PCR-RFLP genotyping for Japanese and Korean populations of Pacific oyster using mitochondrial DNA noncoding region

Production of Pacific oyster Crassostrea gigas in Miyagi and Hiroshima (Japan) has gradually increased, with a marked increase in imported oysters from Goseong (Korea), and then cultured oysters of the Miyagi, Hiroshima, and Goseong populations accounting for most oyster consumption in Japan. In this study, we developed a simple PCR-RFLP analysis of a mitochondrial DNA noncoding region (mtDNA-NCR) for differentiating cultured oysters of these populations. PCR amplification yielded a 818 bp fragment comprising the entire mtDNA-NCR and parts of adjacent tRNA^Cys and tRNA^Asn genes from all the specimens. By use of a wide restriction test using 30 different restriction enzymes, only a single enzyme only, AluI, produced a unique RFLP pattern enabling us to discriminate Miyagi and Hiroshima oysters from Goseong oysters. This difference is probably because of nucleotide alteration at the presumptive AluI recognizing site on position 439 of the mtDNA-NCR. Our simple, robust and cost-effective PCR-RFLP analysis is potentially useful for population genetic investigation of cultured Pacific oyster, particularly when large numbers of specimens must be analyzed.     

Genetic variation of wild and hatchery populations of the catla Indian major carp (Catla catla Hamilton 1822: Cypriniformes, Cyprinidae) revealed by RAPD markers

Genetic variation is a key component for improving a stock through selective breeding programs. Randomly amplified polymorphic DNA (RAPD) markers were used to assess genetic variation in three wild population of the catla carp (Catla catla Hamilton 1822) in the Halda, Jamuna and Padma rivers and one hatchery population in Bangladesh. Five decamer random primers were used to amplify RAPD markers from 30 fish from each population. Thirty of the 55 scorable bands were polymorphic, indicating some degree of genetic variation in all the populations. The proportion of polymorphic loci and gene diversity values reflected a relatively higher level of genetic variation in the Halda population. Sixteen of the 30 polymorphic loci showed a significant (p < 0.05, p < 0.01, p < 0.001) departure from homogeneity and the F(ST) values in the different populations indicated some degree of genetic differentiation in the population pairs. Estimated genetic distances between populations were directly correlated with geographical distances. The unweighted pair group method with averages (UPGMA) dendrogram showed two clusters, the Halda population forming one cluster and the other populations the second cluster. Genetic variation of C. catla is a useful trait for developing a good management strategy for maintaining genetic quality of the species.     

内蒙古地区亚洲小车蝗不同地理种群的RAPD分析

采用随机扩增多态性DNA(RAPD)技术对内蒙古地区亚洲小车蝗Oedaleus asiaticus(B.-bienko)9个不同地理种群90个个体进行扩增,8条随机引物扩增共产生了78条带,多态性片段为62条。对Nei′s基因多样性指数和遗传距离进行分析,结果表明:种群间的遗传分化系数为0.2343,即23.43%的遗传变异存在于种群间,种群内的遗传分化系数为0.7657,即76.57%的遗传多样性存在于种群内,群体内遗传多样性大于群体间遗传多样性。用NJ法对这3个种群的Nei′s遗传距离作聚类分析,结果表明亚洲小车蝗不同种群的遗传分化程度与地理距离具有正相关关系。     

A novel mitochondrial intergenic spacer reflecting population structure of Pacific oyster

Nucleotide sequence divergence in a novel major mitochondrial DNA intergenic spacer (IGS) of Pacific oyster Crassostrea gigas was analyzed for 29 cultured individuals within the Goseong population (Korea). A total of 7 variable sites were detected within the IGS, and the relative frequency of nucleotide alteration was determined to be 1.16%;. All alterations were due to a single nucleotide substitution, and 5 transitions and 2 transversions were observed. Among 29 specimens, only 8 haplotypes could be identified, and 6 of the haplotypes were unique to particular specimens. Pairwise genetic diversity of all 8 haplotypes was calculated to be 0.412+/-0.134 from multiple sequence substitutions based on the two-parameter model. The phylogenetic tree obtained for these haplotypes according to the neighbor-joining method illustrated a single cluster of linkages, which comprised 5 haplotypes associated with 23 specimens, while the other 3 haplotypes associated with 6 specimens were scattered. The results indicate that the IGS is higher polymorphic and thus more suitable as a genetic marker for population structure analysis of Pacific oyster than the mtDNA coding regions, such as cytochrome c oxidase I and 16S ribosomal RNA genes.     

Analysis of Genetic Diversity in Cicer arietinum L Using Random Amplified Polymorphic DNA Markers

PCR-based random amplified polymorphic DNA (RAPD) markers were employed to assess genetic diversity in 23 chickpea genotypes. Forty of the 100 random primers screened revealed polymorphism among the genotypes. Most of the primers revealed single polymorphic band, and only 14.1 2% of the products were polymorphic. Estimates of genetic similarity based on Jaccard’s coefficient ranged from 0.92 to 0.99, indicating narrow genetic variability among the genotypes based on RAPD markers.The 23 chickpea genotypes formed two major clusters in the dendrogram.The low RAPD polymorphism among chickpea genotypes suggests that more number of polymorphic primers need to be analysed to determine genetic relationships. It was observed that RAPD analysis employing 30 polymorphic primers could provide better estimates of genetic relationships in chickpea.     

云南三种稻蝗基因组DNA的RAPD多态性分析

应用RAPD技术对采自云南龙陵两交水和腾冲曲石马鹿冲的24头稻蝗进行了基因组DNA多态性分析,10条随机引物共产生129条清晰、稳定的谱带.基于Jaccard相似系数,用Between-groups Linkage法得出的聚类图与基于Nei's遗传距离分别用UPGMA和NJ法构建的分子系统树基本一致:供试的所有稻蝗个体分为3个聚类簇:龙陵两交水的3、7～8号个体与腾冲曲石马鹿冲(海拔1450m)的8个个体聚为一支,龙陵两交水的1、2、4～6号个体聚为另一支,两支相聚后与腾冲曲石马鹿冲(海拔1 550 m)的8个个体聚为一起.形态学鉴定表明:龙陵两交水的3、7～8号个体为日本稻蝗,1、2、4～6号个体为小稻蝗,腾冲曲石马鹿冲(海拔1450 m)的8个个体均为日本稻蝗,腾冲曲石马鹿冲(海拔1550 m)的个体为有待进一步鉴定的未知种.形态学鉴定结果与聚类结果完全吻合,充分展示了RAPD技术在区分近缘物种方面的独到优势.比较不同采集地的日本稻蝗、小稻蝗的RAPD分析结果,发现云南的这两种稻蝗在同物种不同种群中的遗传多样性最为丰富,从一个侧面显示出我国云南昆虫的物种多样性及物种内部丰富的遗传多样性.     

Comparative Assessment of Genetic Variability in the Populations of Endemic and Endangered Yellow Catfish, Horabagrus brachysoma (Teleostei: Horabagridae), Based on Allozyme, RAPD, and Microsatellite Markers

The comparative assessment of genetic diversity using allozymes, random amplified polymorphic DNA (RAPD), and microsatellite markers was conducted in endemic and endangered yellow catfish (Horabagrus brachysoma) sampled from three locations in Western Ghats river systems of India. Among the three markers, microsatellites show more polymorphism, having 100% polymorphic loci, whereas allozymes show the least (56%). In RAPD, 60.5% of fragments were polymorphic. Observed heterozygosity and F(ST) values were very high in microsatellites, compared with the other markers. Microsatellite and RAPD markers reported a higher degree of genetic differentiation than allozymes among the populations depicted by pairwise F(ST)/G(ST), AMOVA, Nei's genetic distance, and UPGMA dendrogram. The three classes of markers demonstrated striking genetic differentiation between pairs of H. brachysoma populations. The data emphasize the need for fishery management, conservation, and rehabilitation of this species.     

Molecular phylogeny of mangroves. VI. Intraspecific genetic variation in mangrove species Excoecaria agallocha L. (Euphorbiaceae).

Genomic DNA from 84 individuals of Excoecaria agallocha from seven mangrove populations were analysed for random amplified polymorphic DNAs (RAPDs) using 16 random 10-mer primers. Polymorphism within populations varied from 20% to 31%. At the interpopulation level, 111/149 (74%) of RAPDs were polymorphic. Restriction fragment length polymorphism (RFLP) analysis of 21 individuals (3 individuals randomly selected from the 7 populations) using 30 probe-enzyme combinations revealed a high level of interpopulation polymorphism (62.2%) indicating interpopulation genetic divergence. The polymorphic RAPDs and RFLPs were pooled, and clustering was carried out based on mean similarity for individual populations. The dendrogram showed groupings of populations from the West and East Coasts of India into separate clusters, at 60% similarity level. Further, RAPD and RFLP analysis of male and female plants showed approximately the same level of variation in both sexes, and no sex-linked markers were found. These results demonstrate that considerable intrapopulation and interpopulation genetic variations exist in E. agallocha, and that lack of genetic variation is not the reason for the morphological uniformity observed across the range of the species.     

Genetic variation within and among populations of a wild rice Oryza granulata from China detected by RAPD and ISSR markers

Genetic variation within and between five populations of Oryza granulata from two regions of China was investigated using RAPD (random amplified polymorphic DNA) and ISSR (inter-simple sequence repeat amplification) markers. Twenty RAPD primers used in this study amplified 199 reproducible bands with 61 (30.65%) polymorphic; and 12 ISSR primers amplified 113 bands with 52 (46.02%) polymorphic. Both RAPD and ISSR analyses revealed a low level of genetic diversity in wild populations of O. granulata. Furthermore, analysis of molecular variance (AMOVA) was used to apportion the variation within and between populations both within and between regions. As the RAPD markers revealed, 73.85% of the total genetic diversity resided between the two regions, whereas only 19.45% and 6.70% were present between populations within regions and within a population respectively. Similarly, it was shown by ISSR markers that a great amount of variation (49.26%) occurred between the two regions, with only 38.07% and 12.66% between populations within regions and within a population respectively. Both the results of a UPGMA cluster, based on Jaccard coefficients, and pairwise distance analysis agree with that of the AMOVA partition. This is the first report of the partitioning of genetic variability within and among populations of O. granulata at the DNA level, which is in general agreement with a recent study on the same species in China using allozyme analysis. Our results also indicated that the percentage of polymorphic bands (PPB) detected by ISSR is higher than that detected by RAPD. It seems that ISSR is superior to RAPD in terms of the polymorphism detected and the amplification reproducibility. Received: 29 March 2000 / Accepted: 15 May 2000     

有性生殖对栗疫病菌群体结构的影响

采用RAPD方法对来源于栗疫病菌8个不同子囊壳的子囊孢子后代和无性生殖的对照群体各23个菌株进行了群体结构的比较。从RAPD随机引物中筛选出扩增多态性丰富的4条引物,共扩增出条带73条,多态性检测率为100%。研究结果表明,在8个子囊壳和无性生殖群体中的基因多样性,64.27%由群体内部引起,只有35.73%的多样性由群体之间的基因差异引起。各子囊壳群体间存在的基因流动很小(Nm=0.8994)。有性群体和无性群体之间的遗传距离为0.1389,基因流动值为3.4212,说明子囊壳群体和无性生殖群体之间存在一定的系统关系。分析表明栗疫病菌子囊孢子后代在自然界的传播对自然界的病菌的多样性起重要的作用。     

濒危灌木长叶红砂遗传多样性的RAPD分析

应用RAPD分子标记对濒危灌木长叶红砂(Reaumuria trigyna)种群遗传多样性进行了分析.应用18条随机引物对长叶红砂5个种群的95个个体进行扩增,检测到118个位点,其中多态位点105个.结果表明:长叶红砂种群的多态位点比率(P)为88.98%,显示出长叶红砂种群存在较高的遗传多样性.Shannon多样性指数(0.4966)、Nei基因多样性指数(0.3303)和基因分化系数(Gst=0.1425)的分析结果显示,长叶红砂种群遗传变异大多存在于种群内,种群间的遗传分化占14.25%.聚类分析表明,长叶红砂种群遗传距离与地理距离之间无直接相关关系.遗传多样性水平与物种特性和所处不同群落有关,濒危植物并不一定表现为遗传变异水平的降低.     

Use of RAPD fingerprinting for discriminating two populations of Hilsa shad (Tenualosa ilisha Ham.) from inland rivers of Bangladesh

The Random Amplified Polymorphic DNA-Polymerase Chain Reaction (RAPD-PCR) was applied to analyze the genetic variation of the Hilsa shad, Tenualosa ilisha Ham., from the two major inland rivers (Padma and Meghna) in Bangladesh. Twenty-eight random 10-mer primers were primarily scored in 8 individuals from each of the two locations. Fifteen primers, which gave polymorphism, were selected and used in the final analysis of 34 individuals from the two sites. Using these primers, 480 scorable DNA fragments were found, of which 98 (20.41%) were polymorphic. By comparing the RAPD banding patterns, variations were found between and within the populations. A dendrogram was constructed with the polymorphic fragments to analyze the genetic distances between the Hilsa shad populations. The results show two major clusters of Padma and Meghna, assuming different spawning populations with different stocks or races of Hilsa shad in the major Bangladesh rivers.     

珍稀濒危植物天目木兰（Magnolia amoena）遗传多样性的RAPD分析

运用随机扩增多态DNA(RAPD)技术,对天目木兰(Magnolia amoena)居群的遗传多样性进行了研究．从40个10-mer随机引物中筛选出14个能得到清晰、稳定扩增带的引物进行扩增,14个引物共检测了94个位点,其中多态性位点为23,占24．4％,计算了12个居群之间的遗传相似度和遗传距离,并运用UPGMA法进行了聚类分析,结果显示相同严地个体间(居群内)的遗传距离较小,遗传多样性水平很低;不同产地个体间(居群间)遗传距离较大,遗传多样性水平较前者高,即天目木兰个体间遗传多样性水平与它的地理分布有关,天目木兰总体较低的遗传多样性是导致它濒危的原因之一。     

Randomly amplified polymorphic DNA-polymerase chain reaction analysis of two different populations of cultured Korean catfishSilurus asotus

Genetic similarity and diversity of cultured catfishSilurus asotus populations collected from two areas in western Korea were examined using randomly amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR). Out of 20 random primers tested, 5 produced 1344 RAPD bands ranging from 8.2 to 13.6 polymorphic bands per primer. The polymorphic bands in these populations ranged from 56.4% to 59.6%. Polymorphic bands per lane within populations ranged from 4.9% to 5.3%. The similarity within the Kunsan population varied from 0.39 to 0.82 with a mean (± SD) of 0.56 ± 0.08. The level of bandsharing values was 0.59 ± 007 within the catfish population from Yesan. The genetic similarity in cultured catfish populations may have been caused because individuals from two populations were reared in the same environmental conditions or by inbreeding during several generations. However, in view of bandsharing values, polymorphic bands and also the specific major bands that were inter-population-specific, significant genetic differentiation between these populations were present even if bandsharing (BS) values were somewhat numerically different. Therefore, the number of RAPD polymorphisms identified in this study may be sufficient to permit estimating genetic similarity and diversity. However, in future, additional populations, sampling sites and individuals will be necessary to make up for these weak points.     

湖北星斗山台湾杉居群的遗传多样性研究

采用随机扩增多态性DNA(RAPD)方法对孑遗植物台湾杉分布于湖北星斗山的野生和栽培居群共42个个体进行了遗传多样性分析。从60个随机引物中筛选出13个引物,共扩增出155条清晰谱带,多态位点百分率为72.9%。野生居群和栽培居群的多态位点百分率分别为65.81%和59.35%;Nei’s基因多样性分别为0.2001和0.1911;Shannon’s信息指数分别为0.3120和0.2946。野生居群的遗传多样性高于栽培居群,表明这些原生古树的后代出现了一定程度的遗传衰退。用UPGMA方法对42个单株聚类可知,来自野生群体的个体和来自栽培群体的个体明显地各聚为一类。尽管孑遗物种台湾杉在湖北仅有一个野生群体,且为零星分布,总个体数40多株,但仍保持较高的遗传多样性水平,这说明遗传多样性不是导致其种群濒危的主要原因,导致台湾杉种群濒危的原因可能与台湾杉自然种群及群落所处生境的直接破坏及其本身生物学和生态学特性所导致的自然更新不良有着密切的联系。     

Randomly amplified polymorphic DNA analysis of four different populations of the Indian major carp, Labeo rohita (Hamilton)

The level of genetic variation provides the raw material for selective improvement of a stock. Random amplified polymorphic DNA (RAPD) assay was used to assess the genetic variation in three rivers: the Halda, the Jamuna and the Padma as well as in one hatchery population of the commercially important Indian major carp, Labeo rohita. RAPD markers were amplified from DNA samples of 35 fish from each of the four populations using six decamer random primers. The polymorphic loci proportions were 0.33, 0.28, 0.28 and 0.26 and Nei's gene diversity values were 0.06, 0.07, 0.06 and 0.05 for the Halda, the Jamuna, the Padma and the hatchery populations, respectively. The pairwise population differentiation (F_ST) values indicated a low level of genetic differentiation between the population pairs. From the unweighted pair group method of arithmetic mean (UPGMA) dendrogram based on Nei's genetic distances a correlation between genetic affinities and geographical area was found. The populations were segregated into two groups: the Halda in one group and the Jamuna, the Padma and the hatchery in another group. Overall, the RAPD technique can be introduced as a tool in the population genetics of the rohu fish to provide information on their genetic stock structure.     

应用RAPD标记技术对树鼩遗传多样性的分析

目的建立树鼩RAPD（random amplified polymorphic DNA）遗传标记分析方法,了解中缅树鼩种群的遗传多样性。方法采用PCR技术对40条随机引物进行优化,筛选出能有效用于树鼩群体遗传分析的RAPD位点,对48只树鼩个体的基因组DNA进行PCR扩增,并应用Popgene 1.32与RAPDistance Package Version 1.04等软件进行数据处理,分析树鼩的群体遗传特性。结果20个RAPD引物共检测到113个位点,平均每个引物可扩增出5.65个位点,其中多态位点数为69个（占61.1%）。个体间的遗传相似系数平均为0.8307,个体间遗传距离在0.09-0.27之间,平均遗传距离为0.1693。雄性群体的遗传多态度（H0）（0.1864）略高于雌性群体（0.1470）,平均遗传多态度（Hpop）为0.1667;树鼩遗传多态度所占的比例在群体内为48.29%,而雌、雄群体间为51.71%。NJ法进行聚类分析得出T15、T33和T47号树鼩个体聚类成一大类,其余45只树鼩个体聚成另一大类,雌、雄个体呈相互交叉现象。结论实验所筛选的随机引物可有效用于中缅树鼩种群的遗传结构分析。本树鼩群体具有较好的遗传多样性。     

Essential oil composition and preliminary molecular study of four Hungarian Thymus species

Chemical and genetic differences of twenty taxa belonging to four Thymus species were studied in order to determine whether molecular characters and essential oil components could be used as taxonomic markers and to examine the correlation between them. Plant samples, representing different taxa and geographic regions, were collected from experimentally grown populations. Essential oil samples were analysed by GC/MS and cluster analysis of volatile composition resulted in segregation of thymol chemotypes from sesquiterpenic ones. Thymol was characteristic for all the populations of Thymus glabrescens and T. pannonicus as well as for certain taxa belonging to T. praecox and T. pulegioides. Sesquiterpenes occurred in only two taxa of T. glabrescens, in each sample of T. praecox and in three taxa of T. pulegioides. Plant samples were analysed by random amplified polymorphic DNA (RAPD). The obtained dendrogram revealed high gene diversity. The 13 primers resulted 114 polymorphic RAPD bands, and the average percentage of polymorphism was 80.8%. The RAPD dendogram showed separation neither at interspecific nor at interpopulational levels. Therefore, further specific molecular studies involving more taxa are suggested. Partial correlation have been found between molecular and chemical assessments.     

Genetic diversity of nine populations of the black goat (Capra hircus) in Sichuan, PR China

Sixteen polymorphic primers screened from 100 random primers were selected to analyze the randomly amplified polymorphic DNA (RAPD) of 540 domesticated black goats (Capra hircas) from 9 different geographical populations in Sichuan Province of China. After the test, 170 entirely repeatable RAPD markers representing goat polymorphisms were obtained from the 16 polymorphic primers, the lengths of the markers ranging from 0.1 to 2.5 kb. The genetic distance among the black goat populations ranges from 0.1051 to 0.2978. The similarity coefficient (0.9002) between Jintang and Lezhi black goats was the highest in the 9 populations, followed by the coefficient (0.8953) between Jialing and Yinshan goats, while that between Jiangan and Huili goats was found to be the lowest (0.7424). The coefficient of differentiation among population genes (Gst) was 0.2766, indicating a comparatively low degree of differentiation among the black goat populations. A UPGMA dendrogram constructed from similarity coefficients showed that the two populations from Huili and Baiyu, which are found mostly on the Western Sichuan plateau and in mountainous areas, clustered together, and the other seven populations formed another group. It can also be clearly seen that the Huili and Baiyu populations are very special, and must have been closely related in the past, even though their link with the other populations is quite weak as a result of genetic communication. The results of the experiment offer some crucial scientific data useful for the breeding of black goats.     

Random amplified polymorphic DNA markers for discriminating Cochliomyia hominivorax from C. macellaria (Diptera: Calliphoridae)

The screwworm, Cochliomyia hominivorax (Coquerel), is one of the most important pests of livestock in the Western Hemisphere. During early immature stages it is morphologically very similar (first instars are virtually indistinguishable) to the secondary screwworm, C. macellaria (Fabricius). Here, the utility of the random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) was explored as a technique for developing molecular genetic markers for these two species. Of the 120 arbitrary primers screened, 21 primers produced markers that were further investigated. Seven of the 21 primers produced clear and reproducible markers that were tested with DNA of five individuals from four populations of each species; five of these primers showed 12 RAPD markers that differentiated the species in all populations. Analyses of data from these seven primers also suggested that intraspecific polymorphisms exist that could be useful in distinguishing populations of screwworms. Some population genetic tools, such as genetic distance, cluster analysis and bootstrapping, were used to statistically explore these polymorphisms. The resulting statistics showed 100% support for the ability of RAPD-PCR to discriminate between the two species. Bootstrapping with data from one of the genetic distance calculations produced a tree with all individual screwworms in the correct populations, indicating that RAPD-PCR has promise for displaying intraspecific genetic variation that could be used in establishing the general geographic origin of screwworm samples.