles were found per locus, and the expected heterozygosity and fixation index ranged from 0.00 to 0.675 and −0.033 to 0.610, respectively. We were also able to successfully amplify these markers in five additional Antennaria species.ConclusionsThese markers are promising tools to study the population genetics of sexual Antennaria species and to investigate interspecific gene flow, clonal diversity, and parentage of Antennaria polyploid agamic complexes.

Development and characterization of microsatellite loci for <i>Campomanesia xanthocarpa</i> (Myrtaceae) and cross amplification in related species

Campomanesia xanthocarpa is a native tree, of common occurrence in almost all Brazilian Forest formations, which has its fruits and timber with high commercial value. Using an enriched genomic library we isolated and characterized microsatellite loci for C. xanthocarpa (Myrtaceae), in order to estimate genetic diversity parameters for this and related species. Twenty-eight microsatellite loci were identified and ten of them successfully amplified and showed polymorphism in a sample of 96 individuals, from four natural populations. The number of alleles per locus ranged from two to eight, and the observed and expected heterozygosities varied from 0.042 to 1.000 and from 0.294 to 0.855, respectively. These markers were tested and validated in two related species ( C. eugenioides and C. guazumifolia ). The microsatellite markers will be used in further studies of population genetics of C. xanthocarpa , in order to understand the genetic variability and to define the strategies needed for the conservation of the species.

Isolation and characterization of 12 microsatellite loci in Epipedobates anthonyi (Amphibia: Anura: Dendrobatidae) for population genetic analysis.

Tropical anurans are among the most diverse and vulnerable organisms on Earth, yet the evolutionary mechanisms behind their diversity remain relatively unexplored. Epipedobates anthonyi is a poison frog that inhabits southern Ecuador and northern Peru along a broad elevational range (0-1800m). Throughout its range, this species exhibits variation in phenotypic traits, such as color, advertisement calls, and alkaloid composition. The aim of this study is to isolate and characterize microsatellite loci to investigate patterns of genetic variation within the species. Using a next-generation sequencing approach to screen an enriched genomic library, we report twelve polymorphic microsatellite loci. The number of alleles per locus ranged from 7 to 15 per population. For the two populations tested, mean observed heterozygosity was 0.69 and 0.79, and mean expected heterozigosity was 0.84 and 0.85 respectively. Only locus EAN002 showed significant departure of HWE in both populations. None of the loci showed consistent null alleles in both populations. Also, no evidence of linkage disequilibrium was found across loci. In this paper, we report for the first time 12 microsatellite loci for E. anthonyi. These markers will be used to further elucidate evolutionary mechanisms underlying genetic and phenotypic variation across the species' range.

Development of New SSR (Simple Sequence Repeat) Markers for Lentils (Lens culinaris Medik.) from Genomic Library Enriched with AG and AC Microsatellites.

Simple sequence repeat (SSR) markers are the major molecular tools for genetic and genomic researches that have been extensively developed and used in major crops. However, few are available for lentils (Lens culinaris M.), economically an important cool-season legume. The lack of informative simple sequence repeat (SSR) markers in lentil has been a major limitation for lentil molecular breeding studies. Therefore, in order to develop SSR markers for lentil, an enriched genomic libraries for AC and AG repeats were constructed from the Lens culinaris cv Kafkas. A total of 350 clones were inquired for the detection of SSRs. Of 350 clones, 68 had SSR motifs. In polymorphism analysis using 53 newly developed SSRs, a total of 144 alleles across 24 lentil cultivars were detected with an average of 4.64 per locus. The average heterozygosity was 0.588 and polymorphism information contents ranged from 0.194 to 0.895 with an average value of 0.520. These newly developed SSRs will constitute useful tools for molecular breeding, mapping, and assessments of genetic diversity and population structure of lentils.

QTL detection for growth and latex production in a full-sib rubber tree population cultivated under suboptimal climate conditions

BackgroundRubber tree is cultivated in mainly Southeast Asia and is by far the most significant source of natural rubber production worldwide. However, the genetic architecture underlying the primary agronomic traits of this crop has not been widely characterized. This study aimed to identify quantitative trait loci (QTLs) associated with growth and latex production using a biparental population established in suboptimal growth conditions in Brazil.ResultsA full-sib population composed of 251 individuals was developed from crossing two high-producing Asiatic rubber tree cultivars, PR 255 and PB 217. This mapping population was genotyped with microsatellite markers from enriched genomic libraries or transcriptome datasets and single-nucleotide polymorphism (SNP) markers, leading to construction of a saturated multipoint integrated genetic map containing 354 microsatellite and 151 SNP markers. Height and circumference measurements repeated over a six-year period and registration of cumulative latex production during six consecutive months on the same individuals allowed in-depth characterization of the genetic values of several growth traits and precocious latex production. Growth traits, circumference and height, were overall positively correlated, whereas latex production was not correlated or even negatively correlated with growth traits. A total of 86 distinct QTLs were identified, most of which were detected for only one trait. Among these QTLs, 15 were linked to more than one phenotypic trait (up to 4 traits simultaneously). Latex production and circumference increments during the last wintering period were associated with the highest numbers of identified QTLs (eleven and nine, respectively), jointly explaining the most significantly observed phenotypic variances (44.1% and 44.4%, respectively). The most important QTL for latex production, located on linkage group 16, had an additive effect of the male parent PB 217 and corresponded to a QTL at the same position detected in a previous study carried out in Thailand for the biparental population RRIM 600 x PB 217.ConclusionsOur results identified a set of significant QTLs for rubber tree, showing that the performance of modern Asiatic cultivars can still be improved and paving the way for further marker-assisted selection, which could accelerate breeding programs.

English

Ten newly developed microsatellite loci were isolated from an AG20 and CAG20 enriched genomic DNA library of Radix sp. from Iraq. The simple sequence repeats (SSR) comprised 76.0% di-, 20.0% tri- and 4.0% tetra-nucleotide repeats. The number of alleles for all 10 loci ranged from 4 to 18, with a mean of 11.3 alleles per locus. The observed (Ho) and expected heterozygosity (He) values varied from 0.286 to 0.8670.927 (mean 0.640) and from 0.263 to 0.939 (mean 0.753), respectively. The polymorphic information content (PIC) values ranged from 0.336 to 0.923 (mean 0.749). Five loci showed significant deviation from Hardy-Weinberg equilibrium (HWE) and no significant linkage disequilibrium (LD) was observed. Cross transferability was successfully tested across on species from the genus Melanopsis. These polymorphic SSRs will be useful for assessment of the genetic diversity and population genetics of a Radix sp. as well as other related species in Iraq. Key words: Cross-transferability, eenriched genomic DNA library, ffreshwater snail, microsatellitemicrosatellite.

Development and characterization of Novel Microsatellite markers in great snakehead, Channa marulia ( )

The species-specific microsatellite markers in the great snakehead, Channa marulia, were mined from the genome, through an enriched genomic library construction. Total 27 microsatellite loci exhibited polymorphism. The repeat containing sequences were annotated to ensure the genetic neutrality of the loci. Nineteen, consistently scorable loci were validated, and used to estimate the genetic indices in n = 67 individuals sampled from three Indian rivers (Godavari, Mahanadi and Teesta). The polymorphism information content (PIC) values ranged from 0.366 to 0.831, while heterozygosities varied from 0.077 to 0.818. Bayesian model-based clustering grouped the samples into three discrete populations with evidences of little genetic admixture. The results confirmed a set of 19 microsatellite markers, as promising molecular tool, for estimating genetic diversity studies in C. marulia. Successful cross-transferability was observed in three congener species viz. C. punctata, C. gachua, and C. striata. The results can be useful in conservation and management of C. marulia.

Development of 15 microsatellite markers in Acer triflorum (Aceraceae) and cross-amplification in congeneric species.

Premise of the Study Acer (Aceraceae) is an important genus in forest ecosystems in the Northern Hemisphere. In China, 151 species have been reported, and approximately 61 species are endemic. Thus, China is considered to host the greatest diversity of Acer, but markers are needed to evaluate the genetic structure and genetic diversity of these populations of wild Acer species.Methods and ResultsUsing an enriched genomic library, we developed and characterized 15 microsatellite primers for A. triflorum, 10 of which were polymorphic. The number of alleles varied from one to nine. The levels of observed heterozygosity and expected heterozygosity per locus ranged from 0.000 to 1.000 and 0.000 to 0.826, respectively. Most primers also successfully amplified in A. ginnala, A. griseum, A. mandshuricum, A. pseudosieboldianum, A. sinopurpurascens, A. tegmentosum, and A. ukurunduense.ConclusionsThese markers from A. triflorum will provide an opportunity to study genetic diversity and genetic structure in the genus Acer.

Isolation and Characterization of Novel Polymorphic Microsatellite Markers for Cimex hemipterus F. (Hemiptera: Cimicidae).

Due to the growing public health and tourism awareness, Cimex hemipterus Fabricius (Hemiptera: Cimicidae) has gained a great interest in increasing reported infestation cases in tropical regions of the world, including Malaysia. Since the information on the molecular ecology and population biology of this species are tremendously lacking, the isolation and development of molecular markers can be used to determine its genetic structure. In this study, novel microsatellite primers isolated from enriched genomic libraries of C. hemipterus were developed using 454 Roche shotgun sequencing. Seven validated polymorphic microsatellite primers were consistently amplified and characterized from 70 tropical bed bugs collected from seven locations throughout Malaysia. The number of alleles per locus identified ranged from 6 to 14. Comparison of loci for overall and between population were done with mean observed and expected heterozygosity were determined at 0.320 and 0.814, 0.320 and 0.727, respectively. Polymorphic information criteria (PIC) valued the markers as highly informative as PIC >0.5. Overall population, they are possibly in Hardy-Weinberg equilibrium with loci Ch_09ttn, Ch_01dn, and Ch_13dn showing signs of a null allele. There were no scoring errors caused by stutter peaks, no large allele dropout was detected for all loci and showed no evidence of linkage disequilibrium. In conclusion, all seven molecular microsatellite markers identified can be beneficially used to gain more information on the population genetic structure and breeding patterns of C. hemipterus as well as the relationship of dispersal and infestation.

Microsatellites for Phytolacca acinosa (Phytolaccaceae), a traditional medicinal herb.

Premise of the study:Phytolacca acinosa (Phytolaccaceae) is a traditional Chinese herb with multiple medicinal uses and is an important pigment source. Due to excessive human harvesting, the population numbers and sizes have decreased dramatically.Methods and Results:Using an enriched genomic library, we developed and characterized 15 microsatellite primers for P. acinosa, 13 of which were polymorphic. The number of alleles varied from two to seven. The observed heterozygosity and expected heterozygosity per locus ranged from 0.267 to 1.000 and 0.331 to 0.743, respectively. All of the primers that were developed were also successfully applied in P. americana.Conclusions:These markers should be useful in probing the genetic diversity, genetic structure, and mating systems of P. acinosa, which could provide information about protecting and sustainably harvesting this species.

Isolation and Characterisation of 11 Microsatellite Loci in the Ecuadorian Hillstar Oreotrochilus chimborazo

The Ecuadorian Hillstar Oreotrochilus chimborazo is a high Andean hummingbird that inhabits paramo ecosystems from extreme southern Colombia to southern Ecuador. The species is currently divided into three subspecies that differ in morphology and geographic distribution but no molecular differences have been found between them using mitochondrial markers. The aim of this study was to isolate and test microsatellite loci to further explore possible genetic variability between and within these subspecies. Screening an enriched genomic library of O. chimborazo we obtained eleven polymorphic microsatellite loci. These markers will be used to further elucidate evolutionary patterns in the species.

Microsatellite markers for Rhyzopertha dominica (Coleoptera: Bostrichidae)

Rhyzopertha dominica (F.) is a destructive insect pest of stored products. Due to this pest's broad host range, short life cycle, and high reproductive capacity, it has rapidly spread throughout the world. Understanding the population structuring of this important pest is vital for developing and implementing an effective integrated pest management strategy. In this study, we isolated 19 novel polymorphic microsatellite loci from an enriched genomic library based on a biotin/streptavidin capture protocol. The genetic parameters were estimated based on 80 individual R. dominica from two natural populations. The numbers of alleles ranged from 3 to 10, and sixteen loci exhibited polymorphic information contents (PICs) greater than 0.5. The observed (HO) and expected (HE) heterozygosities ranged from 0.033 to 0.900 and 0.310 to 0.882, respectively. Six locus/population combinations significantly deviated from Hardy–Weinberg equilibrium (HWE). These microsatellite markers will provide a valuable resource for studying the population genetic structure of this species.

A Set of 20 New SSR Markers Developed and Evaluated in Mandevilla Lindl.

Mandevilla is an ornamental crop with a bright future worldwide because of its high commercial acceptance and added value. However, as with most ornamental species, there are few molecular tools to support cultivar breeding and innovation. In this work, we report the development and analysis of 20 new Simple Sequence Repeat (SSR) markers in Mandevilla. Microsatellites were isolated from two enriched small-insert genomic libraries of Mandevilla × amabilis. The diversity parameters estimated after their amplification in a group of 11 commercial genotypes illustrate the effect of two opposite drifts: the high relatedness of cultivars belonging to the same commercial group and the high divergence of other cultivars, especially M. × amabilis. Based on their different band patterns, six genotypes were uniquely distinguished, and two groups of sport mutations remained undistinguishable. The amplification of the SSRs in three wild species suggested the existence of unexploited diversity available to be introgressed into the commercial pool. This is the first report of available microsatellites in Mandevilla. The development process has provided some clues concerning the genome structure of the species, and the SSRs obtained will help to create new products and to protect existing and upcoming plant innovations.