Earticle

In Malaysia, Labisia pumila Benth & Hook f, popularly known as ‘Kacip Fatimah’ has been used traditionally to treat various elements of the woman’s health in Malay community. The objective of this study was to develop randomly amplified polymorphic DNA (RAPD) based DNA markers for the identification of L. pumila and to distinguish its three varieties from each other. Total DNA from nine accessions of L. pumila was extracted by CTAB method and polymerase chain reactions (PCR) were carried out to amplify the segments of DNA using different primers to develop DNA barcode using RAPD technique. To find out variety-specific DNA marker/s, twenty different 10-mer primer sequences with annealing temperature from 36-40 °C were evaluated in triplicate. Out of 20 random primers, two primers (OPA-1 and OPA-2/A10) were selected which produced reliable RAPD band patterns. To have DNA based handle, two RAPD amplification products were cloned and sequenced to determine the identity of the DNA. RAPD analysis using two random primers generated 72 discrete bands ranging in size 200 bp-3,000 bp. Fifty nine of these were polymorphic loci (82%) and thirteen were non-polymorphic loci (18%). A total of 32 bands polymorphic loci (72%) were amplified with primer OPA-1 and analyzed by cluster analysis and UPGMA (Unweighted Pair Group Method with Arithmetic) to present a dendogram depicting the degree of genetic relationship among nine accessions of L. pumila. Our results shows the reasonable genetic diversity among the L. pumila varieties and within varieties; and two RAPD marker sequences obtained could be used to identify L. pumila at species level.

Ficus deltoidea Jack is an important and popular medicinal plant species found in the Malaysia. Plants are being collected and used based on morphology and authentication to prevent adulteration is not in practice. In this study, twenty-six accessions of F. deltoidea Jack were collected from Kelantan and Terengganu states of Peninsular Malaysia to examine their genetic similarities and differences using randomly amplified polymorphic DNA (RAPD) technique. Out of 20 arbitrary primers, two primers (D-10 and D-11) were selected which produced reliable DNA polymorphism. D-10 and D-11 primers generated 138 RAPD bands ranging from 250 bp to 3000 bp. Ninety-nine of them were polymorphic loci (72%) and thirty-nine were nonpolymorphic loci (28%). A total of 56 bands with polymorphic loci were amplified with primer D-10 and analyzed by cluster analysis and UPGMA to present a dendrogram depicting the degree of genetic relationship among 26 accessions. Eight RAPD markers were sequenced to determine their identity. RAPD analysis showed the genetic diversity among 26 accessions of F. deltoidea Jack. The RAPD profile and RAPD marker sequences reported in this paper could be used in plant and/or plant material authentication. This study also suggested that RAPD can be a useful technique to study DNA polymorphism in F. deltoidea Jack.

돼지 Duroc 품종의 mitochondria DNA D-loop전체 유전자를 증폭하기 위하여 많은 동물에서 고도로 상동성이 높은 tRNA-Pro와 tRNA-Phe 염기서열 일부를 이용하여 oligonucleotide primer를 제작하였다. 그 결과 Duroc 품종의 D-loop 전체 유전자는 1,145 base pairs 였으며, 그 중간위치에 10bp의 Sus Scrofa-specific sequence (TACACGTGCG)가 10개 존재하고 있었다. 돌연변이 검출을 위하여 가장 변이가 심한 지역을 primer 제작하여 345 bp의 DNA 단편을 증폭하였으며, Single Stranded Conformation Polymorphism(SSCP) 분석은 8% polyacrylamide gel에서 200 V, 16시간 전기영동하여 ethidium bromide (EtBr)로 10분간 염색하여 UV image analyzer로 관찰하였다. 그 결과 두 개의 서로 다른 밴드유형을 관찰하였으며, 21개 부위에서 염기서열 변이가 관찰되었다. 이러한 결과는 유전적 다양성 변이를 검출하는데 SSCP 분석이 유용한 도구라고 사료된다.

The mitochondrial DNA(mtDNA) D-loop region was amplified from Duroc(Sus scrofa) by polymerase chain reaction(PCR). The oligonucleotide primer used to amplify the Sus scrofa mtDNA D-loop region was designed using tRNA-Pro and tRNA-Phe sequence in mtDNA regions highly conserved in many other animal species. There were 1,145 base pairs(bp) in the D-loop region. The middle of the region contained 10 tandem repeat of an 10-bp Sus scrofa-specific sequence, TACACGTGCG. We designed primers for PCR-mediated single stranded conformation polymorphism(SSCP) analysis that amplified a 345 bp fragment, which contained the most variable region according to our sequencing data. SSCP analysis of denatured amplification products was carried out by polyacrylamide(8%) gel electrophoresis followed by ethidium bromide staining. The SSCP analysis identified two band patterns(A and B) and comparision of these two nucleotide sequences identified 21 base substitutions. These results show that SSCP analysis of the D-loop region is useful for detecting the genetic polymorphism.

Broadening the genetic base of Platycodon grandiflorum DC. cultivar to sustain improvement requires assessment of genetic diversity available in P. grandiflorum DC.. The objective of this study was to analyze the genetic variation, genetic relationship among 48 samples collected from East-Asian Area by means of RAPD-PCR (random amplified polymorphic DNA-polymerase chain reaction) markers. From the 18 primers tested, produced total 211 bands with an average of 11.7 bands per primer and obtained 103 polymorphic band with an average of 5.7 bands per primer,s revealed relatively high percentage of polymorphic bands (48.8%). The genetic similarities calculated from RAPD data varied from 0.688 to 0.994 and were clustered to six major groups on a criterion of 0.78 similarity coefficient. The present study has revealed the significant genetic similarity among the samples tested. The analysis of genetic relationships in P. grandiflorum using RAPD-PCR banding data can be useful for the breed improvement.

This study was conducted to detect the specific fragment genes by using RAPD-PCR and RFLP method in the Korean Tiger cattle and Korean Native cow. And then, the specific fragment gene was investigated by the analysis of the genes for detection significance according to the expressing pattern. We found the specific expression gene by the RAPD-PCR analysis in Korean Tiger cattle. It were a detected the differences of the species in the colour and external section. The Korean Tiger cattle were vary low compare to the Korean Native cattle by analysis result of polymorphism and distribution. And the polymorphism over 500bp into the size classification detected was highly pattern and it could be utilize by the resource of the specific gene. There was a found the specific gene by sequencing in the 1855bp gene fragment of Korean Tiger cattle. And the sequencing result of the R9B was different between Korean Native cow and Holstein cattle. Thus, this gene can be apply as the specific gene in the Korean Tiger cattle.

국내에서 사육중인 치와와 31두, 풍산개 20두, 래브라도 리트리버 8두를 대상으로 micro- satellite DNA형의 유전자 빈도에 기초하여 heterozygosity, PIC, 그리고 PE를 분석한 결과를 요약하면 다음과 같다. 치와와의 대립유전자 수는 4∼14개로서 expected heterozygosity와 PIC는 각각 0.432∼0.883 (평균 0.711), 0.397∼0.856(평균 0.659)으로 나타났으며 PEZ1, PEZ3, PEZ6, PEZ10, PEZ12의 marker는 PIC 0.7이상으로 나타났고 14개 marker를 조합시 부권부정율은 0.9999로 관찰되었다. 풍산개의 대립유전자 수는 2∼9개로서 expected heterozygosity와 PIC는 각각 0.262∼0.817 (평균 0.559), 0.222∼0.772 (평균 0.503)으로 나타났고 PEZ1, PEZ6, PEZ13의 marker는 PIC 0.7이상으로서 16개 marker를 조합시 부권부정율은 0.9991로 관찰되었다. 래버라도 리트리버의 대립유전자 수는 3∼5개로서 expected heterozygosity와 PIC는 각각 0.425∼0.808 (평균 0.660), 0.354∼0.717 (평균 0.563)으로 나타났고 PEZ8, PEZ12의 marker는 PIC 0.7이상으로 관찰되었으며 12개 marker를 조합시 부권부정율은 0.9968로 관찰되었다. 이상의 결과는 microsatellite DNA형에 의한 개의 친자감정 및 개체식별에 유용한 자료로 활용할 수 있을 것으로 사료된다.

This study was carried out to investigate a usefulness of the microsatellite DNA markers for individual identification and parentage verification in three dog breeds. A total of 59 random dog (31 Chiwawa, 20 Poongsan, 8 Labrador Retriever) samples were genotyped by using 14 markers (Chiwawa dog), 16 markers (Poongsan dog), and 12 markers (Labrador Retriever dog) among the 17 international standard markers (PEZ1, 3, 5, 6, 8, 10, 11, 12, 13, 15, 16, 17, 20, 21, FHC2010, FHC2054 and FHC2079), respectively. The number of alleles per locus varied from 4 to 14 with a mean value of 6.07 in Chiwawa dog, 2 to 9 with a mean of 4.75 in Poongsan dog, and 3 to 5 with a mean of 4.00 in Labrador Retriever dog. Observed heterozygosity was ranged 0.419∼0.968 (mean 0.755), 0.300∼0.950 (mean 0.597) and 0.125∼0.750 (mean 0.604), and expected heterozygosity was ranged 0.432∼0.883 (mean 0.711), 0.262∼0.817 (mean 0.559) and 0.425∼0.808 (mean 0.660) in these three dog breeds. PIC value was ranged 0.397∼0.856 (mean 0.659), 0.222∼0.772 (mean 0.503) and 0.354∼0.717 (mean 0.563) in these three dog breeds. Of the 17 markers, PEZ1, PEZ3, PEZ6, PEZ10, PEZ12 loci, PEZ1, PEZ6, PEZ13 loci, and PEZ8, PEZ12 loci have relatively high PIC value (＞0.7) in Chiwawa dog, Poongsan dog and Labrador Retriever dog, respectively. The exclusion probability was ranged 0.240∼0.741, 0.111∼0.616, and 0.198∼0.529, and the combination of microsatellite loci was 0.9999, 0.9991, and 0.9968 in Chiwawa dog, Poongsan dog and Labrador Retriever dog, respectively. These results can give basic information for developing parentage verification and individual identification system in these three dog breeds.

소의 흰 반점 관련 후보유전자로 c-KIT receptor 유전자를 선정하여, c-KIT receptor 유전자내의 변이를 탐색하고 변이가 흰반점 표현형과 연관성이 있는지를 분석하였다. 한우, Angus, Brown Swiss, Charolais, Hereford, Hols- tein, Limousin 및 Simmental 등 8개 품종의 DNA 시료를 사용하여 c-KIT receptor 유전자의 intron 6번 영역에서 다형성을 조사하고 분석하였다. c-KIT receptor 유전자의 intron 6번 영역에서는 4개의 염기치환이 발견되어, MspⅠ, BsrBⅠ 및 NdeⅠ 제한효소를 이용하여 PCR- RFLP 분석을 실시하였다. Intron 6번을 포함하는 영역의 PCR 산물 크기는 2,440 bp 이었다. MspⅠ다형성은 PCR-RFLP 분석 결과 3개의 대립유전자가 존재하였으며, 한우품종에서는 3개의 대립유전자 모두가 발견되었고, CC 형태의 유전자형을 제외한 5개의 유전자형 (AA, AB, AC, BC 및 BB)을 확인하였다. Angus, Brown Swiss, Hereford, Holstein 및 Simmental 품종에서는 A 대립유전자만을 갖는 것으로 조사되었고, 한우는 44%만 AA 유전자형을 나타내었다. BsrBⅠ 다형성은 2개의 대립유전자로서 3개의 유전자형이 나타나는 것을 확인하였으며, Charo- lais 및 Hereford 품종이 다른 소 품종에 비하여 A 대립유전자의 빈도가 높게 나타났다. NdeⅠ다형성을 분석한 결과 Brown Swiss 품종에서는 NdeⅠ에 의해 절단되는 형태인 A 대립유전자만 관찰되었으며, Holstein 품종은 92%, Simmental 품종은 72%가 절단되는 형태를 나타내어, 모색이 흰색을 띠는 소 품종에서 절단되는 형태가 많았다. 소 c-KIT receptor 유전자의 intron 6번 영역에서 확인된 4개의 염기치환은 품종에 따라 다른 빈도를 보였으나, 이들 염기치환과 흰 반점과의 연관성에 대한 증거는 발견하지 못하였다. 그러므로 소의 흰 반점과 c-KIT receptor 유전자 내의 변이와의 관련성은 다른 영역에 대한 추가적인 분석과, 이미 보고된 다른 모색관련 유전자의 다형성과의 연관성 분석 등과 같은 연구가 필요한 것으로 판단된다.

We considered KIT gene as a candidate gene for the white-spotting pattern in cattle. This study was carried out to detect genetic variation of c-KIT receptor gene and to investigate association between the mutation and the white-spotting pattern in cattle. PCR-RFLP analysis within intron 6 of c-KIT receptor gene were performed with 8 cattle breeds including Hanwoo, Angus, Brown Swiss, Charolais, Hereford, Holstein, Limousin and Simmental.When PCR product of approximately 2,440 bp including intron 6 of c-KIT receptor gene was sequenced, four nucleotide substitutions were found within intron 6 of the bovine c-KIT receptor gene. In PCR-RFLP analysis, three alleles (A, B and C), two alleles (A and B) and two alleles (A and B) at each locus were identified by MspⅠ, BsrBⅠ and NdeⅠ, respectively. Although frequencies of allele at each locus were different among cattle breeds, we could not get any evidence related with white or white spotting phenotypes in these mutations on intron 6 of c-KIT receptor gene. However, we can not entirely exclude the possibility that c-KIT receptor gene is responsible for white spotting phenotype in cattle. Thus, further studies need to detect other mutations in c-KIT receptor gene and to test association of those mutations and coat color phenotypes in cattle.

돼지 Duroc 품종의 mitochondria DNA D-loop전체 유전자를 증폭하기 위하여 많은 동물에서 고도로 상동성이 높은 tRNA-Pro와 tRNA-Phe 염기서열 일부를 이용하여 oligonucleotide primer를 제작하였다. 그 결과 Duroc 품종의 D-loop 전체 유전자는 1,145 base pairs 였으며, 그 중간위치에 10bp의 Sus Scrofa-specific sequence (TACACGTGCG)가 10개 존재하고 있었다. 돌연변이 검출을 위하여 가장 변이가 심한 지역을 primer 제작하여 345 bp의 DNA 단편을 증폭하였으며, Single Stranded Conformation Polymorphism(SSCP) 분석은 8% polyacrylamide gel에서 200 V, 16시간 전기영동하여 ethidium bromide (EtBr)로 10분간 염색하여 UV image analyzer로 관찰하였다. 그 결과 두 개의 서로 다른 밴드유형을 관찰하였으며, 21개 부위에서 염기서열 변이가 관찰되었다. 이러한 결과는 유전적 다양성 변이를 검출하는데 SSCP 분석이 유용한 도구라고 사료된다.

The mitochondrial DNA(mtDNA) D-loop region was amplified from Duroc(Sus scrofa) by polymerase chain reaction(PCR). The oligonucleotide primer used to amplify the Sus scrofa mtDNA D-loop region was designed using tRNA-Pro and tRNA-Phe sequence in mtDNA regions highly conserved in many other animal species. There were 1,145 base pairs(bp) in the D-loop region. The middle of the region contained 10 tandem repeat of an 10-bp Sus scrofa-specific sequence, TACACGTGCG. We designed primers for PCR-mediated single stranded conformation polymorphism(SSCP) analysis that amplified a 345 bp fragment, which contained the most variable region according to our sequencing data. SSCP analysis of denatured amplification products was carried out by polyacrylamide(8%) gel electrophoresis followed by ethidium bromide staining. The SSCP analysis identified two band patterns(A and B) and comparision of these two nucleotide sequences identified 21 base substitutions. These results show that SSCP analysis of the D-loop region is useful for detecting the genetic polymorphism.

The genetic background of the garlic (Allium sativum L.) is not well understood, since it is cultivated exclusively by vegetative propagation. To understand its genetic background, a local clutivar, Danyang, was chosen, and several basic characteristics of its chromosomal DNA were examiend. Its G＋C content was 40.6%, and the relative proportion of fast reassociated sequences, intermediate reassociated sequences, and slow reassociated sequences were 12%, 40%, and 48%, respectively. The genome size, calculated based on reassociation kinetic experiments, was 1.11$\times$1010 bp or 12.16 pg per haploid genome. To compare the genetic variation among four local cultivars, Munkyung, Seosan, Euiseong, and Danyang, random amplified polymorphic DNA (RAPD) analysis was performed. By using slightly longer primers, 18-24 nucleotides in size, than traditional primers used for such analysis, more reliable RAPD results were obtained. 15 primers gave rise to amplified bands, and the results could be grouped into two categories. The patterns of amplified products produced by 12 primers, group A, were polymorphic. These results were analyzedusing a NTSYS-PC (Numerical Taxonomy and Multivariate Analysis System), and a dendrogram grouping the four local cultivars was produced. The three primers of group B gave rise to a monomorphic band pattern from four local garlic clutivars, indicating that these primers possibly recognize garlic specific sequences. These primers were useful in identifying genetic variations among the Allium species.

Insulin-like growth factor binding protein-3 (IGFBP-3) gene is important for regulation of growth and development in mammals. The present investigation was carried out to study DNA polymorphism by PCR-RFLP of IGFBP-3 gene and its effect on fibre traits of Chinese Inner Mongolian cashmere goats. The fibre traits data investigated were cashmere fibre diameter, combed cashmere weight, cashmere fibre length and guard hair length. Four hundred and forty-four animals were used to detect polymorphisms in the hircine IGFBP-3 gene. A 316-bp fragment of the IGFBP-3 gene in exon 2 was amplified and digested with HaeIII restriction enzyme. Three patterns of restriction fragments were observed in the populations. The frequency of AA, AB and BB genotypes was 0.58, 0.33 and 0.09 respectively. The allelic frequency of the A and B allele was 0.75 and 0.25 respectively. Nucleotide sequencing revealed a C>G transition in the exon 2 region of the IGFBP-3 gene resulting in R158G change which caused the polymorphism. Least squares analysis revealed a significant effect of genotypes on cashmere weight (p<0.0001), cashmere fibre length (p<0.001) and hair length (p<0.05) of the animals. The effect of genotypes on cashmere fibre diameter was not statistically significant (p>0.05). The animals of AB and BB genotypes showed higher cashmere weight, cashmere fibre length and hair length than the animals possessing AA genotype. These results suggested that polymorphisms in the hircine IGFBP-3 gene might be a potential molecular marker for cashmere weight in cashmere goats.

The PCR- restriction fragment length polymorphism (RFLP) in and around TM4 of SLC11A1 gene and its association with the incidences of brucellosis in Hariana breed (Bos indicus) and Holstein Friesian crossbred (Bos indicus${\times}$Bos taurus) cattle was examined. A fragment of 954 bp encoding the TM4 was amplified, and RFLP was identified by digestion of the amplicon independently with AluI and TaqI. The amplicon (GenBank Acc. No. AY338470 and AY338471) comprised of a part of exon V (<59 bp) and VII (62>), and entire intron 5 (423 bp), exon VI (71 bp) and intron 6 (339 bp). Digestion with AluI revealed the presence of two alleles viz, A (281, 255, 79 and 51 bp) and B (541, 255, 79 and 51 bp). The frequency of A allele was estimated as 0.80 and 0.73 in Hariana and crossbred cattle, respectively. Due to presence of a polymorphic TaqI site at intron 5, two alleles: T (552 and 402 bp) and Q (231, 321 and 402 bp) were identified. The frequency of T allele was estimated as 0.96 and 0.97, respectively. For association study, on the basis of serological tests and history of abortion, the animals were grouped into "affected" and "non-affected". However, no association could be established with the observed RFLPs.

The distributions of G to A substitution ($G^{-75}{\rightarrow}A$) mutation in the human apolipoprotein A1 (APOAI) gene promoter region and glutathione S-tran-sferase Mu1 (GSTM1) gene deletion were examined in subjects with Korean population. The $G^{-75}{\rightarrow}A$ mutation of APOA1 was genotyped by the polymerase chain reaction (PCR) and subsequent digestion of the PCR product using either Mspl or Mval (n=206). The observed numbers of GG, GA and AA genotypes were 132, 63 and 11, respectively. The allele frequencies of G and A were 0.794 and 0.206, respectively. The GSTM1 gene deletion was simply examined by the PCR amplification (n=106). The observed numbers of null type ($GSTM1^*0/GSTM1^*0$) and positive type were 55 and 51, respectively. The allele frequency of $GSTM1^*0$ was 0.720.

Most expressed HLA loci exhibit a remarkable degree of allelic polymorphism, which derives from sequence differences predominantly localized to discrete hypervariable regions of the amino-terminal domain of the molecule. In this study, the HLA-DRB1 genotypes were determined in eighteen control cell lines and 112 unrelated Koreans using the PCR-SSP (Polymerase Chain Reaction-Sequence Specific Primer) technique. 29 specific primer pairs in assigning the DRB1 gene were used. The results of control cells correlated well with the data which was previously reported. The heterozygosity and homozygosity of the DRB1 gene were 0.786 and 0.214, respectively. In a total of 41 different DRB1 alleles and 83 genotypes, the most frequent allele and genotype were DRB1*04 and DRB1*0901/1501, respectively. This study shows that the PCR-SSP technique is relatively simple, fast and a practical tool for the determination of the HLA-DRBI genotypes. Moreover, these results-allele and genotype frequency and heterozygosity of the HLA DRB1 gene-could be useful for database study before being applied to individual identification and transplantation immunity.

Mitochondrial DNA haplotypes from the displacement-loop (D-loop) region (436 bp) were genotyped and sequenced in Japanese Black beef cattle raised in the same herd. Correlation coefficients between mitochondrial DNA haplotypes, maternal lineage, birth weight, preweaning average daily gain, weaning weight, post weaning average daily gain and yearling weight were computed. The objective was to study the relationship between maternal and postnatal growth traits and to investigate if postnatal growth of calves to yearling age could be accurately predicted from mitochondrial DNA haplotypes. Results of the phylogenetic analysis revealed 17 maternal lineages and four mitochondrial DNA haplotypes. There were strong, positive and highly significant (p<0.001) correlations among maternal traits ranging from 0.52 to 0.98. Similarly, among postnatal growth traits, most of the correlations were also strong, positive and highly significant (p<0.001); the highest correlation of 0.94 was between preweaning average daily gain and weaning weight. However, correlations between mitochondrial DNA haplotypes and postnatal growth traits were very low, mostly negative and non-significant (p>0.05) ranging from -0.05 to 0.1. Prediction of postnatal growth from mitochondrial DNA yielded very low $R^{2}$ values ranging from 0.002 to 0.019. It was concluded that mitochondrial DNA polymorphism has no significant association with postnatal growth from birth to yearling age, and by implication, nuclear rather than cytoplasmic DNA, accounts for most of the genetic variation observed in postnatal growth of Japanese Black cattle. Therefore, mitochondrial DNA genotyping at an early age has no bearing on the accurate prediction of the future growth performance of calves.

We used a portion of mitochondrial COI gene sequences (438 bp) to investigate the phylogenetic and population genetic characteristics of the diamondback moth, Plutella xylostella, in Korea. A total of 27 mtDNA haplotypes were obtained from the samples collected from eight localities mostly in southern part of Korea. One haplotype, DB24, obtained from Muju in Chunrabuk Province formed an independent phylogenetic group in the PAUP and PHYLIP analyses, separated by 1.8~2.7% of sequence divergence from others. This distinct haplotype appears to be one that carried by immigrants from outside study areas, but further study is required. Even excluding DB24, sequence divergence was moderate to substantial (0.2~1.4%) and haplotype diversity within populations was substantially higher (mean H = 0.95), compared with other relevant studies. Although most haplotypes were confined within one locality, female gene flow was substantial or high (Nm = 10.6-infinite). The same pattern was observed in the hierarchical genetic analysis, showing that local populations were composed of heterogeneous haplotypes, and the genetic distance between localities were not statistically significant. These findings allowed us to conclude that the local diamondback moth populations was formed with randomly dispersed individuals, survived from the diversified use of insecticides over geographic areas.