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Development of Method using LC-ESI-MS/MS and KASP for Identification of Gymnema sylvestre in Food (식품에서 당살초 판별을 위한 LC-ESI-MS/MS 분석법과 KASP 마커 개발)

  • Park, Boreum;Lee, Sun Hee;Eom, Kwonyong;Noh, Eunyoung;Moon Han, Kyoung;Hwang, Jinwoo;Kim, Hyungil;Baek, Sun Young
    • Journal of Food Hygiene and Safety
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    • v.37 no.2
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    • pp.46-54
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    • 2022
  • Known for its effectiveness in weight loss and diabetes prevention, Gymnema sylvestre products can be found in the US, Japanese, and Indian markets. However, the recommended dosage or safety of these products has not yet been proven. Therefore, development of an analytical method for detecting the content of Gymnema sylvestre in food products is required. Accordingly, this study proposes an analysis method that can examine Gymnema sylvestre in food using LC-ESI-MS/MS and KASP (Kompetitive Allele-Specific PCR) markers. In LC-ESI-MS/MS, a simultaneous analysis method for gymnemic acid and deacylgymnemic acid was optimized using negative ionization mode, and its validation test was completed for solid and liquid samples. In addition, KASP markers were prepared by finding the specific SNP of G. sylvestre in ITS2 and matK through DNA barcodes. The two KASP markers returned positive FAM fluorescence result when combined with G. sylvestre, and this aspect was confirmed in raw G. sylvestre as well. The applicability of the method was tested on 21 different food and healthy functional products containing G. sylvestre purchased on the internet. As a result, although there was a difference in the ratios of gymnemic acid and deacylgymnemic acid in LC-ESI-MS/MS, the index component was detected in all 21 products samples. In the KASP analysis, 9 products returned positive FAM result, and the rest of the products were found to be containing G. sylvestre extract. This study is the first study to use the dual system of LC-ESI-MS/MS and KASP for the analysis of G. sylvestre. The study has confirmed that these two methods are applicable to the examine G. sylvestre content in food products.

Characterization of Dopamine Receptor D4 Gene Polymorphisms in Horses (말에서 Dopamine Receptor D4 유전자의 변이 특성 분석)

  • Choi, Jae-Young;Choi, Yeonju;Lee, Jongan;Shin, Sang-Min;Yoon, Minjung;Kang, Yong-Jun;Shin, Moon-Cheol;Yoo, Ji-Hyun;Kim, Hyeonah;Cho, In-Cheol;Yang, Byoung-Chul;Kim, Nam-Young
    • Journal of Life Science
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    • v.32 no.1
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    • pp.29-35
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    • 2022
  • This study was conducted to analyze the genetic polymorphisms of dopamine receptor D4 (DRD4) in horse breeds and its association with substrate characteristics in Jeju crossbreds (Jeju Horse × Thoroughbred). Polymorphisms in DRD4 are candidate genes associated with temperament in various mammals, including humans. Single nucleotide polymorphism (SNP) G292A in the exon 3 region of the horse DRD4 has a reported association with curiosity and vigilance in thoroughbreds. Sanger sequencing was used to identify polymorphisms of the mutations in DRD4 in three horse breeds. The SNP frequency in Jeju horses was significantly different from the frequency in other breeds. Character evaluation, conducted in the Jeju crossbreds and scored using a temperament test and contact test, revealed a high correlation between each test. Comparison of the polymorphism in the DRD4 of horses and the results of the character evaluation revealed lower scores for all temperaments in horses carrying allele A. Comparison of the SNP of G292A and blood dopamine levels in Jeju crossbreds showed 2.87 times higher levels for the GA type than for the GG type. This study identified an association between DRD4 polymorphism and various test methods for evaluating horse temperament and levels of neurotransmitters. Further research could validate the use of this gene as a genetic marker for character evaluation.

Development of Near-Isogenic Line of japonica Rice Cultivar Saenuri without Lipoxygenase-3 (새누리 벼 품종 배경 lipoxygenase-3 결핍 자포니카 근동질계통 개발)

  • Park, Hyun-Su;Lee, Keon-Mi;Kim, Ki-Young;Kim, Jeong-Ju;Shin, Woon-Cheol;Baek, Man-Kee;Kim, Choon-Song;Park, Seul-Gi;Lee, Chang-Min;Suh, Jung-Pil;Cho, Young-Chan
    • Korean Journal of Breeding Science
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    • v.51 no.3
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    • pp.190-200
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    • 2019
  • It is reported that the absence of lipoxygenase-3 (LOX-3) may contribute to a reduction in stale flavor after the storage of rice. To improve the quality of stored rice of the Korean japonica rice cultivar, we conducted a breeding program to develop near-isogenic rice without LOX-3 in the genetic background of Saenuri, a mega variety of Korea. In the first step of the breeding program, we used a donor parent of LOX-3 null, Daw Dam, and a recurrent japonica parent, Sindongjin, to develop HR27873-AC12 by backcross (BC1), color test for introgression of lox-3, and anther culture for rapid fixation. In the second step, we used the donor parent, HR27873-AC12, and the recurrent parent, Saenuri, to develop HR28896-31-3-1-1 by backcross (BC1), marker-assisted selection (MAS) for lox-3, and phenotypic selection (PS) for agronomic traits. Finally, in the third step, we developed HR30960-186-2-1-2-1 (Jeonju624), derived from a cross between Saenuri and HR28896-31-3-1-1, by MAS for lox-3 and PS with high selection pressure for agronomic characteristics. Jeonju624 was confirmed with the introgression of lox-3 by molecular marker. Jeonju624 was a mid-late maturing rice with similar agronomic characteristics to Saenuri, lodging tolerance with short culm, erect plant architecture, and resistance to bacterial blight and rice stripe virus. The yield components of Jeonju624 were mostly similar to Saenuri, except for the 1,000-grain weight of brown rice. The appearance of the grain of Jeonju624 was better than that of Saenuri, and the characteristics of cooked rice were similar to those of Saenuri. In the genetic background analysis using 406 KASP (Kompetitive Allele-Specific PCR) markers, Jeonju624 was confirmed to be the near-isogenic line (NIL) of Saenuri with a 95.8% recovery rate. Jeonju624 is the NIL of Saenuri without LOX-3, and overcomes the linkage drag of Daw Dam with similar agronomic characteristics and genetic background to Saenuri. Jeonju624 can be utilized as a practical cultivar to improve the quality of stored rice, breeding material for the introgression of lox-3, and genetic material to elucidate the effect of introgressed genes.

Strain Improvement of the Genus Pleurotus by Protoplast Fusion (원형질체(原形質體) 융합(融合)에 의한 느타리버섯속(屬)의 품종개발(品種開發))

  • Yoo, Young-Bok;You, Chang-Hyun;Cha, Dong-Yeul
    • The Korean Journal of Mycology
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    • v.21 no.3
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    • pp.200-211
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    • 1993
  • Somatic hybrids of Pleurotus florida ASI 2016 and Pleurotus ostreatus ASI 2018 were obtained by protoplast fusion. The 40 fusants($P1{\sim}P40$) was examined for the yield on fermented and pasteurized rice straw in a tray. The carpophore yield of them were showed as the range of $27.0{\sim}155.2$, based on parental values of 100(ASI 2018), The pilei of fusants between orange white colored P. florida and dark grey colored P. ostreatus had mixed colors in the young stage. Other breeding programmes were performed to improve new varieties with high yield and good quality. A new oyster mushroom variety, Wonhyeongneutaribeosus(P72), was developed at the Agricultural Sciences Institute, Rural Development Administration in 1990. This P.florida-ostreatus-ostreatus hybrid P72 was selected from 38 protoplast fusion products($P41{\sim}P78$) between P.florida-ostreatus recombinant P5-M 43-arg rib and P. ostreatus ASI 2-13-0 2001-19-pro orn. The yield indexes of 38 hybrids ranged $40.5{\sim}152.7$ compared with the parental values of 100(ASI 2001). Hybrid P72 was characterized by the large fruiting bundle of semispherical shape with long stipe and by the small and circular pileus, resulting in lower harvesting cost. A significant increase in carpophore production was observed in somatic hybrids of protoplasts due to heterosis. A comparision of hybrid with parents P72 was made using isozyme analysis. The esterase banding patterns could be characterized by new bands in the hybrids. Seven fusion products of four crosses between P.florida ASI 2016 and P. ostreatus ASI 2018 were analysed with respect to the distribution of progenies and segregation of gene markers by random basidiospore analysis. Segregation of alleles should yield progeny of four genotypes in a Mendelian ratio of 1 : 1 : 1 : 1 for prototrophs, auxotrophs of one parental type, auxotrophs of the other parental type, and auxotrophic recombinants, respectively. However, five fusants of them did not detect one parental, P.ostreatus, type. Basidiospores could yield progeny of 16 genotypes in the cross of one of the recombinant P5-M43-arg $rib{\times}P. ostreatus$ ASI 2-13-pro orn but the segregants of three fusants were not detected clearly. The allele ratio of loci could be expected 1 : 1 : 1 : 1 for arg, rib, pro and orn. The ratio, however, would be changed to 4 : 1 : 1 : 1 with increasing proportion of argo In almost all the fusants, prototrophic recombinants were recovered in large numbers against auxotrophic markers. Parental genotypes were recovered with the recombinant progeny amounting to $38.68{\sim}99.56%$. The analysis provides proof of heterokaryosis and strong evidence for haploidy of vegetative nuclei, a sexual cycle consisting of nuclear fusion and meiosis.

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A Case-Control Study on Effects of Genetic Polymorphisms of GSTM1, GSTT1, CYP1A1 and CYP2E1 on Risk of Lung Cancer (GSTM1과 GSTT1, 그리고 CYP1A1, CYP2E1 다형성이 폐암발생에 미치는 영향에 대한 환자-대조군연구)

  • Nan, Hong-Mei;Kang, Jong-Won;Bae, Jang-Whan;Choe, Kang-Hyeon;Lee, Ki-Hyeong;Kim, Seung-Taik;Won, Choong-Hee;Kim, Yong-Min;Kim, Heon
    • Journal of Preventive Medicine and Public Health
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    • v.32 no.2
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    • pp.123-129
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    • 1999
  • Objectives: This study was performed to investigate sweets of genetic polymorphisms of glutathione S-transferase M1 (GSTM1), glutathione S-transferase M1 (GSTT1), cytochrome P450 1A1 (CYP1A1) and cytoehrome P450 2E1 (CYP2E1) on lung cancer development. Methods: Ninety-eight lung cancer patients and 98 age-sex matched non-cancer patients hospitalized in Chungbuk National University Hospital form March 1997 to August 1998, were the subjects of this case-control study. Direct interview was done and genotypes of GSTM1, GSTT1, CYP1A1 and CYP2E1 were investigated using multiplex PCR or PCR-RFLP methods with DNA extracted from venous blood. Effects of the polymorphisms of GSTM1, GSTT1, CYP1A1 and CYP2E1, lifestyle factors including smoking, and their interactions on lung rancor were statistically analyzed. Results: GSTM1 was deleted in 67.01% of the cases and 58.16% of the controls, and the odds ratio(95% CI) was 1.46(0.82-2.62). GSTT1 deletion was 58.76% for the lung cancer patients and 50.00% for the controls[OR:1.43(0.81-2.51)]. The frequencies of lle/lle, lle/Val and Val/Val of the CYP1A1 polymorphisms were 59.18-18%, 35.71%, and 5.10% for the cases, and 52.04%, 45.92%, 2.04% for the controls, respectively. Risk of lung cancer was not associated with polymorphism of CYP1A1 ($x^2trend=0.253$, p-value>0.05). The respective frequency of c1/c1 c1/c2, c2/c2 genotypes for CYP2E1 were 50.00%, 42.86%, 7.14% for the lung cancer patients, and 66.33%, 30.61%, 3.06% for the controls $(x^2trend=5.783,\;p<0.05)$. c2 allele was a significant risk factor for lung cancer. We also observed a significant association of cigarette smoking history with lung cancer risk. The odds ratio(95% Cl) of cigarette smoking was 3.03(1.58-5.81). In multiple logistic analysis including genotypes of GSTM1, GSTT1, CYP1A1 and CYP2E1, and smoking habit, only snaking habit came out to be a significant risk factor for lung cancer. Conclusion: Genetic polymorphisms of GSTM1, GSTT1, CYP1A1 and CYP2E1 are not so strongly associated with lung cancer as lifestyle factors including cigarette smoking.

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Genetic Diversity of Korean Native Chicken Populations in DAD-IS Database Using 25 Microsatellite Markers (초위성체 마커를 활용한 가축다양성정보시스템(DAD-IS) 등재 재래닭 집단의 유전적 다양성 분석)

  • Roh, Hee-Jong;Kim, Kwan-Woo;Lee, Jinwook;Jeon, Dayeon;Kim, Seung-Chang;Ko, Yeoung-Gyu;Mun, Seong-Sil;Lee, Hyun-Jung;Lee, Jun-Heon;Oh, Dong-Yep;Byeon, Jae-Hyun;Cho, Chang-Yeon
    • Korean Journal of Poultry Science
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    • v.46 no.2
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    • pp.65-75
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    • 2019
  • A number of Korean native chicken(KNC) populations were registered in FAO (Food and Agriculture Organization) DAD-IS (Domestic Animal Diversity Information Systems, http://www.fao.org/dad-is). But there is a lack of scientific basis to prove that they are unique population of Korea. For this reason, this study was conducted to prove KNC's uniqueness using 25 Microsatellite markers. A total of 548 chickens from 11 KNC populations (KNG, KNB, KNR, KNW, KNY, KNO, HIC, HYD, HBC, JJC, LTC) and 7 introduced populations (ARA: Araucana, RRC and RRD: Rhode Island Red C and D, LGF and LGK: White Leghorn F and K, COS and COH: Cornish brown and Cornish black) were used. Allele size per locus was decided using GeneMapper Software (v 5.0). A total of 195 alleles were observed and the range was 3 to 14 per locus. The MNA, $H_{\exp}$, $H_{obs}$, PIC value within population were the highest in KNY (4.60, 0.627, 0.648, 0.563 respectively) and the lowest in HYD (1.84, 0.297, 0.286, 0.236 respectively). The results of genetic uniformity analysis suggested 15 cluster (${\Delta}K=66.22$). Excluding JJC, the others were grouped in certain cluster with high genetic uniformity. JJC was not grouped in certain cluster but grouped in cluster 2 (44.3%), cluster 3 (17.7%) and cluster8 (19.1%). As a results of this study, we can secure a scientific basis about KNC's uniqueness and these results can be use to basic data for the genetic evaluation and management of KNC breeds.

Studies on Genetic Analysis by the Diallel Crosses in $F_2$ Generation of Cowpea(Vigna sinensis savi.) (동부 Diallel Cross$ F_2$세대의 유전분석에 관한 연구)

  • Kim, J.H.;Ko, M.S.;Chang, K.Y.
    • KOREAN JOURNAL OF CROP SCIENCE
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    • v.28 no.2
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    • pp.216-226
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    • 1983
  • Genetic studies on the $F_2$ generation of a set of half diallel crosses involving six cowpea varieties were conducted. by the randomized block design with three replications to determine combining ability, gene action and the relationships between parents and their $F_2$ hybrids. The 12 agronomic characters namely, days to flowering, days from flowering to maturity, days to maturity, diameter of stem, length of internode, number of branches per plant, length of pod, number of pods per plant, number of grains per pod, number of grains per plant, 100 grain weight and grain weight per plot were observed, and the $F_2$ generation of this diallel set of crosses was analysed for each character according to the method by Jinks and Hayman. The results obtained are summarized as follows: 1. Vr-Wr graphical analyses; The following seven characters, days to flowering, number of branches per plant, length of pod, number of pods per plant, number of grains per plant, 100 grain weight and grain weight per plot appeared to be partially dominant, and over dominance was found for days from flowering to maturity, days to maturity, length of internode and number of grains per pod. But diameter of stem indicated partial dominance near complete dominance. 2. Estimates of genetic variance components; In the degree of dominance,. eight characters, that is, days to flowering, days from flowering to maturity, days to maturity, length of internode, number of pods per plant, number of grains per pod, number of grains per plant and grain weight per plot were expressed larger than 1. And the characters, days from flowering to maturity, number of branches per plant and number of grains per plant as the degree of mean dominance ($H_1$/D) were found to be negative value over other characters. On the other hand, apprent asymmetry of dominance-recessive allele ($H_2$ /$4H_1$) produced comparatively estimates with lower value on days from flowering to maturity, length of internode, number of branches per plant and number of grains per pod. 3. Analyses of combining ability; Mean square value of GCA(general combining ability) appeared to be more important than those of SCA (specific combining ability) for most characters, and among them, grain weight per plot showed the highest mean square value in GCA and SCA. 4. Effect of combining ability; Variety 178 was expressed as the highest GCA effects in five characters (days to flowering days to maturity, number of pods per plant, number of grains per plant and grain weight per plot). SCA effects were differed from parents, characters and crosses, but crosses between TVu 1857 $\times$ TVu 2885 and TVu 2702 $\times$ J78 were shown to be highly with SCA effects on yield.

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