Isolation and Characterization of Microsatellite Markers in Tsaiya Duck

Hsiao, M.C.,Liu, H.C.,Hsu, Y.C.,Hu, Y.H.,Li, S.H.,Lee, S.R. Asian Australasian Association of Animal Productio 2008 Animal Bioscience Vol.21 No.5

An enrichment library of GATA-repeats from genomic DNA was constructed in this study to isolate and characterize microsatellite loci in Tsaiya duck (Anas platyrhynchos). Thirty-three microsatellite markers were developed and used to detect polymorphisms in 30 Tsaiya ducks. A total of 177 alleles were observed and all loci except APT022 were polymorphic. The number of alleles ranged from 2 to 9 with an average of 5.5 per microsatellite locus. The observed and expected heterozygosity of these polymorphic markers ranged from 0.07 to 0.93 with an average number of 0.60 and 0.10 to 0.86 with an average number of 0.61, respectively. Among the polymorphic markers, the observed heterozygosities of 23 loci were higher than 0.50 (69.70%). The polymorphism information content (PIC) in the 32 loci ranged from 0.09 to 0.83 with an average of 0.57. Seven of the 33 duck microsatellite loci had orthologs in the chicken genome, but only APT004 had a similar core repeat to chickens. These microsatellite markers will be useful in constructing a genetic linkage map for the duck and a comparative mapping with the chicken can also provide a valuable tool for studies related to biodiversity and population genetics in this duck species.

한국인집단의 3p (D3S1228, D3S1029, D3S1038 및 THRB)와 9p (IFNA, D9S171, D9S126 및 D9S169)의 Microsatellite 다형현상

안광숙 대전대학교 기초과학연구소 1999 自然科學 Vol.10 No.2

한국인을 대상으로 3번과 9번 염색체의 단완에 있는 8가지 유전자좌(D3S1228, D3S1029, D3S1038, THRB, IFNA, D9S171, D9S126, 및 D9S169)에서 polymerase chain reaction 과 polyacrylamide gel electrophoresis 방법을 이용하여 microsatellite의 다형현상을 분석하였다. 각 유전자좌에서 발견된 대립인자의 수는 3개 (D9S126) 내지 9개 (D3S1029)가 관찰되었으며, 각 유전자좌에 따라 산출된 heterozygosity 빈도는 0.17 (D9S126) 에서 0.73 (D9A169)의 범위에 속하여 매우 polymorphic 한 것을 알 수 있었다. 본 연구에서 사용한 microsatellite marker는 유전적으로 매우 다양함에 따라 유전적 지표로서 친자확인, 개인동정, 유전자 지도작성에 널리 이용될 수 있음을 확인하였다. In Korean population, we analyzed the polymorphism of microsatellite at 3p(D3S1228, D3S1029, D3S1038, THRB) and 9p (IFNA, D9S171, D9S126, D9S169) by the use of polymerase chain reaction and polyacrylamide gel electrophoresis. The number of alleles at locus were range from 3 (D9S126) to 9 (D3S1029) and the frequencies of heterozygosity were from 0.17 (D9S126) to 0.73 (D9A169) with a mean value of 0.50. In the present study, 8 loci were high polymorphic and had genetic variation. This result suggests that microsatellite markets used in this study are very useful as genetic markers for parentage testing, forensic identification, and medical diagnostics.

인삼 (Panax ginseng C.A. Meyer)의 Microsatellite 마커에 대한 유전적 다형성과 특성 규명

박선화(Sun Wha Park),현영세(Young Se Hyun),정기화(Ki Wha Chung) 고려인삼학회 2009 Journal of Ginseng Research Vol.33 No.3

인삼에 대한 microsatellite 개발은 다른 분자적 마커들에 비해 늦게 이루어져, 최근에 와서야 인삼의 microsatellite 들이 보고되고 있는 실정이다. 본 연구에서는, 분리된 microsatellite들 중에서 5 개의 다형성 마커를 선별하여 국내 경작지나 시장에서 유통되는 인삼을 대상으로 유전적 다형성을 조사하고, 각 마커의 특성을 규명하였다. 유전자형 분석은 변성 PAGE와 silver staining법으로 하거나 형광표지 primer로 표지한 PCR 산물을 자동 염기서열 분석기로 분석하였다. 본 연구에서 개발한 5개의 microsatellite 마커들의 평균 대립유전자 수는 3.2 개였으며, 평균 GD는 0.367 였다. 전체적으로 볼 때, PG1419가 가장 높은 다형성을 보였으며 (PIC: 0.460, GD: 0.543), PG770은 가장 낮은 다형성을 나타내었다 (PIC: 0.070, GD: 0.078). 각 좌위들의 예상 이형 접합도 (Hexp)는 0.077에서 0.541 (mean = 0.313)로 계산되었으나, 관측 이형접합도 (Hobs)는 0.040에서 0.130 (mean = 0.083)으로 훨씬 낮게 관찰되었으며, 유전자형의 분포는 Hardy-Weinberg 평형상태에서 벗어남을 보였다. 본 연구에서 개발한 인삼의 microsatellite 마커들은 인삼의 분자적 마커의 데이터베이스 확립의 기초 자료로 활용될 뿐 아니라, 인삼의 분자적 구별법 및 QTL 좌위의 염색체지도 작성에 유용하게 활용될 것이다. Ginseng (Panax ginseng C.A. Meyer) is one of the most important medicinal plants in East Asia. Microsatellite or simple sequence repeat (SSR) markers are used in obtaining genetic analysis and authentication in many plants. The present study examined five microsatellites in conjunction with P. ginseng in Korea. The total observed allelic number was 17 (mean = 3.4), and gene diversities varied from 0.078 to 0.543 with an average of 0.314. Through a combined analysis of five loci in 100 ginseng samples, 44 different combined genotypes were observed. Expected and observed heterozygosites ranged from 0.077 to 0.541 (mean = 0.313) and 0.040 to 0.130 (0.083), respectively. All examined loci exhibited deficiency of heterozygosity and deviation from the Hardy-Weinberg equilibrium. Such results may be explained by the non-random mating and inbreeding that has occurred for several hundred years. These microsatellite markers could be used for the study of molecular genetics and the establishment of DNA marker database, as well as authentication of ginseng species and chromosomal mapping of QTL loci in P. ginseng.

Assessment of high-resolution melting (HRM) profiles as predictors of microsatellite variation: an example in Providence Petrel (Pterodroma solandri)

Anicee J. Lombal,Theodore J. Wenner,Christopher P. Burridge 한국유전학회 2015 Genes & Genomics Vol.37 No.12

High-resolution melting (HRM) analysis is an emerging technology to screen microsatellites for polymorphism. A potential issue surrounding this method is that amplicon sizes for HRM should typically be short (80–100 bp) for highest sensitivity to reveal polymorphism via the presence of two peaks in the curve of the derivative of fluorescence over temperature (dF/dT). In contrast, microsatellite amplicons are typically 100–400 bp. Therefore, we compared HRM analysis melting temperature range (DTm) and multiple dF/dT peaks for predicting microsatellite polymorphism. We assessed polymorphism at 27 microsatellite loci, with estimated lengths of 122–321 bp, in Providence Petrel (Pterodroma solandri). We validated HRM assessment using traditional capillary electrophoresis (CE). While 100 % of loci exhibiting multiple peaks in the dF/dT curve were confirmed as polymorphic by CE, 16 % improvement in sensitivity (83 vs. 67 %) was achieved by using DTm, and 25 % (92 vs. 67 %) by using DTm in addition to multiple dF/dT peaks. We suggest HRM melting temperature range as new predictor of polymorphism that can be used to rapidly assess microsatellites polymorphism.

Microsatellite와 SNP Marker를 이용한 한국재래닭의 유전적 연관지도 작성

서동원(Dong Won Seo),박희복(Hee Bok Park),최누리(Nu Ri Choi),진실(Shil Jin),유채경(Chae Kyoung Yoo),술타나(Hasina Sultana),허강녕(Kang Nyeong Heo),조철훈(Cheorun Jo),이준헌(Jun Heon Lee) 한국가금학회 2015 韓國家禽學會誌 Vol.42 No.1

& & 닭은 전체 염기서열의 길이가 포유류에 비해 3분의 1 정도로 비교적 작은 유전체를 가지고 있기 때문에 인간의 주요한 단백질 공급원으로써의 중요성뿐 아니라, 동물의 형질연관 유전자 연구 및 발생유전학적 연구에 좋은 모델 동물이다. 따라서 본 연구에서는 한국재래닭 이용성을 증대시키는 목적으로 한국재래닭의 유전적 구조를 이해하고, 다양한 응용연구의 토대를 마련하기 위하여 131개의 microsatellite(MS) 마커와 8개의 SNP 마커 유전자형을 이용하여 한국재래닭의 유전적 연관지도를 작성하였다. 그 결과, 한국재래닭의 유전 연관지도의 총 길이는 2729.4 cM으로 확인되었고, 연관지도 작성에 사용된 각 마커 간의 거리는 평균 19.64 cM으로 계산되었다. 모든 마커의 유전적 거리와 물리적 거리의 순서는 GGA8의 ADL0278과 MCW0351의 물리적 거리의 순서가 바뀐 결과만 제외하고, 이전의 연구결과와 매우 유사한 결과를 나타내었다. 또한 macrochromosome과 microchromosome의 재조합률을 비교해 본 결과, macrochromosome이 microchromosome보다 3.7배 크게 나타나, 이전에 보고와 유사한 결과를 나타내었다. 유전 연관지도의 암수에 따른 차이에서는 GGA1, 7, 13, 27의 네 개의 염색체에서 암, 수의 성별에 따른 차이를 나타내었다. 각 마커의 평균 대립유전자 수와 이형접합도(Hexp), 다형성(PIC) 수치는 각각 5.5, 0.63, 0.58로 유전적 지도를 작성하기에 충분하 다형성을 가진 것을 확인하였다. 따라서 본 연구의 결과는 한국재래닭의 유전적 구조를 이해하고, 유전체 QTL 연구와 같은 응용연구에 기초자료로써 유용하게 사용될 수 있을 것으로 사료된다. & & Chicken is one of the major livestock, especially for supplying proteins to human. The chicken genome size is approximately one-third compared with that of the human genome and regarded as a valuable model animal for genetics and development biology. In this study, we constructed the genetic linkage map for Korean native chicken (KNC) using 131 microsatellite (MS) and 8 single nucleotide polymorphism (SNP) markers. As a result, the total map length was calculated as 2729.4 cM and the average genetic distance between markers was 19.64 cM. The marker orders and genetic distances were well matched with the consensus linkage map except for the physical order of ADL0278 and MCW0351 in GGA8. In addition, the recombination rates in marcrochromosomes were 3.7 times higher than that of microchromosomes. The average numbers of alleles, expected heterozygosity (Hexp) and polymorphic information content (PIC) values were calculated as 5.5, 0.63 and 0.58, respectively. These results will give useful information for the understanding of genetic structure and QTL studies in KNC.

Microsatellite를 이용한 한국 보리 유전자원의 유연관계 분석

백형진,윤문섭,김행훈,이정란,박광래,조양희,최원열 한국육종학회 2004 한국육종학회지 Vol.36 No.5

This study was conducted to understand the genetic diversity and relationships of Korean cultivated barley groups classified as landrace, improved and introduced varieties by microsatellite variation (SSRP, Simple Sequence Repeats Polymorphism). With the

Characterization of 20 microsatellite loci by multiplex PCR in swimming crab, Portunus trituberculatus

이혜진,이도현,윤승종,김대현,김상규,현영세,민기식,정기화 한국유전학회 2013 Genes & Genomics Vol.35 No.1

The swimming crab, Portunus trituberculatus (Crustacea: Decapoda: Brachyura), is one of the most important fishery resources in East Asia. The aim of this work is to develop microsatellite multiplex PCR systems for 20 microsatellites which were isolated from a microsatellite-enriched genomic library and to explore the genetic diversity of P. trituberculatus captured in the coastal regions of the Yellow Sea in Korea. We prepared four multiplex PCR systems (two hexplex and tetraplex systems each) which could be used for the genotyping of 20 markers. Most markers in this system are highly informative with mean polymorphism information contents and observed heterozygosity of 0.913 and 0.930. The total number of alleles observed in this study was 724, and the mean allelic number per locus was 36.2. Significant deviations from the Hardy–Weinberg equilibrium (HWE) were observed at six loci in the Wonsando Island population, but all the loci deviated from the HWE in the Jeonjangpo population. The genetic differentiation between the two populations and the phylogenetic tree constructed by the unweighted pair group method with the arithmetic average suggested that there is no significant genetic difference between Wonsando Island and Jeonjangpo populations. The prepared multiplex PCR systems and population genetic data will be helpful to study phylogeographic analysis as well as to prepare strategies of stock management and aquaculture of P. trituberculatus.

한국인집단의 D14S299 유전자좌내 microsatellite의 다형현상

박귀성,강혜자,안광숙 대전대학교 기초과학연구소 2000 自然科學 Vol.11 No.1

The allele frequency of D14S299 microsatellite locus was analyzed in 242 normal blood from Korean population. Using the polymerase chain reaction and polyacrylamide gel electrophoresis. we examined the allele and genotype frequency. The size of 0145299 PCR fragments ranged from 299 bp to 331 bp. The most frequent allele in the Korean subjects was allele 30 (44.9%) followed by allele 31 (25.4%), allele 32 (12.4%), and minor alleles (17.3%). The heterozygosity of D14S299 was 0.710. The polymorphism information content (PIC) and power of discrimination (POE) were 0.67 and 0.880, respectively in the present study. The genotype distribution and allele frequency of D14S299 microsatellite in the Korean subjects was similar to those of Japanese and Chinese populations including in the same Asian Population.

Reverse Random Amplified Microsatellite Polymorphism Reveals Enhanced Polymorphisms in the 3’ End of Simple Sequence Repeats in the Pepper Genome

민웅기,한정헌,강원희,이흥렬,김병동 한국분자세포생물학회 2008 Molecules and cells Vol.26 No.3

Microsatellites or simple sequence repeats (SSR) are widely distributed in eukaryotic genomes and are informative genetic markers. Despite many advantages of SSR markers such as a high degree of allelic polymorphisms, co-dominant inheritance, multi-allelism, and genome-wide coverage in various plant species, they also have shortcomings such as low polymorphic rates between genetically close lines, especially in Capsicum annuum. We developed an alternative technique to SSR by normalizing and alternating anchored primers in random amplified microsatellite polymorphisms (RAMP). This technique, designated reverse random amplified microsatellite polymorphism (rRAMP), allows the detection of nucleotide variation in the 3’ region flanking an SSR using normalized anchored and random primer combinations. The reproducibility and frequency of polymorphic loci in rRAMP was vigorously enhanced by translocation of the 5’ anchor of repeat sequences to the 3’ end position and selective use of moderate arbitrary primers. In our study, the PCR banding pattern of rRAMP was highly dependent on the frequency of repeat motifs and primer combinations with random primers. Linkage analysis showed that rRAMP markers were well scattered on an intra-specific pepper map. Based on these results, we suggest that this technique is useful for studying genetic diversity, molecular fingerprinting, and rapidly constructing molecular maps for diverse plant species.

한국인 집단에서 D14S299 및 Cellular Stress Response(CSR)유전자의 Microsatellite 다형현상

류성호,박귀성,안광숙 한국유전학회 2002 Genes & Genomics Vol.24 No.2

Allele frequencies at microsatellite loci D14S299 and CSR were analysed by polymerase chain reaction from Korean subjects. According to repeat number of microsatellite, nine alleles were identified at D14S299 locus and PCR amplified fragments ranged from 299 to 331 bps in size. The most frequent allele in this study was allele 30 (44.9%) followed by allele 31 (25.4%) and few more alleles. By the results of statistical analysis, the heterozygosity of D14S299 was 0.710, and the polymorphism information content (PIC) and the power of discrimination (POD) were 0.670 and 0.880, respectively. The genotype distribution and allele frequency of D14S299 microsatellite in Korean population were similar to those of Japanese and Chinese populations. Genes involved in cellular stress response (CSR) possess various numbers of dinucleotide (CA) repeats. Frequencies of two most commonly occurring alleles, a2 and a3, were 31.2% and 31.5%, respectively. Allele frequencies agreed with the Hardy-Weinberg expectation. The observed heterozygosity was 0.69, and the values of PIC and POD were 0.730 and 0.866, respectively. We found D14S299 and CSR were genetically polymorphic in the Korean population and useful microsatellite markers for forensic practices.