Isolation and characterization of polymorphic microsatellite loci in Castanopsis chinensis Hance (Fagaceae)

Twelve novel microsatellite loci were isolated and characterized from enriched genomic libraries of Castanopsis chinensis. Four previously reported microsatellites from Castanopsis cuspidata were cross-amplified in C. chinensis. Forty-two sample trees from a wild population were tested for polymorphism using a set of the 16 polymorphic microsatellites. The average allele number of these microsatellites was 4.6 per locus, ranging from 2 to 7. The ranges of observed and expected heterozygosity were 0.262–1.000 and 0.238–0.818, respectively. Significant deviations from Hardy–Weinberg equilibrium were detected at five loci and no linkage disequilibrium was observed.     

Isolation and characterization of microsatellite loci in Paspalum notatum Flüggé (Poaceae)

Paspalum notatum is a forage grass recognized as one of the major constituents of the native grasslands in the New World. The knowledge of the genetic diversity and structure of P. notatum populations is fundamental for the conservation and germplasm management of this species. About 11 microsatellite markers were isolated from P. notatum and characterized in 25 accessions. The average number of alleles per locus was 7.9 and the PIC ranged from 0.36 to 0.89. The data demonstrated that the most of markers are suitable to detect polymorphism and to study the genetic diversity in the P. notatum species. Moreover, the transferability of these microsatellite were tested on other three congeneric species.     

Isolation and charaterization of 12 dinucleotide microsatellite loci from Belenger’s jewfish (Johnius belengerii Cuvier 1830)

We describe the first isolation of 12 polymorphic microsatellite markers from Belenger’s jewfish (Johnius belengnerii Cuvier 1830). From a (GT)n-enriched genomic library, 54 microsatellites were selected for designing microsatellite primers, of which 36 gave working primer pairs. 12 of these loci were polymorphic in a test population of 21 individuals with alleles ranging from 3 to 18, and expected and observed heterozygosities from 0.5772 to 0.9449 and from 0.4286 to 0.9231, respectively. No significant linkage disequilibrium between pairs of loci was found, however, loci Jobe24 significantly deviated from Hardy–Weinberg equilibrium after Bonferroni correction. These polymorphic microsatellite loci should provide sufficient level of genetic diversity to investigate population structure in Belenger’s jewfish.     

Isolation and characterization of polymorphic microsatellite loci from a dinucleotide-enriched genomic library of spotted maigre (Nibea albiflora)

In the present study, we reported 13 polymorphic microsatellite loci isolated from a dinucleotide-enriched genomic library of Nibea albiflora. The number of alleles, observed and expected heterozygosity per locus in 44 individuals ranged from 2 to 13, from 0.0909 to 0.9773 and from 0.0886 to 0.9073, respectively. Three loci significantly deviated from Hardy–Weinberg equilibrium after Bonferroni correction. Analysis of linkage disequilibrium showed non-significant among the two pairs of loci. As a result, 13 microsatellite loci probably located on different chromosome pairs and these polymorphic microsatellite loci should provide sufficient level of genetic diversity to investigate the fine-scale population structure and evaluate the breeding strategy in Nibea albiflora. Shichao Xing, Changwei Shao contributed equally to this work.     

Development and characterization of 11 polymorphic compound tetranucleotide microsatellite loci for the Leisler’s bat, Nyctalus leisleri (Vespertilionidae, Chiroptera)

Eleven polymorphic microsatellite marker loci were developed from a Leisler’s bat (Nyctalus leisleri) genomic enriched library. Assessment of the usefulness of these markers for population genetics studies of Leisler’s bats was carried out by screening 100 specimens sampled from eight locations in Ireland and two in Northeastern France. Both moderately and highly polymorphic marker loci were identified. Five to 28 alleles were found to be segregating per locus with observed heterozygosities values ranging from 28.4 to 94%. Initial evaluation indicates that these microsatellites will be useful for genetic based studies aiming, for instance, at parentage and population structure of Leisler’s bats.     

Isolation and characterization of marine biofilm forming bacteria from a ship’s hull

Background

Diverse aquatic microorganisms are capable of colonizing living and non-living surfaces leading to the formation of biofilms. Commonly visualized as a slimy layer, these biofilms are filled with hundreds of other microorganisms compared to free living planktonic cells. Microbial surface colonization and surface-associated metabolic activities also exert several macroscale deleterious effects, including biofouling, biocorrosion and the persistence and transmission of harmful or pathogenic microorganisms and virulence determinants. The present study deals with the isolation and screening of marine bacteria for biofilm formation. The screened isolates were characterized and identified as Pychrobacter celer, Pychrobacter alimentarius and Kocuria rhizophila by 16S rRNA sequencing.

Methods

Biofilm forming bacteria were isolated by spread plate technique and subjected to screening by microtiter plate assay. The potent biofilm formers were identified by molecular characterization using 16S rRNA gene sequencing.

Results

Twelve bacterial isolates were obtained by pour plate technique and subjected to biofilm assay. Among the 12 isolates three isolates which showed maximum biofilm formation were subjected to molecular characterizationby 16S rRNA gene sequencing method. The isolates were identified as Pychrobacter celer, Pychrobacter alimentarius and Kocuria rhizophila. The EPS produced by the three biofilm forming bacteria was extracted and the protein and carbohydrate content determined.

Conclusion

Among the isolates screened, isolate 8 (Kocuria rhizophila) produced maximum protein and carbohydrate which was also in accordance with the results of microtiter plate assay.

     

Isolation and characterization of polymorphic microsatellite loci from a repeat-enriched genomic library of stone flounder (Kareius bicoloratus) and cross-species amplification

Stone flounder (Kareius bicoloratus) is a rare fish species in China. Here, we reported 11 polymorphic microsatellite loci isolated from a repeat-enriched genomic library of stone flounder. The number of alleles, observed and expected heterozygosity per locus in 32 individuals ranged from 3 to 6, from 0.1613 to 0.8667 and from 0.1549 to 0.7932, respectively. Two loci significantly deviated from Hardy–Weinberg equilibrium after Bonferroni correction and no significant linkage disequilibrium between pairs of loci was found. Cross-species amplification of these microsatellite loci in additional three fish species was performed. These polymorphic microsatellite loci would be useful for investigating genetic population structure and construction of genetic linkage map in Kareius bicoloratus. Yong-Sheng Tian and Gui-Dong Miao are contributed equally.     

Isolation and characterization of microsatellite loci in Peganum harmala (Peganaceae), an important resist-drought and medicinal plant

Peganum harmala is a herb grows spontaneously in arid and rocky areas. From ancient time, it has been claimed to be an important medicinal plant in rough environment. In this study, we developed 12 microsatellite loci from P. harmala by the combining biotin capture method for the first time. A total of 31 microsatellite sequences were recovered through screening the library and 12 of them are polymorphic. The number of alleles per locus in 36 sampled individuals ranged from 3 to 8, expected heterozygosity and observed heterozygosity ranged from 0.1381 to 0.6821 and from 0.3573 to 0.8739, respectively. In addition, all markers have been crossly checked in the other congeneric species. These microsatellite markers would provide a useful tool for investigating the genetic diversity and study the population genetic structure in detail.     

Isolation and characterization of 14 polymorphic microsatellite loci in the ark shell Scapharca subcrenata (Bivalvia: Arcidae)

The ark shell Scapharca subcrenata is an important fishery resource, but has been suffering from severe population decline due to the deterioration of the coastal environment and over-exploitation. To investigate the genetic variation and structure among populations of S. subcrenata, 14 polymorphic microsatellite loci were developed. The number of alleles per polymorphic locus ranged from 11 to 29 and the observed and expected heterozygosity varied from 0.400 to 0.923 and from 0.705 to 0.965, respectively. These microsatellites will help advance the investigation of the genetic population structure and genetic diversity in this species.     

Isolation and characterization of ten polymorphic microsatellite in the endangered tree Erythrophleum fordii oliv

Erythrophleum fordii oliv is a famous hardwood tree distributed in South China and Vietnam. It is now endangered due to heavy exploitation and deforestation. Ten polymorphic microsatellite loci were isolated and characterized from it. The number of alleles per locus varied from two to six. The expected and observed heterozygosities ranged from 0.3802 to 0.6903, and 0.2083 to 0.7719, respectively. These microsatellite markers will provide genetic information upon which strategies for effective conservation and management could be based.     

Development of twelve novel polymorphic microsatellite DNA markers for the Boeseman’s rainbowfish (Melanotaenia boesemani) and tests for their cross-utility in 21 rainbowfish species from West Papua (Indonesia)

We developed 12 microsatellite markers for the endangered Boeseman’s rainbowfish (Melanotaenia boesemani). Twenty-eight individuals from the type locality at Ayamaru Lake were examined, and all loci were polymorphic with a number of alleles per locus varying from 3 to 18. Average observed and expected heterozygosities were 0.681 and 0.678, respectively. Cross-species amplification was successfully obtained for 21 Melanotaenia species, with a number of alleles per locus ranging between 1 and 20. Average observed and expected heterozygosities varied between 0.105 and 0.708 and 0.118–0.755, respectively. Only 21 inbreeding coefficient (Fis) values presented a significant homozygote excess among the 264 locus-by-locus calculated values. Tests for genotyping errors revealed that four of these 21 significant Fis values could be explained by the presence of null alleles. These new microsatellite markers appear highly reliable for further conservation purposes or population genetic studies of the many rainbowfish endangered species.     

Isolation and characterization of eight microsatellite loci for the vulnerable Hainan Eld’s deer (<Emphasis Type="Italic">Cervus eldi hainanus</Emphasis>) in China

We report on the isolation and characterization of eight microsatellite markers in the Hainan Eld’s deer (Cervus eldi hainanus) from genomic DNA-enriched libraries. Thirty-three microsatellites were screened from the libraries, and 8 of the screened microsatellites were polymorphic. The number of observed alleles for each locus in 47 individuals ranged from 2 to 9, and the expected and observed heterozygosity was 0.141–0.792 and 0.128–0.957, respectively. Three loci (CEH-2, CEH-6 and CEH-8) of eight deviated from Hardy-Weinberg expectation and no significant linkage association was found among all these loci. These microsatellite markers provide useful tool for population genetic studies of the Eld’s deer.     

Genetic diversity and assessment of 23 microsatellite markers for parentage testing of Santa Inês hair sheep in Brazil

Santa Inês is the most common hair sheep breed in Brazil and probably has the highest genetic diversity among sheep breeds in this country. Successful breeding programs for Brazilian sheep breeds are not common for various reasons, including a lack of control of parentage in the flocks. We developed an allele frequency database for 23 STR loci for the Santa Inês breed based on 285 animals sampled from five populations distributed across the central-western and north-eastern regions of Brazil. The marker set included seven microsatellites used in the 2011 International Society for Animal Genetics sheep genotyping comparison tests and all eight microsatellites currently approved by the Brazilian Agricultural Ministry laboratory accreditation guidelines for sheep identification. The microsatellites had an average of 10 alleles and a mean expected heterozygosity of 0.745. Combined paternity exclusion probabilities when no parent or one parent was known were >99.99%. A small proportion (5.8%) of the existing genetic variation was found to be among the Santa Inês populations, possibly derived from genetic drift and selection. We found that the marker panel proposed by the Agricultural Ministry, although generally useful, should be enhanced by including more markers for improved exclusionary power in parentage testing. This database provides a useful tool for parentage testing of this major Brazilian breed, contributing to improved management and breeding of existing herds.     

Isolation and characterization of 12 microsatellite loci for Michelia coriacea (Magnoliaceae), a critically endangered endemic to Southeast Yunnan, China

By using a modified biotin–streptavidin capture method, a total of 12 microsatellite loci were developed and characterized for Michelia coriacea (Magnoliaceae), a critically endangered endemic to Southeast Yunnan, China. The number of alleles (A) ranged from two to six in 30 samples of this species. The ranges of observed (H _O ) and expected (H _E ) heterozygosities were 0.033–0.8000 and 0.033–0.7910, respectively. Cross-species amplification in M. yunnanensis showed that a subset of these markers holds promise for congeneric species. These polymorphic SSR markers would be useful tools for population genetics studies on M. coriacea and other congeneric species.     

Isolation and characterization of mesenchymal stem cells derived from bone marrow of patients with Parkinson’s disease

Mesenchymal stem cells (MSCs) are capable of self-renewing and differentiating into multiple tissues; they are expected to become a source of cells for regenerative therapy. Compared to allogeneic MSCs, autologous MSCs from patients needing cell-based therapy may be an ideal alternative stem cell source. However, characterizations of MSCs from a disease state remains extremely limited. Therefore, we have isolated and characterized MSCs from Parkinson's disease (PD) patients and compared them with MSCs derived from normal adult bone marrow. Our results show that PD-derived MSCs are similar to normal MSCs in phenotype, morphology, and multidifferentiation capacity. Moreover, PD-derived MSCs are capable of differentiating into neurons in a specific medium with up to 30% having the characteristics of dopamine cells. At last, PD-derived MSCs could inhibit T-lymphocyte proliferation induced by mitogens. These findings indicate that MSCs derived from PD patients' bone marrow may be a promising cell type for cellular therapy and somatic gene therapy applications.     

Isolation and characterization of dinoflagellate and chrysophyte cytochrome-f (553–4)

Cytochrome-f (553–4) was isolated from mass cultures of the six dinoflagellates, Amphidinium carterae (2 strains), Cachonina niei, Glenodinium sp., Peridinium foliaceum, Gonyaulax polyedra, and one chrysophyte, Cricospherae carterae. Sonication of whole cell suspensions released the water-soluble protein, which was then purified by vacuum dialysis, salt fractionation and column chromatography. Reduced forms of isolated cytochromes had absorption maxima at 270–6, 316–8, 415–6, 522–3 and 553–4 rim. The α-absorption peak was asymmetrical. MW's, as determined by SDS polyacrylamide gel electrophoresis, ranged from 10 700 to 13 500. Amino acid analysis of C. niei cytochrome-f revealed 102 residues, with a composite MW of 10836. Purified cytochromes had isoelectric points ranging from 3·45 to 4·25 and oxidation-reduction potentials ranging from +0·374 to +0·351 V.     

Isolation and characterization of β-galactosidase fromLactobacillus crispatus

β-Galactosidase was isolated from the cell-free extracts ofLactobacillus crispatus strain ATCC 33820 and the effects of temperature, pH, sugars and monovalent and divalent cations on the activity of the enzyme were examined.L. crispatus produced the maximum amount of enzyme when grown in MRS medium containing galactose (as carbon source) at 37°C and pH 6.5 for 2 d, addition of glucose repressing enzyme production. Addition of lactose to the growth medium containing galactose inhibited the enzyme synthesis. The enzyme was active between 20 and 60°C and in the pH range of 4–9. However, the optimum enzyme activity was at 45°C and pH 6.5. The enzyme was stable up to 45°C when incubated at various temperatures for 15 min at pH 6.5. When the enzyme was exposed to various pH values at 45°C for 1 h, it retained the original activity over the pH range of 6.0–7.0. Presence of divalent cations, such as Fe²⁺ and Mn²⁺, in the reaction mixture increased enzyme activity, whereas Zn²⁺ was inhibitory. TheK _m was 1.16 mmol/L for 2-nitrophenyl-β-d-galactopyranose and 14.2 mmol/L for lactose.     

Isolation and characterization of wheat ω-gliadin genes

The DNA sequences of two full-length wheat ω-gliadin prolamin genes (ωF20b and ωG3) containing significant 5′ and 3′ flanking DNA sequences are reported. The ωF20b DNA sequence contains an open reading frame encoding a 30,460-Dalton protein, whereas the ωG3 sequence would encode a putative 39,210-Dalton protein except for a stop codon at amino-acid residue position 165. These two ω-gliadin genes are closely related and are of the ARQ-/ARE-variant type as categorized by the derived N-terminal amino-acid sequences and amino-acid compositions. The ω-gliadins were believed be related to the ω-secalins of rye and the C-hordeins of barley, and analyses of these complete ω-gliadin sequences confirm this close relationship. Although the ω-type sequences from all three species are closely related, in this analysis the rye and barley ω-type sequences are the most similar in a pairwise comparison. A comparison of ω-gliadin flanking sequences with respect to that of their orthologs and with respect to wheat gliadin genes suggests the conservation of flanking DNA necessary for gene function. Sequence data for members of all major wheat prolamin families are now available. Received: 24 August 2000 / Accepted: 15 December 2000