Genetic heterogeneity of the tropical abalone (Haliotis asinina) revealed by RAPD and microsatellite analyses

Genetic heterogeneity of the tropical abalone, Haliotis asinina was examined using randomly amplified polymorphic DNA (RAPD) and microsatellite analyses. One hundred and thirteen polymorphic RAPD fragments were generated. The percentage of polymorphic bands of H. asinina across overall primers was 85.20%. The average genetic distance of natural samples within the Gulf of Thailand (HACAME and HASAME) was 0.0219. Larger distance was observed when those samples were compared with HATRAW from the Andaman Sea (0.2309 and 0.2314). Geographic heterogeneity and F(ST) analyses revealed population differentiation between H. asinina from the Gulf of Thailand and the Andaman Sea (p < 0.0001). Three microsatellite loci (CUHas1, CUHas4 and CUHas5) indicated relatively high genetic diversity in H. asinina (total number of alleles = 26, 5, 23 and observed heterozygosity = 0.84, 0.42 and 0.33, respectively). Significant population differentiation was also found between samples from different coastal regions (p < 0.0001). Therefore, the gene pool of natural H. asinina in coastal Thai waters can be genetically divided to 2 different populations; the Gulf of Thailand (A) and the Andaman Sea (B).     

SCAR markers for discriminating species of two genera of medicinal plants, Liriope and Ophiopogon

The development of DNA markers that can closely discriminate between Liriope and Ophiopogon species is vital for efficient and accurate identification of these species, and to ensure the quality, safety, and efficacy of medicines made from these plants. We developed species-specific molecular markers for these two genera. Forty RAPD primers were tested to detect polymorphism; species-specific RAPD bands were gel-purified, cloned, and sequenced. Primers for sequence-characterized amplified regions (SCARs) were then designed, based on nucleotide sequences of specific RAPD primers. SCAR markers SA06 and SB05, specific to Ophiopogon japonicus, amplified 460- and 553-bp DNA fragments, respectively. The marker SA12 amplified a 485-bp fragment specific to Liriope platyphylla. This is the first report of a species-specific SCAR marker for this group. These markers will be useful for rapid identification of closely related Liriope and Ophiopogon species.     

Development of reliable PCR-based markers linked to downy mildew resistance genes in lettuce

Summary Sequence characterized amplified regions (SCARs) were derived from eight random amplified polymorphic DNA (RAPD) markers linked to disease resistance genes in lettuce. SCARs are PCR-based markers that represent single, genetically defined loci that are identified by PCR amplification of genomic DNA with pairs of specific oligonucleotide primers; they may contain high-copy, dispersed genomic sequences within the amplified region. Amplified RAPD products were cloned and sequenced. The sequence was used to design 24-mer oligonucleotide primers for each end. All pairs of SCAR primers resulted in the amplification of single major bands the same size as the RAPD fragment cloned. Polymorphism was either retained as the presence or absence of amplification of the band or appeared as length polymorphisms that converted dominant RAPD loci into codominant SCAR markers. This study provided information on the molecular basis of RAPD markers. The amplified fragment contained no obvious repeated sequences beyond the primer sequence. Five out of eight pairs of SCAR primers amplified an alternate allele from both parents of the mapping population; therefore, the original RAPD polymorphism was likely due to mismatch at the primer sites.     

穿山甲标本和甲片的DNA提取及PCR扩增

为验证经处理后的穿山甲(Manis spp.)标本和甲片是否可以用于种间分子鉴定标记的开发及个体识别工作,本文在样品的预处理、消化、提取后纯化等方面对传统提取方法进行了改进,分别从穿山甲剥制标本、干皮标本及甲片中提取总DNA;然后用Cyt b基因扩增通用引物、12S rRNA基因全序列扩增引物、RAPD引物及微卫星引物进行了PCR扩增,并对部分扩增结果进行了序列测定.结果表明,除剥制标本的脚底皮张组织外,其他样品基本都可以提取出DNA.以此为模板的PCR扩增中,2种线粒体基因引物扩增出明显目的条带,RAPD引物扩增出种间特异条带,测序结果可用于种间特异性引物及SCAR引物的开发;微卫星引物在甲片样品中扩增稳定,可用于个体识别工作.     

马铃薯瓢虫与茄二十八星瓢虫SCAR标记的建立及应用

为了探索快速鉴定马铃薯瓢虫Henosepilachna vigintioctomaculata(Motschulsky)和茄二十八星瓢虫Henosepilachna vigintioctopunctata(Fabricius)的分子生物学方法,本研究在随机扩增多态性DNA(random amplified polymorphic DNA,RAPD)的基础上,分别设计了可以鉴别两个物种的序列特征扩增区域(sequence characterized amplified regions,SCAR)标记。从随机合成的60条引物中筛选出来2条特异性引物(分别为OPI-6和OPJ-15),引物OPI-6在马铃薯瓢虫中扩增出约750 bp的特异性条带,引物OPJ-15在茄二十八星中扩增出约750 bp的特异性条带,根据测序结果设计了两对SCAR引物对筛选结果进行验证,发现根据OPI-6的测序结果所设计的SCAR引物(OPI-6 test)仅能在马铃薯瓢虫中扩增出645 bp的条带,而根据OPJ-15的测序结果所设计的SCAR引物(OPJ-15 test)仅能在茄二十八星瓢虫中扩增出436 bp的条带。这两对SCAR引物能够准确、稳定且快速地区分马铃薯瓢虫与茄二十八星瓢虫,对这两种害虫的精准防控具有重要意义。     

烟草SCAR标记技术的建立

目的:通过烟草随机扩增多态性DNA(RAPD)标记技术建立烟草特征序列扩增区域(SCAR)标记技术,用于烟草品种鉴定。方法:对12个烟草品种的复烤叶片DNA进行RAPD分析,得到2个RAPD特异片段S1和S2,通过切胶回收,连接pUCm-T载体克隆转化,片段测序,设计特异性引物S1-1/S1-2和S2-1/S2-2,对SCAR-PCR扩增退火温度进行优化。结果:2个RAPD标记成功地转化为稳定快捷的SCAR标记,可将红花大金元和NC102等2个品种从12个烟草品种中快捷准确地鉴别出来。结论:SCAR标记可作为准确稳定的DNA水平的烟草品种鉴定方法,可对种植、复烤和配方品种的烟叶或叶片进行鉴别。     

Generation and characterization of SCARs by cloning and sequencing of RAPD products: a strategy for species-specific marker development in bamboo

BACKGROUND AND AIMS: The aim of this study was to develop species-specific molecular markers for Bambusa balcooa and B. tulda to allow for their proper identification, in order to avoid unintentional adulteration that affects the quality and quantity of paper pulp production. METHODS: Two putative, species-specific RAPD markers, Bb836 for B. balcooa and Bt609 for B. tulda were generated using a PCR-based RAPD technique. Species-specificity of these two markers was confirmed through Southern hybridization in which RAPD gels were blotted and hybridized with radiolabelled cloned RAPD markers. Southern hybridization analyses were also performed to validate homology of the co-migrating Bb836 and Bt609 marker bands amplified from 16 different populations of B. balcooa and B. tulda, respectively. Sequence-characterized amplified region (SCAR) markers were developed from Bb836 and Bt609 sequences, using 20-mer oligonucleotide primers designed from both the flanking ends of the respective RAPD primers. KEY RESULTS: As anticipated, Bb836 hybridized with an amplified band from B. balcooa and Bt609 hybridized only with an amplified product from B. tulda; the two markers did not hybridize with the amplified products of any of the other 14 bamboo species studied. The two pairs of SCAR primers amplified the target sequences only in the respective species. The species-specific SCAR fragments were named as 'Balco836' for B. balcooa and 'Tuldo609' for B. tulda. The species-specific 'Balco836' was amplified from the genomic DNA of 80 individuals of 16 populations of B. balcooa studied. Similarly, the presence of 'Tuldo609' was noted in all the 80 individuals representing 16 populations of B. tulda assessed. These SCAR fragments contained no obvious repetitive sequence beyond the primers. CONCLUSION: These two molecular markers are potentially useful for regulatory agencies to establish sovereign rights of the germplasms of B. balcooa and B. tulda. In addition, this is the first report of species-specific SCAR marker development in bamboo.     

The application of RAPD markers in stock discrimination of the four-wing flyingfish, Hirundichthys affinis in the central western Atlantic

The polymerase chain reaction–random amplified polymorphic DNA (PCR–RAPD) technique was used to examine genetic variability and population structuring in the four-wing flyingfish, Hirundichthys affinis within the central western Atlantic. Three random decamer primers and pairs of these primers were used to amplify nuclear DNA from 360 fish sampled from six populations (at five locations) across the region. A total of 58 polymorphic RAPD markers were identified, 20 of which were population-specific and six of which were subregional or stock-specific markers. Cluster analysis of similarity indices indicated the presence of three genetically distinct subregional stocks located in the eastern Caribbean, southern Netherlands Antilles and Brazil, respectively. Estimates of gene diversity (φ) and gene flow ( Nm ) are consistent with this three-stock hypothesis. Furthermore, partially restricted gene flow was apparent among spatially and temporally separate sampled populations within the eastern Caribbean subregional stock, indicating the possible presence of different spawning groups. These results are entirely consistent with those obtained from PCR–RFLP analysis of the mtDNA D-loop in the same fish, indicating the presence of barriers to dispersal and interbreeding in both sexes. We conclude that the PCR–RAPD technique is suitable for determining population stock structure in this species and that a three-stock approach to managing H. affinis within the central western Atlantic would be appropriate.     

Optimization of primer screening for evaluation of genetic relationship in rose cultivars

Optimization of primer screening for evaluation of genetic relationship in 34 cultivars of rose through random amplified polymorphic DNA (RAPD) markers was investigated. Four series of decamer primers were used for screening and optimization of RAPD analysis between which A and N series performed good amplification of fragments as compared with other series. The primers OPN-07 and OPN-15 produced maximum number of DNA fragments in Rosa hybrida cv. Anuraag. Some primer either did not produce amplification or produced very poor amplification. Further, ten selected primers were used for genetic analysis of 34 rose cultivars. The primer OPN-15 amplified 21 fragments in all cultivars tested. A total of 162 distinct DNA fragments (bands) ranging from 100 to 3400 base pairs were amplified by using 10 selected random primers. The cluster analysis indicated that these rose cultivars formed nine clusters.     

Examining genetic relationships of Chinese Pleurotus ostreatus cultivars by combined RAPD and SRAP markers

Combined randomly amplified polymorphic DNA (RAPD) and sequence-related amplified polymorphism (SRAP) were used to assess the genetic diversity of Pleurotus ostreatus strains cultivated in China. For the RAPD and SRAP analyses, 479 and 282 polymorphic bands were obtained from 20 P. ostreatus strains using 20 and 13 selected primers or primer pairs, respectively. A combined RAPD/SRAP dendrogram grouped the 20 strains into five clades with a coefficient of 0.690. The comparison of RAPD and SRAP was evaluated in the present study. The combined RAPD/SRAP markers provided reliable information regarding the relationships among the P. ostreatus strains.     

Genetic Diversity of Pleurotus pulmonarius Revealed by RAPD,ISSR, and SRAP Fingerprinting

Pleurotus pulmonarius is one of the most widely cultivated and popular edible fungi in the genus Pleurotus. Three molecular markers were used to analyze the genetic diversity of 15 Chinese P. pulmonarius cultivars. In total, 21 random amplified polymorphic DNA (RAPD), 20 inter-simple sequence repeat (ISSR), and 20 sequence-related amplified polymorphism (SRAP) primers or primer pairs were selected for generating data based on their clear banding profiles produced. With the use of these RAPD, ISSR, and SRAP primers or primer pairs, a total of 361 RAPD, 283 ISSR, and 131 SRAP fragments were detected, of which 287 (79.5 %) RAPD, 211 (74.6 %) ISSR, and 98 (74.8 %) SRAP fragments were polymorphic. Unweighted Pair-Group Method with Arithmetic Mean (UPGMA) trees of these three methods were structured similarly, grouping the 15 tested strains into four clades. Subsequently, visual DNA fingerprinting and cluster analysis were performed to evaluate the resolving power of the combined RAPD, ISSR, and SRAP markers in the differentiation among these strains. The results of this study demonstrated that each method above could efficiently differentiate P. pulmonarius cultivars and could thus be considered an efficient tool for surveying genetic diversity of P. pulmonarius.     

Search for retroviral related DNA polymorphisms using RAPD PCR in schizophrenia

Random amplification of polymorphic DNA (RAPD) is widely used to detect polymorphisms in many organisms. Individual (or strain) specific amplified bands are generated with single or pairs of primers in PCR reactions and can serve as genetic markers. We have used this method to generate a large number of reproducible bands with single primers, random and retroviral related, on 92 human DNA samples. Theoretically, RAPD PCR presents a logical approach for assessing variability among individuals. We used ten retroviral related primers (12, 20 and 22 bp) and eight random primers (10 bp) to assess individual differences in the context of testing the retroviral hypothesis for schizophrenia. Three pairs of discordant monozygotic twins, four pairs of discordant full sibs and 53 schizophrenic individuals with 25 of their unrelated matched controls were analyzed. Ten of these primers resulted in a total of approx. 850 amplified bands (65-110 bands per primer). Almost all of these bands were identical among each individual analyzed. However, the results are inconclusive with respect to the retroviral hypothesis for schizophrenia. The general lack of RAPD polymorphism in this study may argue for mechanisms other than rearrangements such as inversions, associated with the evolution of the human genome.     

Development of sequence amplified characterized region (SCAR) markers of helicoverpa armigera: a new polymerase chain reaction-based technique for predator gut analysis

A method is described for the development of DNA markers for detection of Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae) in predator gut analysis, based on sequence characterized amplified regions (SCARs) derived from a randomly amplified polymorphic DNA (RAPD) band. A 1200-bp DNA fragment of H. armigera, absent in the predator band pattern and in other closely related prey species, was identified by RAPD analysis. This fragment was cloned and its extremes sequenced to design extended strand-specific 20-mer oligonucleotide primers. Three pairs of SCAR primers, which amplified three different DNA fragments, were used to study the effect of fragment length on detection of prey in the predator gut. Using the pair of primers that amplified the longest fragment of H. armigera DNA, a single band of 1100 bp was obtained, but its detection was not possible in the predator gut. Detection of the ingested prey was possible with the other two pairs of SCAR primers, obtaining bands of 600 and 254 bp, respectively. Detection of H. armigera DNA in the gut of the predator Dicyphus tamaninii was evaluated immediately after ingestion (t = 0) and after 4 h. Detection of H. armigera DNA after 4 h was only possible using the pair of primers that amplified the shortest fragment (254 bp). The test for specificity, using these last pair of primers, showed that H. armigera was the only species detected. The detection threshold was defined at a 1:8192 dilution of a H. armigera whole egg in all samples.     

Random Amplified Polymorphic DNA Analysis of Heterodera cruciferae and H. schachtii populations

Heterodera schachtii and H. cruciferae are sympatric in California and frequently occur in the same field upon the same host. We have investigated the use of polymerase chain reaction (PCR) amplification of nematode DNA sequences to differentiate H. schachtii and H. cruciferae and to assess genetic variability within each species. Single, random oligodeoxyribonucleotide primers were used to generate PCR-amplified fragments, termed RAPD (random amplified polymorphic DNA) markers, from genomic DNA of each species. Each of 19 different random primers yielded from 2 to 12 fragments whose size ranged from 200 to 1,500 bp. Reproducible differences in fragment patterns allowed differentiation of the two species with each primer. Similarities and differences among six different geographic populations of H. schachtii were detected. The potential application of RAPD analysis to relationships among nematode populations was assessed through cluster analysis of these six different populations, with 78 scorable markers from 10 different random primers. DNA from single cysts was successfully amplified, and genetic variability was revealed within geographic populations. The use of RAPD markers to assess genetic variability is a simple, reproducible technique that does not require radioisotopes. This powerful new technique can be used as a diagnostic tool and should have broad application in nematology.     

Determination of RAPD Markers in Rice and their Conversion into Sequence Tagged Sites (STSs) and STS-Specific Primers

We produced 102 randomly amplified polymorphic DNA (RAPD) markersmapped on all 12 chromosomes of rice using DNAs of cultivarsNipponbare (japonica) and Kasalath (indica) and of F2 populationgenerated by a single cross of these parents. Sixty random primers10 nucleotides long were used both singly and in random pairsand about 1,400 primer-pairs were tested. Using both agarosegel and polyacrylamide gel electrophoresis enabled us to detectpolymorphisms appearing in the range from <100 bp to 2 kb.The loci of the RAPD markers were determined onto the frameworkof our RFLP linkage map and some of these markers were mappedto regions with few markers. Out of the 102 RAPD markers, 20STSs (sequence-tagged sites) and STS-specific primer pairs weredetermined by cloning, identifying and sequencing of the mappedpolymorphic fragments.     

Development of Species-Specific Markers of the Tropical Oyster (Crassostrea belcheri) in Thailand

Randomly amplified polymorphic DNA (RAPD) analysis was used to identify species-specific markers of 5 oyster species in Thailand: Crassostrea belcheri, Crassostrea iredalei, Saccostrea cucullata, Saccostrea forskali, and Striostrea (Parastriostrea) mytiloides. Species-specific markers were found in C. belcheri, C. iredalei, and S. cucullata but not in S. forskali and S. mytiloides. Three C. belcheri–specific RAPD fragments were cloned and sequenced. A primer set was designed from each of the recombinant clones (pPACB1, pPACB2, and pPACB3). The polymerase chain reaction products showed expected sizes of 536, 600, and 500 bp, respectively, with the sensitivity of detection approximately 30 pg of C. belcheri total DNA template. The specificity of pPACB1 was examined against 135 individuals of indigenous oyster species in Thailand and against outgroup references S. commercialis (N= 12) and Perna viridis (N= 12). Results indicated the species-specific nature of primers developed from pPACB1. This primer set can be used for broodstock selection and determination of C. belcheri larvae to assist the selective breeding program for this commercially important species. Received December 8, 1999; accepted March 16, 2000.     

兴安、长白及华北落叶松RAPD分子标记的物种特异性鉴定

以中国北方地区主要乡土落叶松树种兴安落叶松（Larix gmelini）、长白落叶松（L.olgensis）和华北落叶松（L．principis—rupprechtii）的针叶及种子胚乳为研究材料,采用RAPD分子标记技术对3种落叶松进行不同物种的种间鉴别。结果表明,通过引物筛选得到了4个可以鉴别3种落叶松的RAPD引物,其中有2个引物在落叶松针叶和种子胚乳基因组中都扩增出相同的条带。引物OPB-11在兴安和长白落叶松基因组DNA中1500bp处扩增出特异条带,而在华北落叶松中没有：引物OPX-14在兴安落叶松基因组DNA中1200bp处扩增出特异条带,而在长白落叶松中没有：还有2个引物可分别作为3种落叶松苗木和种子鉴别的辅助标记。本研究从分子水平上为落叶松的种间鉴别提供了新的鉴定方法。     

Comparative Evaluation of RAPD and AFLP Based Genetic Diversity in Brinjal (Solanum melongena)

The present investigation was carried out with an objective of evaluating genetic diversity in brinjal (Solanum melongena) using DNA markers. A total of 38 brinjal accessions including one wild-species, Solanum sisymbrifolium were characterized using random amplified polymorphic DNA (RAP D) and amplified fragment length polymorphism (AFLP) techniques. Out of 45 primers employed to generate RAPD profiles, reproducible patterns were obtained with 32 primers and 30 (93.7%) of these detected polymorphism. A total of 149 bands were obtained, out of which 108 (72.4%) were polymorphic. AFLP analysis was carried out using four primer combinations. Each of these primers was highly polymorphic. Out of 253 fragments amplified from these four primer combinations, 237 (93.6%) were polymorphic. The extent of pair-wise similarity ranged from 0.264 to 0.946 with a mean of 0.787 in RAPD, in contrast to a range of 0.103 to 0.847 with a mean of 0.434 in AFLP. The wild species clustered separately from the brinjal genotypes. In the dendrogram constructed separately using RAPD and AFLP markers, the brinjal genotypes were grouped into clusters and sub-clusters, and the varieties released by IARI remained together on both the dendrograms. All the 30 RAPD primers in combination and each of the four primer pairs in AFLP could distinguish the brinjal accessions from each other. AFLP was thus found to be more efficient than RAPD in estimation of genetic diversity and differentiation of varieties in brinjal.     

Genome- and species-specific markers and genome relationships of diploid perennial species in Triticeae based on RAPD analyses.

Eight different genomes (E, H, I, P, R, St, W, and Ns) represented by 22 diploid species of the tribe Triticeae were analyzed using the random amplified polymorphic DNA (RAPD) technique. The genome relationships were obtained based on 371 RAPD fragments produced with 30 primers. The four species of the genus Psathyrostachys (having various Ns genomes) were closely related. The genomes Ee and Eb had a similarly close relationship and were distinct from all other genomes analyzed. Genomes P, R, and St were grouped in one cluster and genomes H and I in another. Genome W had a distant relationship with all other genomes. These results agree with the conclusions from studies of chromosome pairing and isozyme and DNA sequence analyses. Twenty-nine and 11 RAPD fragments are considered to be genome- and species-specific markers, respectively. One to six genome-specific markers were identified for each genome. These RAPD markers are useful in studies of genome evolution, analysis of genome composition, and genome identification.     

A PCR Assay to Identify and Distinguish Single Juveniles of Meloidogyne hapla and M. chitwoodi

Random amplified polymorphic DNA (RAPD) bands that distinguish Meloidogyne hapla and M. chitwoodi from each other, and from other root-knot nematode species, were identified using a series of random octamer primers. The species-specific amplified DNA fragments were cloned and sequenced, and then the sequences were used to design 20-mer primer pairs that specifically amplified a DNA fragment from each species. Using the primer pairs, successful amplifications from single juveniles were readily attained. A mixture of four primers in a single PCR reaction mixture was shown to identify single juveniles of M. hapla and M. chitwoodi. To confirm specificity, the primers were used to amplify DNA from several isolates of M. hapla that originated from different crops and locations in North America and also from isolates of M. chitwoodi that differed in host range. In characterizing the M. hapla isolates, it was noted that there was a mitochondrial DNA polymorphism among isolates for cleavage by the restriction endonuclease DraI.