Cross-amplification In Other Species Research Articles

Differentiation of cattle and buffalo meat using multiplex polymerase chain reaction

Authentication of origin of animal species is of paramount importance owing to economic, social, religious, forensic and public health considerations. In the present study, a multiplex polymerase chain reaction (mPCR) was developed by designing unique species-specific forward and a common reverse primers flanking mitochondrial D-loop region for the authentication of cattle (Bos indicus / B. taurus) and buffalo (Bubalus bubalis) species. Novel mPCR amplified 382 bp product specific to cattle and 336 bp specific to buffalo. Possibility of cross amplification in other species was rules out by testing as many as 25 other animal species. The novel mPCR assay developed in this study for the authentic identification and differentiation of meats of cattle and buffalo could be applied for the differentiation of samples derived from cattle and buffalo in diagnostic laboratories thereby ensuring prompt labelling of products, protecting consumer sentiments and enforcement of laws.

Eleven microsatellite loci developed from the Chinese Hesperid butterflies, Polytremis fukia (Lepidoptera: Hesperiidae), and cross-amplification in other species of Polytremis

Eleven polymorphic microsatellite loci were isolated and characterized from a Chinese endemic species, Polytremis fukia Evans. The isolated loci were polymorphic, with 5–10 alleles in 21 individuals. The analyses revealed that they had different multilocus genotypes, with the expected heterozygosity ranging from 0.69 to 0.86 and observed heterozygosity ranging from 0.48 to 0.65. All loci were in Hardy-Weinberg equilibrium. Linkage disequilibrium was not detected in any pair of loci. Ten loci were amplified successfully in at least one of the eight related species. These microsatellite markers reported here are the first for P. fukia and will be used to investigate the population genetic structure and gene flow in P. fukia as well as congeneric and related species.

Development and characterization of microsatellite markers for Echinopsis rhodotricha and cross-amplification in other species of Cactaceae

Located in central South America, the Chaco is a large subtropical dry forest characterized by plants with xeromorphic features, including numerous cacti. In the Brazilian Chaco at the northeasternmost part of the Chaquenian region, Echinopsis rhodotricha (Cactaceae, Trichocereeae) is one of the species threatened by intense deforestation caused by expanding pastureland. This study characterizes the microsatellite loci isolated from E. rhodotricha and cross-amplification in thirteen other cactus species. Twelve microsatellite loci were developed from an enriched genomic library. Eight of these were polymorphic and characterized in 48 individuals from three E. rhodotricha populations. The loci showed a mean of 3.2 alleles per locus and overall levels of expected and observed heterozygosities ranging from 0.00 to 0.83 and 0.00 to 0.66, respectively. Five loci showed significant departures from the Hardy–Weinberg equilibrium and also exhibited signs of null alleles. Cross-amplification in other Cactaceae species was successful, ranging from one (Ferocactus latispinus, Cacteae and Harrisia adscendens, Trichocereeae) to twelve loci (Echinopsis calochlora, Trichocereeae). The development of these microsatellite markers will contribute to investigations of population structure, genetic diversity, and gene flow in E. rhodotricha populations, as well as in other cactus species, providing information useful for the creation and delimitation of conservation areas in the Brazilian Chaco region.

Development and characterization of microsatellite markers for Vriesea simplex (Bromeliaceae) and cross-amplification in other species of Bromeliaceae

Ten polymorphic microsatellite loci were isolated and characterized for Vriesea simplex epiphytic and endemic species from the Brazilian Atlantic Rainforest. The number of alleles per locus ranged from 2 to 16. The observed and expected heterozygosity ranged from 0.000 to 0.414 and 0.068 to 0.795, respectively. All loci departed significantly from Hardy–Weinberg equilibrium. Linkage disequilibrium was not detected in any pair of loci. Transferability of 10 loci was positive across ten other Bromeliaceae species. The loci will be used for studying population genetic structure, mating system variation, and the origin and maintenance of reproductive barriers to gene exchange among sympatric Vriesea species.

A sensitive and specific multiplex PCR approach for sex identification of ursine and tremarctine bears suitable for non‐invasive samples

We report a new approach for molecular sex identification of extant Ursinae and Tremarctinae bears. Two Y-specific fragments (SMCY and 318.2) and one X-specific fragment (ZFX) are amplified in a multiplex PCR, yielding a double test for male-specific amplification and an internal positive control. The primers were designed and tested to be bear-specific, thereby minimizing the risk of cross-amplification in other species including humans. The high sensitivity and small amplicon sizes (100, 124, 160 base pairs) facilitate analysis of non-invasively obtained DNA material. DNA from tissue and blood as well as from 30 non-invasively collected hair and faeces yielded clear and easily interpretable results. The fragments were detected both by standard gel electrophoresis and automated capillary electrophoresis.

Isolation and characterization of polymorphic microsatellite loci for the gopher tortoise (Gopherus polyphemus) and cross-amplification in other species of Gopherus

All members of the tortoise genus Gopherus have some degree of conservation concern. We report on the development of thirty-two new microsatellite loci for gopher tortoise, which were tested on 31 individuals from Ft. Benning, Georgia, USA. These loci possessed an average number of 5.09 alleles, an average observed heterozygosity of 0.574 and an average expected heterozygosity of 0.589. We also tested these loci in three congeners: Gopherus morafkai, Gopherus flavomarginatus and Gopherus berlandieri. The new microsatellite loci should prove useful in conservation efforts of Gopherus polyphemus and other Gopherus species.

Characterization of novel microsatellite loci for Hetaerina americana damselflies, and cross-amplification in other species

Hetaerina damselflies are distributed throughout the neotropics. We developed eleven microsatellite loci for the damselfly Hetaerina americana. Microsatellites were tested for polymorphism on a panel of 24 individuals. The number of alleles ranged from 2 to 6, observed heterozygosity from 0.080 to 0.701, and the fixation index from −0.266 to 1.000. Cross-amplification was tested in 7 different species in the genus Hetaerina from the United States and Mexico. These microsatellite loci will be useful for studies of population structure and gene flow in H. americana.

Development of new microsatellite loci for the genus Polistes from publicly available expressed sequence tag sequences

Over the last 20 years, microsatellites have revolutionized the study of cooperation in the social insects. The Polistes paper wasps have been an important model system for investigations of cooperative behavior. Recently, an expressed sequence tag (EST) library has been developed for P. metricus, allowing researchers to investigate the genetic basis of cooperative behavior in primitive social insect societies for the first time. We searched these freely available EST sequences for microsatellite motifs. This represents a relatively new approach to the development of microsatellite loci that allows for the development of a greater number of loci at less expense. We designed 32 PCR primer pairs, of which 23 amplified PCR products and 18 were polymorphic. These loci exhibited high levels of polymorphism, comparable to anonymous loci isolated via screens of partial genomic libraries. Thus, they are appropriate for population genetic studies as well as the reconstruction of colony genetic structure. A screen of the entire EST database found a total of 708 di-, tri-, tetra- and penta-nucleotide repeats with large repeat units typical of polymorphic loci and at least 30 bp of flanking sequence for primer design. This pool of potential loci represents a new genetic tool for P. metricus, as well as Polistes more generally, as there is great promise for cross amplification in other species.

Characterization of novel microsatellite loci for Myzomela cardinalis and M. rubrata honeyeaters, and cross-amplification in other species

Myzomela honeyeaters are distributed from eastern Austalia and Indonesia throughout the islands of the tropical Pacific, a biodiversity hotspot that is particularly vulnerable due to small population sizes, habitat destruction, and high levels of isolation between islands. We developed fourteen microsatellite loci for Myzomela honeyeaters from the tropical Pacific. Microsatellites were tested for polymorphism on a panel of 104 individuals. The number of alleles ranged from 3 to 23, observed heterozygosity from 0.067 to 0.913, and the fixation index from −0.143 to 0.634. Cross-amplification was tested in 18 different species and subspecies in the genus Myzomela from throughout the Pacific, Australia, and eastern Indonesian archipelago. These new microsatellites could potentially be useful in making informed conservation decisions regarding Myzomela honeyeaters on the islands of the tropical Pacific.