Identification of DNA polymorphism in cultivated groundnut using random amplified polymorphic DNA (RAPD) assay.

Construction of a genetic linkage map is necessary to apply marker-assisted selection tools in a crop improvement program. Except for the recent studies from two laboratories, most of the previous studies have shown little or no DNA polymorphism in cultivated groundnut (Arachis hypogaea L.). In the present study, 70 selected genotypes, representing variability for several morphological, physiological, and other characters, were studied for polymorphism employing random amplified polymorphic DNA (RAPD) assay with 48 oligonucleotide primers. Of the 48 oligonucleotide primers only 7 (14.6%) yielded polymorphic amplification products. The total number of bands from the 7 primers was 408, of which 27 were polymorphic. Detection of polymorphism in cultivated groundnut opens up the possibility of development of its molecular map by judicious selection of genotypes that show DNA polymorphism. This approach will be useful for developing marker-assisted selection tools for genetic enhancement of groundnut for desirable traits.     

体细胞克隆山羊微卫星DNA分析

用10对山羊微卫星DNA多态性引物对2只体细胞克隆济宁青山羊、青山羊供体细胞、受体奶山羊母羊以及具有亲缘关系的3只对照济宁青山羊进行微卫星DNA分析.结果表明有5对山羊微卫星DNA多态性引物,即SR-CRSP1, SR-CRSP5, SR-CRSP6, SR-CRSP7和SR-CRSP24,扩增产物有明显的多态性.扩增产物经过6%变性聚丙烯酰胺凝胶电泳后银染,结果2只体细胞克隆山羊的微卫星DNA指纹与供体细胞完全相同,而且不同于其受体母亲也不同于其他所有同品种不同个体的对照青山羊.证明体细胞克隆山羊基因组来源于供体细胞.     

莲藕品种DNA指纹图谱的绘制

采用RAPD技术对14个莲藕品种进行遗传多态性分析,用5个Operon引物和80个SBS的RAPD引物进行筛选,从中选出来自SBS的RAPD-C13和RAPD-D15扩增出的8条多态性条带,绘制了14个品种的DNA指纹图谱,在该图谱中每个品种均有各自特异的DNA指纹。     

Male-specific DNA sequences in pigs

One hundred primers (Operon kits OPAA, OPAO, OPAV, OPC, and OPE series) were used for random amplified polymorphic DNA (RAPD) fingerprinting to determine male-specific fragments. Seventy-four percent of the primers yielded Yorkshire polymorphic fragments. One of these primers, OPAV-18, produced a novel 1098-bp DNA fragment found only in tested males. This male-specific fragment was isolated and constructed into plasmids for nucleotide sequencing. Two primers (5'-TTGCTCACGG TAGATAACAA GAGAG-3' and 5'-TTGCTCACGG ACCAGGTAGG GAATG-3') were designed according to the cloned male-specific sequence to amplify the male-specific band using polymerase chain reaction (PCR) for pig sexing. Sex-specific bands in the PCR gel products were represented in males but none were found in females when Yorkshire, Duroc, and Landrace genomic DNA samples were amplified with these two primers by PCR. The PCR products in the gel were transferred to nylon membranes and hybridized with a 32P-dCTP labeled probe of the cloned male-specific DNA fragment. There was a clear hybridization signal in samples from all of the male pigs, but not from those of female pigs. Male and female genomic DNA samples from these pigs were spotted onto nylon membranes and hybridized with the male-specific probe. The probe hybridized strongly to males only. A high degree of sequence homology was found among the novel male-specific DNA sequences in Yorkshire, Duroc and Landrace. The sex of these three breeds of pigs could be easily and effectively determined using these two primers.     

应用DNA指纹图谱技术鉴定整肠生菌株BL63516的研究

目的建立应用DNA指纹图谱技术鉴定微生态制剂——整肠生菌株BL63516的方法,提高菌种鉴别水平。方法应用RAPD（随机扩增多态性）方法,采用50条随机引物对7株地衣芽胞杆菌进行基因组DNA指纹图谱分析,选择多态性好、重复性好、稳定性强的随机引物,对BL63516与其他地衣芽胞杆菌进行区分。结果发现选用引物$87或$88分别对7株地衣芽胞杆菌进行基因组DNA指纹图谱分析,BL63516菌株扩增的DNA片段的大小、数量均与其他地衣芽胞杆菌有明显差异。结论此方法具有可重复性,方便、快速和准确的优势,可用于微生态制剂整肠生菌株的鉴别。     

三尖杉科植物RAPD分析及其系统学意义

利用随机扩增多态性ＤＮＡ（ＲＡＰＤ）技术分析了三尖杉科植物三尖杉Ｃｅｐｈａｌｏｔａｘｕｓ ｆｏｒｔｕｎｅｉ Ｈｏｏｋ．ｆ．、粗榧Ｃｅｐｈａｌｏｔａｘｕｓ ｓｉｎｅｎｓｉｓＬｉ、海南粗榧Ｃｅｐｈａｌｏｔａｘｕｓ ｈａｉｎａｎｅｎｓｉｓＬｉ和篦子三尖极Ｃｅｐｈａｌｏｔａｘｕｓ ｏｌｉｖｅｒｉＭａｓｔ。,经筛选Ｏｐｅｒｏｎ公司的４组８０个引物,其中１１４个引物的谱带清晰呈多态性。采用ＵＰＧＭＡ法对各样本     

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.     

家蚕胚胎细胞系的DNA指纹图谱分析

在建立可靠的家蚕细胞系基因组DNA制备和PCR扩增技术体系的基础上,筛选具有稳定多态性位点的RAPD和ISSR引物,建立家蚕细胞系基因组DNA的ISSR和RAPD分子标记技术体系,检测家蚕细胞系的DNA分子标记多态性,构建细胞系的DNA指纹图谱。筛选出了26个ISSR引物和43个RAPD引物,通过PCR扩增在家蚕胚胎细胞系和传代昆虫细胞系等9个样品中分别获得了797条和1205条多态性条带,多态性达到89．9%和76．6%,不同细胞系的DNA多态性有较大差异,三个家蚕胚胎细胞系具有各自特有的DNA标记。测定了9个样品间的Nei's相似系数和遗传距离,构建了系统发育树,结果表明本实验室建立的3个家蚕胚胎细胞系和家蚕“夏芳×秋白”聚为一簇,亲缘关系较近,而来自不同物种的五个传代昆虫细胞系聚为一簇,它们之间的遗传距离比3个家蚕胚胎细胞系之间的遗传距离更小。     

Sensitive and specific detection of Xanthomonas campestris pv. pelargonii with DNA primers and probes identified by random amplified polymorphic DNA analysis.

The random amplified polymorphic DNA method was used to distinguish strains of Xanthomonas campestris pv. pelargonii from 21 other Xanthomonas species and/or pathovars. Among the 42 arbitrarily chosen primers evaluated, 3 were found to reveal diagnostic polymorphisms when purified DNAs from compared strains were amplified by the PCR. The three primers revealed DNA amplification patterns which were conserved among all 53 strains tested of X. campestris pv. pelargonii isolated from various locations worldwide. The distinctive X. compestris pv. pelargonii patterns were clearly different from those obtained with any of 46 other Xanthomonas strains tested. An amplified 1.2-kb DNA fragment, apparently unique to X. campestris pv. pelargonii by these random amplified polymorphic DNA tests, was cloned and evaluated as a diagnostic DNA probe. It hybridized with total DNA from all 53 X. campestris pv. pelargonii strains tested and not with any of the 46 other Xanthomonas strains tested. The DNA sequence of the terminal ends of this 1.2-kb fragment was obtained and used to design a pair of 18-mer oligonucleotide primers specific for X. campestris pv. pelargonii. The custom-synthesized primers amplified the same 1.2-kb DNA fragment from all 53 X. campestris pv. pelargonii strains tested and failed to amplify DNA from any of the 46 other Xanthomonas strains tested. DNA isolated from saprophytes associated with the geranium plant also did not produce amplified DNA with these primers. The sensitivity of the PCR assay using the custom-synthesized primers was between 10 and 50 cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Species-Specific Random Amplified Monomorphic DNA

Intra- and interspecific variability of total DNA isolated from haploid megagametophytes of coniferous species was examined using polymerase chain reaction with random primers. Based on this technique, one can with certainty detect heterozygosity at gene loci carrying null alleles and thus reveal cryptic intraspecific genetic variation. Large population samples were used. Along with random amplified polymorphic DNA, i.e., widely known fragments (amplicons) polymorphic within a species, we found invariant loci lacking individual or geographic variability but differentiating species within genera and other taxa. This DNA was termed RAMD (random amplified monomorphic DNA) to distinguish it from polymorphic DNA. Our findings suggest that genetic monomorphism of species and the dual structure of the eukaryotic genome can be detected at the DNA level as was previously shown for protein gene markers.     

Polymorphic microsatellite DNA amplification customized for chimpanzee paternity testing

DNA segments containing GT/AC dinucleotide repeats in the chimpanzee (Pan troglodytes) genome were screened. Thirteen transformedE. coli colonies were identified with the (GT)₁₀ probe to have chimpanzee DNA fragments containing (GT)_n repeats. These potentially polymorphic (variable n) DNA segments were sequenced. Primers for the polymerase chain reaction (PCR) amplifying these DNA segments were designed. Six pairs of primers yielded polymorphic PCR products. Three of them revealed considerable length polymorphisms and heterozygosities in a group of captive chimpanzees. For studies on chimpanzees in the wild and in captivity, these primers should be useful for paternity testing, for investigating genetic variations, and for improving the genetic maintenance of breeding colonies. The strategy adopted in the present study to obtain PCR primers amplifying polymorphic microsatellite DNA segments may well be applicable to almost all eukaryotic organisms.     

Genetic segregation of random amplified polymorphic DNA in diploid cultivated alfalfa.

Polymerase chain reaction was used, with single 10-mer primers of arbitrary sequence, to amplify random regions of genomic DNA from a diploid cultivated alfalfa backcross population. Segregation of the random amplified polymorphic DNA (RAPD) fragments was analysed to determine if RAPD markers are suitable for use as genetic markers. Of the 19 primers tested, 13 amplified a total of 37 polymorphic fragments, of which 28 (76%) segregated as dominant Mendelian traits. RAPD markers appear useful for the rapid development of genetic information in species like alfalfa where little information currently exists or is difficult to obtain.     

水稻广亲和基因（WCG）近等基因系的随机引物PCR分析

应用随机引物ＰＣＲ（ＲａｎｄｏｍＰｒｉｍｅｒＰＣＲ）技术分别在水稻广亲和基因（ＷＣＧ）的近等基因系和色素原基因（Ｃ）的分离群体库中寻找与ＷＣＧ和Ｃ基因连锁的分子标记。对于ＷＣＧ近等基因系,在２２６个随机引物中初筛到２２个显示多态性片段的引物。根据理论值计算,在２２个多态性片段中预期有２０个与ＷＣＧ连锁。在这些连锁标记中距ＷＣＧ最近的可达０．５ｃＭ。同样在分离群体库的筛选中有１０个扩增产物与Ｃ基因连锁。     

Rapid DNA extraction methods and new primers for randomly amplified polymorphic DNA analysis of Giardia duodenalis.

A randomly amplified polymorphic DNA (RAPD) procedure using simple genomic DNA preparation methods and newly designed primers was optimized for analyzing Giardia duodenalis strains. Genomic DNA was extracted from in vitro cultivated trophozoites by five freezing-thawing cycles or by sonic treatment. Compared to a conventional method involving proteinase K digestion and phenol extraction, both freezing-thawing and sonication were equally efficient, yet with the advantage of being much less time- and labor-intensive. Five of the 10 tested RAPD primers produced reproducible polymorphisms among five human origin G. duodenalis strains, and grouping of these strains based on RAPD profiles was in agreement among these primers. The consistent classification of two standard laboratory reference strains, Portland-1 and WB, in the same group confirmed previous results using other fingerprinting methods, indicating that the reported simple DNA extraction methods and the selected primers are useful in RAPD for molecular characterization of G. duodenalis strains.     

Randomly amplified DNA polymorphisms in dogs are reproducible and display Mendelian transmission

Many inherited diseases occur in pure-bred dogs, but diagnosis at the level of DNA is impossible because the canine genome is largely unknown. Random amplification of polymorphic DNA (RAPD) provides many polymorphisms, but the reproducibility and Mendelian inheritance are not beyond doubt. An optimized polymerase chain reaction (PCR) was developed for canine DNA with respect to the annealing temperature and the concentrations of MgCl₂, template DNA and primers. RAPD amplification products were in the range of 100–1500 base pairs. With six primers, 21 different reactions with different electrophoretic patterns were obtained, yielding 9–29 products per reaction. In DNA from dogs of 16 different breeds, 14% of the products were polymorphic; when only beagles were included the rate of polymorphism was 10%. All of the reaction products were completely reproducible in 16 DNA samples. Mendelian transmission was analysed in six beagle families (42 dogs). The segregation of polymorphic amplification products in 21 reactions performed on DNA from all beagles was nearly complete; in only two of the 630 reactions was there a product that could not be traced back to either of the parents. The reproducibility and Mendelian behaviour of polymorphisms generated by RAPD in dogs makes this tool very suitable for development of DNA markers of canine inherited diseases.     

Detection of section-specific random amplified polymorphic DNA (RAPD) markers in Lilium

Random amplified polymorphic DNA (RAPD) markers were utilized for the identification of Lilium species and inter-specific hybrids. The optimum annealing temperature of the polymerase chain reaction (PCR) for the RAPD assay in Lilium was 54 °C, which is relatively higher than the temperature used for other genera reported by previous researchers. Among 76 primers used to amplify genomic DNA by PCR, 18 primers (24%) generated polymorphic DNA fragments in Lilium species and hybrids. Cultivars were also identified by RAPD markers. Some amplified fragments were unique to species of each section and to hybrids derived from these species; that is, they were the section-specific DNA markers. Sections, Sinomartagon, Leucolirion b, Leucolirion a and Archelirion could be identified by 6 section-specific markers amplified with five primers. Seven inter-section hybrids showed the section-specific bands of both parental sections, indicating that these markers would be useful for identifying the parental sections of inter-section hybrids.     

Strain typing of Lentinula edodes by random amplified polymorphic DNA assay

Abstract Single 10-base primers were used to generate randomly amplified polymorphic DNA (RAPD) markers in the shiitake mushroom, Lentinula edodes . Seven primers produced polymorphisms in all 15 strains tested, producing 12–19 bands ranging from 0.34 to 2.52 kb. Thirteen of the 15 strains had unique DNA fingerprints, whereas L. edodes ATCC 28759 and ATCC 28760 exhibited identical RAPD profiles for all the primers. Molecular-genetic markers obtained with the RAPD assay can be used to differentiate strains of L. edodes and have potential applications in mushroom breeding and strain improvement programs.     

Random amplified polymorphic DNA analysis in Hordeum species.

Random amplified polymorphic DNA analysis was performed by applying a set of 13 arbitrary 10-mer primers to 19 Hordeum species and subspecies. High levels of variation in fragment pattern were observed both within and among species with most of the primers used. Genetic similarities between accessions and species were calculated from the fragment patterns. The resulting phenograms confirmed previous relationships among the Hordeum species.     

Polymorphic microsatellite DNA markers in the penduline tit, Remiz pendulinus

To describe the exceptional mating system of the penduline tit, Remiz pendulinus, we aim to combine field observation records with DNA analysis based on polymorphic microsatellite DNA markers. Here we describe features of nine loci and their corresponding polymerase chain reaction primers. The observed number of alleles varied from four to seven and the observed heterozygosity ranged from 0.419 to 0.802. Neither of the loci is sex-linked and as linkage disequilibrium analysis showed they assort independently. Seven of the nine loci were polymorphic in the Cape penduline tit, Anthoscopus minutus.     

Amplification of DNA markers from evolutionarily diverse genomes using single primers of simple-sequence repeats

The abundance and scattered distribution of simple-sequence repeats (SSR) in eukaryotic genomes prompted us to explore the use of SSR-based oligonucleotide primers in single primer amplification reactions. In a pilot experiment, 23 primers were used across a panel of evolutionarily diverse eukaryotic genomes, including grapes, lettuce, tomato, pine, maize, salmon, chicken, Holstein cows and humans. The primers were 16–20 bases in length and represented SSRs of di-, tri-, tetra-, and pentanucleotide repeats. The results showed that tetranucleotide repeat primers were most effective in amplifying polymorphic patterns. Of 11 such primers tested, 70% produced polymorphic patterns from the DNA of one or more species. Primers representing a combination of two tetranucleotide repeats, or compound microsatellites, were equally effective. The polymorphisms contained in such fingerprints were able to identify individuals of vertebrate species as well as lines or varieties of plants. Inheritance of the polymorphic bands was studied in a maize recombinant inbred population, DE811 x B73. Thirty-two polymorphic bands, derived from two amplification patterns, were mapped as dominant markers on an existing RFLP map of the same population. The bands were distributed across nine of the ten chromosomes.