• Asian-Australasian Journal of Animal Sciences
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• 제33권2호
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• pp.219-229
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• 2020

Objective: Considering the physiological and clinical importance of leptin receptor (LEPR) in regulating obesity and the fact that porcine LEPR expression is not known to be controlled by lncRNAs and miRNAs, we aim to characterize this gene as a potential target of SSC-miR-323 and the lncRNA TCONS_00010987. Methods: Bioinformatics analyses revealed that lncRNA TCONS_00010987 and LEPR have SSC-miR-323-binding sites and that LEPR might be a target of lncRNA TCONS_00010987 based on cis prediction. Wild-type and mutant TCONS_00010987-target sequence fragments and wild-type and mutant LEPR 3'-UTR fragments were generated and cloned into pmiRRB-REPORT^TM-Control vectors to construct respective recombinant plasmids. HEK293T cells were co-transfected with the SSC-miR-323 mimics or a negative control with constructs harboring the corresponding binding sites and relative luciferase activities were determined. Tissue expression patterns of lncRNA TCONS_00010987, SSC-miR-323, and LEPR in Anqing six-end-white (AQ, the obese breed) and Large White (LW, the lean breed) pigs were detected by real-time quantitative polymerase chain reaction; backfat expression of LEPR protein was detected by western blotting. Results: Target gene fragments were successfully cloned, and the four recombinant vectors were constructed. Compared to the negative control, SSC-miR-323 mimics significantly inhibited luciferase activity from the wild-type TCONS_00010987-target sequence and wild-type LEPR-3'-UTR (p<0.01 for both) but not from the mutant TCONS_00010987-target sequence and mutant LEPR-3'-UTR (p>0.05 for both). Backfat expression levels of TCONS_00010987 and LEPR in AQ pigs were significantly higher than those in LW pigs (p<0.01), whereas levels of SSC-miR-323 in AQ pigs were significantly lower than those in LW pigs (p<0.05). LEPR protein levels in the backfat tissues of AQ pigs were markedly higher than those in LW pigs (p<0.01). Conclusion: LEPR is a potential target of SSC-miR-323, and TCONS_00010987 might act as a sponge for SSC-miR-323 to regulate LEPR expression.

• Asian-Australasian Journal of Animal Sciences
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• 제32권7호
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• pp.922-929
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• 2019

Objective: Mutations in low-density lipoprotein receptor (LDLR), which encodes a critical protein for cholesterol homeostasis and lipid metabolism in mammals, are involved in cardiometabolic diseases, such as familial hypercholesterolemia in pigs. Whereas microRNAs (miRNAs) can control LDLR regulation, their involvement in circulating cholesterol and lipid levels with respect to cardiometabolic diseases in pigs is unclear. We aimed to identify and analyze LDLR as a potential target gene of SSC-miR-20a. Methods: Bioinformatic analysis predicted that porcine LDLR is a target of SSC-miR-20a. Wild-type and mutant LDLR 3'-untranslated region (UTR) fragments were generated by polymerase chain reaction (PCR) and cloned into the pGL3-Control vector to construct pGL3 Control LDLR wild-3'-UTR and pGL3 Control LDLR mutant-3'-UTR recombinant plasmids, respectively. An miR-20a expression plasmid was constructed by inserting the porcine premiR-20a-coding sequence between the HindIII and BamHI sites in pMR-mCherry, and constructs were confirmed by sequencing. HEK293T cells were co-transfected with the miR-20a expression or pMR-mCherry control plasmids and constructs harboring the corresponding 3'-UTR, and relative luciferase activity was determined. The relative expression levels of miR-20a and LDLR mRNA and their correlation in terms of expression levels in porcine liver tissue were analyzed using reverse-transcription quantitative PCR. Results: Gel electrophoresis and sequencing showed that target gene fragments were successfully cloned, and the three recombinant vectors were successfully constructed. Compared to pMR-mCherry, the miR-20a expression vector significantly inhibited wild-type LDLR3'-UTR-driven (p<0.01), but not mutant LDLR-3'-UTR-driven (p>0.05), luciferase reporter activity. Further, miR-20a and LDLR were expressed at relatively high levels in porcine liver tissues. Pearson correlation analysis revealed that porcine liver miR-20a and LDLR levels were significantly negatively correlated (r = -0.656, p<0.05). Conclusion: LDLR is a potential target of miR-20a, which might directly bind the LDLR 3'-UTR to post-transcriptionally inhibit expression. These results have implications in understanding the pathogenesis and progression of porcine cardiovascular diseases.

• Animal cells and systems
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• 제12권4호
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• pp.269-277
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• 2008

Siberian flying squirrel, an endangered species in South Korea, is distributed through major mountain regions of South Korea. The number of Siberian flying squirrel(Pteromys volans) in South Korea has decreased and their habitats are fragmented and isolated because of anthropogenic activities. So far no molecular genetic data has, however, been available for their conservation and management. To obtain better information concerning genetic diversity and phylogenetic relationships of the Siberian flying squirrel in South Korea, we examined 14 individuals from South Korea, 7 individuals from Russia, and 5 individuals from northeastern China along with previously published 29 haplotypes for 1,140 bp of the mtDNA cytochrome b gene. The 14 new individuals from South Korea had 7 haplotypes which were not observed in the regions of Russia and Hokkaido. The level of genetic diversity(0.616%) in the South Korean population was lower than that in eastern Russia(0.950%). The geographical distribution of mtDNA haplotypes and reduced median network confirmed that there are three major lineages of Siberian flying squirrel, occupying; Far Eastern, northern Eurasia, and the island of Hokkaido. The South Korean population only slightly distinct from the Eurasia, and eastern Russian population, and is part of the lineage Far Eastern. Based on these, we suggest that the South Korean population could be considered to belong to one partial ESU(Far Eastern) of three partial ESUs but a different management unit. However, the conservation priorities should be reconfirmed by nuclear genetic marker and ecological data.

• Animal cells and systems
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• 제16권3호
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• pp.230-236
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• 2012

The Korean hare, Lepus coreanus, is an important mammal in ecosystem food chains, and is distributed across the entire Korean peninsula and northeastern China. Polymorphic microsatellite loci were developed using the biotinenrichment technique for use in population genetics studies. Five trinucleotide and four dinucleotide microsatellite loci were selected and tested on 22 Korean hare specimens collected from Gangwon Province and Gyeongsangbuk Province in South Korea. The number of alleles across the two sampling regions ranged from three to nine with a mean of 6.1. Mean observed and expected heterozygosities and polymorphic information content were 0.540, 0.627 and 0.579, respectively. Only one locus, Lc06, showed departure from Hardy-Weinberg equilibrium after applying the Bonferroni correction. Four microsatellites, Lc01, Lc03, Lc12, and Lc19, satisfied the criteria to serve as a core set of markers recommended for population genetics studies. These new microsatellite markers will be widely applicable to future genetic studies for management and conservation of the Korean hare and related species, including assessment of the genetic diversity and population structure of L. coreanus.

• Asian-Australasian Journal of Animal Sciences
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• 제26권12호
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• pp.1665-1671
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• 2013

Real-time quantitative PCR (qRT-PCR) is one of the important methods for investigating the changes in mRNA expression levels in cells and tissues. Selection of the proper reference genes is very important when calibrating the results of real-time quantitative PCR. Studies on the selection of reference genes in goat tissues are limited, despite the economic importance of their meat and dairy products. We used real-time quantitative PCR to detect the expression levels of eight reference gene candidates (18S, TBP, HMBS, YWHAZ, ACTB, HPRT1, GAPDH and EEF1A2) in ten tissues types sourced from Boer goats. The optimal reference gene combination was selected according to the results determined by geNorm, NormFinder and Bestkeeper software packages. The analyses showed that tissue is an important variability factor in genes expression stability. When all tissues were considered, 18S, TBP and HMBS is the optimal reference combination for calibrating quantitative PCR analysis of gene expression from goat tissues. Dividing data set by tissues, ACTB was the most stable in stomach, small intestine and ovary, 18S in heart and spleen, HMBS in uterus and lung, TBP in liver, HPRT1 in kidney and GAPDH in muscle. Overall, this study provided valuable information about the goat reference genes that can be used in order to perform a proper normalisation when relative quantification by qRT-PCR studies is undertaken.

• Animal Systematics, Evolution and Diversity
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• 제24권1호
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• pp.25-32
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• 2008

The Gold-spotted pond frog, Rana plancyi chosenica, designated as a vulnerable species by IUCN Red list. This species is a typical example facing local population threats and extinction due to human activities in South Korea. A strategic conservation plan for this endangered species is urgently needed. In order to provide information for future conservation planning, accurate information on the genetic diversity and taxonomic status is needed for the establishment of conservation units for this species. In this study, we used a molecular genetic approach using the mitochondrial cytochrome b gene and control region sequences to find the genetic diversity of gold-spotted pond frogs within South Korea. We sequenced the mitochondrial DNA cytochrome b gene and control region of 77 individuals from 11 populations in South Korea, and one from Chongqing, China. A total of 15 cytochrome b gene haplotypes and 34 control region haplotypes were identified from Korean gold-spotted pond frogs. Mean sequence diversity among Korean gold-spotted pond frogs was 0.31% (0.0-0.8%) and 0.51% (0.0-1.0%), respectively. Most Korean populations had at least one unique haplotype for each locus. The Taean, Ansan and Cheongwon populations had no haplotypes shared with other populations. There was a sequence divergence between Korean and Chinese gold-spotted pond frogs (1.3% for cyt b; 2.9% for control region). Analysis of genetic distances and phylogenetic trees based on both cytochrome b and control region sequences indicate that the Korean gold-spotted pond frog are genetically differentiated from those in China.

• Journal of Ecology and Environment
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• 제42권3호
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• pp.111-119
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• 2018

Background: Juniperus chinensis L. populations are distributed locally on several areas including coastal cliffs which are difficult to access in the central eastern Korea. Wild populations inhabit relatively barren environments such as rocky areas and cliffs, which are very sensitive to even minor environmental disturbances including artificial interventions and natural disturbances, and thus demonstrate great fluctuations in the population size and density. This study aims to analyze the genetic diversity, differentiation, and genetic structure of each population in order to provide useful data required to establish a substantial conservation strategy of J. chinensis. Results: The genetic diversity of J. chinensis at the population level (P = 78.7%, h = 0.282, S.I. = 0.420) was somewhat higher compared with those measured in the same genus, Juniperus. The genetic differentiation degree among nine populations established naturally in central eastern Korea was 11.50% and that among sub-populations within the same area was 5.52%. On the other hand, genetic variation of individuals within the populations was 82.93%. But frequency of the main allele was different among loci. In particular, fixation of allele frequency and occurrence of rare allele in the highly isolated population suggest a likelihood that genetic drift would occur in populations of this plant. As the result of analysis on the genetic structure of nine populations, nearby populations and isolated populations tended to form separate clusters from each other as the hypothetical number of clusters (K) increase. Conclusions: This result implies that if the population size of J. chinensis is reduced due to environmental change and artificial and/or natural disturbances in the future, it could affect negatively on the genetic diversity of the plant species. In order to maintain and conserve genetic diversity of J. chinensis, ecological network, which can help genetic exchange among the local populations, should be prepared, and conservation strategies in situ as well as ex situ are also required with continuous monitoring.

• Journal of Forest and Environmental Science
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• 제24권1호
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• pp.27-34
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• 2008

Level and distribution of genetic diversity in seven populations of Albizia lucida Benth. in eastern region of the Indian sub-continent were estimated using ISSR markers. Relatively higher level of genetic diversity within populations was observed in seven populations of A. lucida (mean of 0.38). From the result of AMOVA, majority of genetic diversity was allocated within populations (96.2%) resulting in a moderate degree of population differentiation. The observed distribution pattern of I-SSR variant among the populations was coincided with the typical pattern of long-lived woody tree species. Genetic relationships among the populations, reconstructed by UPGMA method, revealed two genetic groups. The population of Anugul and Bargarh turned out to be the most closely related despite a distance location between them. These formations will be of great value in the development of conservation plans for species exhibiting high levels of genetic differentiation due to fragmentation, such as indication of conservation unit size, which populations should be chosen as priority in conservation plans and which samples should be introduced in areas with a low number of individuals of A. lucida.

• Asian-Australasian Journal of Animal Sciences
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• 제33권12호
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• pp.1905-1911
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• 2020

Objective: Tibetan pigs, an excellent species unique to China, face serious threats, which in turn affects the development and utilization of the outstanding advantages of plateau hypoxia adaptability and reduces their genetic diversity. Therefore, a discussion of measures to conserve this genetic resource is necessary. The method, based on genetic diversity, genetic divergence and total genetic contribution rate of population, reflects the priority conservation order and varies depending on the three different purposes of conservation. Methods: We analyzed mitochondrial DNA control region (D-loop) variation in 1,201 individuals from nine Tibetan pig populations across five provinces and downloaded 564 mtDNA D-loop sequences from three indigenous pig breeds in Qinghai, Sichuan, and Yunnan Provinces distributed near the Tibetan pigs. Results: We analyzed three different aspects: Changdu Tibetan pigs have the highest genetic diversity, and from the perspective of genetic diversity, the priority conservation is Changdu Tibetan pigs. Hezuo Tibetan pigs have the highest genetic contribution, so the priority conservation is Hezuo Tibetan pigs in the genetic contribution aspect. Rkaze Tibetan pigs were severely affected by indigenous pig breeds, so if considering from the perspective of introgression, the priority conservation is Rkaze Tibetan pigs. Conclusion: This study evaluated genetic diversity and comprehensively assessed conservation priority from three different aspects in nine Tibetan pig populations.

• Asian-Australasian Journal of Animal Sciences
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• 제23권12호
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• pp.1552-1556
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• 2010

The aim of this study was to evaluate, through the use of microsatellite markers, the current genetic diversity and the relationships of 375 individuals from 8 local sheep breeds reared in typical breeding farms in the northwest of China, and moreover, to offer a contribution towards genetic conservation decisions for the studied breeds. The expected heterozygosities and allelic richness for the 8 breeds varied from 0.474 to 0.623 and from 3.8 to 5.4, respectively. All the populations showed a significant deficit in heterozygosity and a relatively low level of genetic diversity. Furthermore, the high positive FIS value (ranging from 0.255 to 0.556) indicated inbreeding to be one of the main causes for high genetic homogeneity and lack of heterozygosity in all breeds. The clustering analysis performed with the DISPAN package showed that Aletai, Kazak, Bashibai and Bayinbuluke were grouped together, and Hetian, Qira black and Duolang were grouped together, which indicated that the relationship among breeds displayed some degree of consistency with their geographical distribution, production and origin. These findings indicate that improved conservation measures must be undertaken to avoid further losses of genetic diversity and minimize inbreeding represented by these breeds.