PCR-based Isolation Of Microsatellite Arrays Research Articles

Microsatellite Marker Mining Using PCR-Based Isolation of Microsatellite Arrays (PIMA) Method on Blue-Spotted Mudskipper, Boleophthalmus Boddarti

Microsatellites are small and are codominant markers that can be amplified with polymerase chain reaction. Both prokaryotic and eukaryotic organisms possess large amounts of the microsatellites repeat. Many microsatellites have high mutation rates that generate the high levels of allelic diversity necessary for genetic studies of processes acting on ecological time scales. The high variability of microsatellites provided the foundation for their successful application in a wide range of fundamental and applied fields of biology. However, de novo isolation is needed for most species hence in this study we tried to mine the microsatellite marker using PCR-based isolation of microsatellite arrays (PIMA) on Blue spotted mudskipper, Boleophthalmus boddarti a fish uniquely restricted to coastal and estuarine habitat was also commercially important. Out of three trials, seven microsatellite repeats were detected but only three repeat types (AAG)4, (TCAG)3 and (CT)4 can be useful as microsatellite marker following PHOBOS V3.3.12 analysis. Meanwhile, the detection of octa (AATACAT)2, penta (TGACA)2 and heptanucleotides (GGAGATA)2 were unable to be continued as functional microsatellite marker as there were missense variants and interruptions detected either on forward or reverse strand of the repeat. Thus, PIMA method could be considered as tedious and detected low yields of microsatellite markers. Nevertheless, the conventional method for generating microsatellite markers from PCR based methods could be done with in silico mining of microsatellite sequences from DNA sequence databases or next generation sequencing (NGS).

Trypanosoma rangeli displays a clonal population structure, revealing a subdivision of KP1(−) strains and the ancestry of the Amazonian group

Assessment of the genetic variability and population structure of Trypanosoma rangeli, a non-pathogenic American trypanosome, was carried out through microsatellite and single-nucleotide polymorphism analyses. Two approaches were used for microsatellite typing: data mining in expressed sequence tag /open reading frame expressed sequence tags libraries and PCR-based Isolation of Microsatellite Arrays from genomic libraries. All microsatellites found were evaluated for their abundance, frequency and usefulness as markers. Genotyping of T. rangeli strains and clones was performed for 18 loci amplified by PCR from expressed sequence tag/open reading frame expressed sequence tags libraries. The presence of single-nucleotide polymorphisms in the nuclear, multi-copy, spliced leader gene was assessed in 18 T. rangeli strains, and the results show that T. rangeli has a predominantly clonal population structure, allowing a robust phylogenetic analysis. Microsatellite typing revealed a subdivision of the KP1(−) genetic group, which may be influenced by geographical location and/or by the co-evolution of parasite and vectors occurring within the same geographical areas. The hypothesis of parasite–vector co-evolution was corroborated by single-nucleotide polymorphism analysis of the spliced leader gene. Taken together, the results suggest three T. rangeli groups: (i) the T. rangeli Amazonian group; (ii) the T. rangeli KP1(−) group; and (iii) the T. rangeli KP1(+) group. The latter two groups possibly evolved from the Amazonian group to produce KP1(+) and KP1(−) strains.

Development and characterization of nine polymorphic microsatellite markers from the sword prawn Parapenaeopsis hardwickii using PCR-based isolation of microsatellite arrays (PIMA)

Nine polymorphic microsatellite makers were isolated and characterized in 20 individuals of Parapenaeopsis hardwickii collected from the fishing ground near Ilan, Taiwan. Number of alleles per locus ranged from 3 to 8, observed and expected heterozygosities ranged from 0.5263 to 0.7632, and from 0.4225 to 0.8044, respectively. Only one locus showed a Hardy–Weinberg equilibrium deviation. No linkage disequilibrium was found among these studied loci. This set of microsatellites provides a suitable tool for population genetic structure and fishery management of this species in Asian coastal waters.

Isolation and Characterization of Polymorphic Microsatellite Loci from Metapenaeopsis barbata Using PCR-Based Isolation of Microsatellite Arrays (PIMA)

The red-spot prawn, Metapenaeopsis barbata, is a commercially important, widely distributed demersal species in the Indo-West Pacific Ocean. Overfishing has made its populations decline in the past decade. To study conservation genetics, eight polymorphic microsatellite loci were isolated. Genetic characteristics of the SSR (simple sequence repeat) fingerprints were estimated in 61 individuals from adjacent seas of Taiwan and China. The number of alleles, ranging from 2 to 4, as well as observed and expected heterozygosities in populations, ranging from 0.048 to 0.538, and 0.048 and 0.654, respectively, were detected. No deviation from Hardy–Weinberg expectations was detected at either locus. No significant linkage disequilibrium was detected in locus pairs. The polymorphic microsatellite loci will be useful for investigations of the genetic variation, population structure, and conservation genetics of this species.

Development and characterization of a novel set of microsatellite markers for Arisaema serratum (Araceae)

We developed novel microsatellite markers in Arisaema serratum, a perennial herb that possesses pitfall flowers and exhibits labile sex expression, to facilitate research on parentage and pollination biology in this species. By using procedures for enrichment of desired microsatellite-containing fragments and PCR-based isolation of microsatellite arrays, we detected 18 novel microsatellite loci. Thirteen were highly polymorphic: the number of alleles per locus ranged from six to 46, the observed heterozygosities ranged from 0.320 to 0.940, and the expected heterozygosities ranged from 0.440 to 0.976. Nine of the 13 markers successfully amplified regions in congeneric species. These highly polymorphic markers will facilitate further studies on the mode of pollination and other aspects of reproductive biology in A. serratum.

Isolation and characterization of polymorphic microsatellite markers from Japanese grenadier anchovy (Coilia nasus)

We isolated and characterized 12 novel polymorphic microsatellite loci for the Japanese grenadier anchovy (Coilia nasus) using the method of PCR-based isolation of microsatellite arrays. Sixteen individuals of C. nasus from Ariake Bay in Japan were genotyped using 24 PCR primers to characterize the polymorphism at each locus. The average number of the observed alleles per locus was 11.08 (ranging from 8 to 20). The observed heterozygosity values ranged from 0.562 to 1.0, and the expected heterozygosity values ranged from 0.647 to 0.969. Only one of the twelve loci deviated significantly from Hardy–Weinberg equilibrium, and linkage disequilibrium was not found in any pairs of the loci. These markers can be used to investigate the genetic structure of the C. nasus population.

Polymorphic Microsatellite Loci Isolated from the Squalidus argentatus Using PCR-Based Isolation of Microsatellite Arrays (PIMA)

Squalidus argentatus (Sauvage and Dabry de Thiersant 1874) is a small-sized freshwater fish which is distributed in Mainland China, Hainan Island and Taiwan. The populations of S. argentatus have dropped sharply probably due to overharvesting and water pollution recently. Eleven polymorphic microsatellite markers were developed for the cyprinid fish S. argentatus. These new markers were tested on 43 individuals collected from Yangtze River and Qiantang River. The number of alleles, observed and expected heterozygosity per locus, in two populations ranged from 3 to 14, from 0.333 to 0.954 and from 0.480 to 0.928, respectively. Only two loci are significantly deviated from Hardy–Weinberg expectations due to the heterozygote deficiency. No significant linkage disequilibrium was detected between the pairwise comparisons of these loci. These polymorphic microsatellite loci will enable us to study the genetic variation, population structure, and conservation genetics of this species in the future.

Short Communication Isolation and characterization of microsatellite markers for the White Cloud Mountain minnow (Tanichthys albonubes) in wild and cultured populations

We developed 12 microsatellite loci for the endangered minnow species, Tanichthys albonubes, using PCR-based isolation of microsatellite arrays. These new markers were tested in 26 individuals from a wild population collected from Guangzhou in China and 26 individuals from a cultured strain. The number of alleles ranged from two to nine and the expected heterozygosity from 0.177 to 0.853. The wild population had significantly higher allelic richness than the cultured strain, with a mean allelic richness of 5.52 (range = 3.69-8.64) and 3.13 (range = 1.99-5.73) for the wild population and the cultured strain, respectively. No evidence of a recent bottleneck was detected in the wild population, but it was found in the cultured strain based on the BOTTLENECK test. These primers can be used to understand the demography and to examine genetic differences between the cultured T. albonubes strains and wild populations to help determine conservation and reintroduction strategies.

Novel Polymorphic Microsatellite Markers in Scylla paramamosain and Cross-Species Amplification in Related Crab Species

Abstract Scylla paramamosain is a commercially important crab species in China. Here we reported 12 novel polymorphic microsatellite markers isolated in S. paramamosain using PCR-based isolation of microsatellite arrays (PIMA). The analysis of genetic variability was performed in a sample of 29 individuals. A total of 82 alleles were detected with an average of 6.8 alleles per locus. The number of alleles, polymorphism information content (PIC), observed and expected heterozygosity per locus ranged from three to 12, from 0.42 to 0.88, from 0.38 to 1.00 and from 0.48 to 0.91, respectively. Three loci significantly deviated from Hardy-Weinberg equilibrium after Bonferroni correction (P < 0.0042) and no significant linkage disequilibrum between pairs of loci was found. Cross-species amplification of these markers was evaluated in three closely related crab species S. tranquebarica, S. olivacea, and S. serrata. This study will potentially be useful for stock management, constructing of a genetic linkage map, ...

Development of polymorphic microsatellite loci for Iranian river buffalo (Bubalus bubalis )

Microsatellite loci were developed using PCR-based isolation of microsatellite arrays (PIMA) for Iranian river buffalo. Blood samples of eighty unrelated individuals from four buffalo populations (Khuzestan, Mazandaran, Guilan and Azarbayejan) were taken and following DNA extraction, isolation of microsatellite loci initiated using enrichment with random amplified polymorphic DNA (RAPD) primers. RAPD-PCR fragments were ligated into PTZ57R TA cloning vector and transformed into DH5
competent cells. Obtained colonies were screened for presence of repetitive elements by repeatspecific and M13 forward and reverse primers. After designing primer pairs for repeat containing fragments, they were tested in all buffalo populations. Two microsatellite loci (RBBSI and RBBSII) were informative and polymorphic. Number of alleles for RBBSI and RBBSII in 80 individuals was 5 and 6, respectively. Expected heterozygosity ranged from 0.65 to 0.81. Significant deviation from Hardy-Weinberg equilibrium expectation occurred for both loci in all populations, but 37.5% of locus/population combination showed the deviation. We postulate that the two newly isolated microsatellite loci during this study could be useful for population genetic studies in Bubalus bubalis.

Isolation and characterization of polymorphic microsatellite loci in Candidia barbata (Cyprinidae) using PCR-based isolation of microsatellite arrays (PIMA)

Candidia barbata is an endemic cyprinid fish in Taiwan and suffers habitat destruction caused by human overexploitation. In this study, a total of 14 polymorphic microsatellite loci from C. barbata were isolated and characterized using an optimized protocol to construct a microsatellite-enriched genomic library. The analysis of variability was performed in 30 specimens of Taiwan. The mean number of alleles across loci was 4.92 ± 1.44. The levels of expected and observed heterozygosities varied from 0.1266 to 0.5079, and from 0.0667 to 0.9667, respectively. Frequencies of null alleles of the 14 loci are not significantly greater than zero. No linkage disequilibrium was detected between loci in either population.

PERMANENT GENETIC RESOURCES: Isolation and characterization of microsatellite loci in the North Island brown kiwi, Apteryx mantelli

We report the isolation and characterization of eight polymorphic and five monomorphic microsatellites in North Island brown kiwi (NIBK, Apteryx mantelli), using two polymerase chain reaction (PCR) techniques employing either short-tandem repeat primers (STR method) or random PCR-based isolation of microsatellite arrays (PIMA method). Microsatellite polymorphism was subsequently determined using 65 individuals. There were two to seven alleles for each polymorphic locus with heterozygozity ranging between 0.04 and 0.86. These primers will be used in future studies to determine the level of extra-pair copulation, dispersal patterns, and genetic diversity within and between wild populations of NIBK.

Isolation and characterization of microsatellite loci in the commercially important mudshrimp Austinogebia edulis (Upogebiidae) using PCR-based isolation of microsatellite arrays (PIMA)

Populations of mudshrimp, Austinogebia edulis, in the intertidal mud flat of western Taiwan have severely declined due to habitat destruction and overfishing in the past decades. Nine polymorphic microsatellite loci were isolated for this ecologically threatened species by using a polymerase chain reaction-based procedure. The number of alleles per locus ranged from five to 14 in 20 specimens from Central West Taiwan. Observed and expected heterozygosities ranged from 0 to 0.2500 (averaged at 0.0944) and 0.7333 to 0.9385 (averaged at 0.8524), respectively. There were significant departures from Hardy–Weinberg expectations. Significant LD was discovered in most loci. These primers may provide a tool for understanding population structure in A. edulis.

Isolation and characterization of microsatellite loci in Pedicularis verticillata L. using PCR-based isolation of microsatellite arrays (PIMA)

Pedicularis verticillata L. is a highly valuable herb for traditional Chinese medical treatment. In this report, 11 microsatellite loci from P. verticillata were isolated. The simple sequence repeat (SSR) markers were screened in 23 samples of wild populations of P. verticillata, and eight samples from its sister P. ikomai. The number of alleles ranged from 4 to 13, and value of expected (HE) and observed (H0) heterozygosity was 0.62609–0.89662 and 0–0.95652, respectively. All loci were significantly deviated from Hardy–Weinberg expectations due to the heterozygote deficiency, indicating a dramatic loss of genetic polymorphisms in the restrictedly distributed species. The markers amplified well in the two species are useful for examining genetic diversity and population genetic structure, which, in turn, can provide information for establishing conservation strategy for these endangered species.

Isolation and characterization of 11 microsatellite loci from Camellia sinensis in Taiwan using PCR-based isolation of microsatellite arrays (PIMA)

We report 11 novel microsatellite primer pairs for the wild tea, Camellia sinensis (L.) O. Kuntze forma formosensis Kitamura. These simple sequence repeat markers were tested in 24 samples collected from wild tea populations, and in cultivars and C. japonica. The number of alleles ranged from 4 to18. The expected (H(E)) and observed (H(O)) heterozygosity were 0.687-0.946 and 0.042-0.792, respectively. All loci were significantly deviated from Hardy-Weinberg expectations due to the heterozygote deficiency, indicating a dramatic loss of genetic polymorphisms in the rare species. Significant LD was discovered in most loci. These primers may provide a tool for understanding demography and population structure in wild tea.

Isolation and characterization of microsatellite loci in Acrossocheilus paradoxus (Cyprinidae) using PCR-based isolation of microsatellite arrays (PIMA)

Isolation and characterization of microsatellite loci in Acrossocheilus paradoxus (Cyprinidae) using PCR-based isolation of microsatellite arrays (PIMA)

A polymorphic, telomeric-like sequence microsatellite in the Neotropical fish Prochilodus

A microsatellite locus from the Neotropical fish genus Prochilodus was isolated using PCR-based isolation of microsatellite arrays. Of 470 positive clones, 15 were sequenced, and 10 of them showed an (AATTT)_n repeat. Primers were designed, and analysis of polymorphism revealed 11 alleles in three Prochilodus species. Fluorescence in situ hybridization analysis showed signals predominantly in the telomeric regions of several chromosomes. The description of this microsatellite may contribute to studies of the population structure of this fish group.