Polymorphisms revealed by PCR with single,short-sized,arbitrary primers are reliable markers for mouse and rat gene mapping

Ten single, arbitrarily designed oligodeoxynucleotide primers, with 50–70% (G+C) content, were used to amplify by polymerase chain reaction (PCR) sequences with DNA templates from several mouse species (Mus spretus, Mus musculus musculus, and Mus musculus domesticus), as well as DNA from the laboratory rat (Rattus norvegicus). Eight of these ten primers, used either individually or associated in pairs, generated a total of 13 polymorphic products which were used as genetic markers. All of these polymorphic sequences but one were mapped to a particular mouse chromosome, by use of DNA panels prepared either from interspecific backcross progeny of the type (C57BL/6 x Mus spretus)F₁ x C57BL/6 or DNA samples prepared from two sets of recombinant inbred (RI) strains (AKXL and BXD). Six rat-specific DNA segments were also assigned to a particular chromosome with DNA panels prepared from 18 rat/mouse somatic cell hybrids segregating rat chromosomes. From these experiments we conclude that, under precisely standardized PCR conditions, the DNA molecules amplified with these arbitrarily designed primers are useful and reliable markers for genetic mapping in both mouse and rat.     

河南地方野生小鼠与近交系小鼠的扩增片段长度多态性分析

目的 从分子水平上阐明河南省兰考地区捕获的野生小鼠(LK)与4个小鼠品系(B6,BALB/c,DDK,PWK)之间的遗传差异及遗传关系,从而进一步确定该种野生小鼠的种属及培育野生来源近交系小鼠品系.方法 利用25对引物,对野生小鼠及4个小鼠品系进行扩增片段长度多态性(AFLP)分析.结果 检测到2035条扩增条带,多态...     

Mouse microsatellites from a flow-sorted 4:6 Robertsonian chromosome

Twenty microsatellites were generated from a previously characterized gt10 library containing C57BL/6J mouse DNA from a flow-sorted 4:6 Robertsonian chromosome. These sequences were analyzed for size variation between different strains of mice with the polymerase chain reaction (PCR) and mapped by use of either strain distribution patterns (SDPs) in recombinant inbred (RI) strains, or intra- and interspecific backcrosses. Eighty-five percent of the sequences showed allelic variations between different inbred strains of mice and the wild mouse, Mus spretus, and 70% were variant between inbred strains. Eight (62%) of the 13 repeats that have been mapped lie on Chromosomes (Chr) 4 and 6. This approach is an effective way of generating informative markers on specific chromosomes.     

Fingerprinting genomes by use of PCR with primers that encode protein motifs or contain sequences that regulate gene expression

PCR primers of arbitrary nucleotide sequence have identified DNA polymorphisms useful for genetic mapping in a large variety of organisms. Although technically very powerful, the use of arbitrary primers for genome mapping has the disadvantage of characterizing DNA sequences of unknown function. Thus, there is no reason to anticipate that DNA fragments amplified by use of arbitrary primers will be enriched for either transcribed or promoter sequences that may be conserved in evolution. For these reasons, we modified the arbitrarily primed PCR method by using oligonucleotide primers derived from conserved promoter elements and protein motifs. Twenty-nine of these primers were tested individually and in pairwise combinations for their ability to amplify genomic DNA from a variety of species including various inbred strains of laboratory mice and Mus spretus. Using recombinant inbred strains of mice, we determined the chromosomal location of 27 polymorphic fragments in the mouse genome. The results demonstrated that motif sequence-tagged PCR products are reliable markers for mapping the mouse genome and that motif primers can also be used for genomic fingerprinting of many divergent species.     

Development of polymorphic microsatellite from RAPD bands of black sea bream Acanthopagrus schlegeli

Eight pairs of simple sequence repeat markers were developed from random amplified polymorphic DNA product in black sea bream Acanthopagrus schlegeli. Twenty microsatellites were selected for designing microsatellite primers, of which eight gave working primer pairs. They had between three and seven alleles. Observed and expected heterozygosities varied from 0.65 to 0.90, and from 0.58 to 0.82, respectively. Eight additional fish species assessed for cross‐species amplification revealed between two and five positive amplifications and between zero and three polymorphic loci per species.     

New murine polymorphisms detected by random amplified polymorphic DNA (RAPD) PCR and mapped by use of recombinant inbred strains

Oligonucleotide primers of random sequence that were 12 bases in length, 58% in GC content, and lacking internal palindromes were designed. By random amplified polymorphic DNA (RAPD) PCR, these primers were used to survey for DNA variations between the progenitors of the mouse AXB and BXA recombinant inbred sets (A/J and C57BL/6J). We identified 17 DNA variants detected by 10 primers. Map positions for these variants were determined by comparing their strain distribution patterns in the AXB, BXA recombinant inbred sets with strain distribution patterns of previously published loci. When necessary, BXD and NXSM recombinant inbred sets were also used. These 17 new loci mapped to 12 chromosomes. The 10 primers were also used to survey 20 inbred mouse strains including the progenitors of other recombinant inbred sets and four mouse strains recently inbred from the wild (CAST/Ei, MOLF/Ei, PERA/Ei, and SPRET/Ei).     

Additional microsatellite markers for mouse genome mapping

Mouse sequence information from the EMBL and GenBank databases, published sequences and genomic clones have been analyzed for simple repetitive elements or microsatellites. Each microsatellite has been amplified by the polymerase chain reaction (PCR) as a single locus marker. PCR primers were designed from unique sequence flanking each repeat. Size variation of PCR products less than 750 base pairs (bp) between mouse strains has been determined using ethidium bromide-stained acrylamide or agarose gels. A further 74 newly characterized microsatellites are presented in this paper, bringing to 185 the total we have analyzed. Of these, 157/185 (85%) have more than one allele, 143/178 (80%) vary in length between C57BL/6J and Mus spretus, and 82/168 (49%) vary between DBA/2J and C57BL/6J. Microsatellites provide informative single locus probes for linkage analysis in the construction of a genetic map of the mouse genome.     

Novel DNA polymorphism in the mouse tumor necrosis factor receptors type 1 and type 2

The introduction of the polymerase chain reaction (PCR) provides an entirely new means of analyzing DNA polymorphism and makes practical the analysis of length variation in simple-sequence tandem repeats of dinucleotides. In the process of cloning and sequencing the mouse genomic DNA for tumor necrosis factor (TNF) receptors type 1 and type 2, we identified two simple dinucleotide repeats within the noncoding regions of TNF receptor type 1 and three such sequences within TNF receptor type 2. PCR analysis of these sequences, using genomic DNA from 21 different inbred and wild mouse strains, as demonstrated by running the amplified products on sequencing gels, showed that the repeats are highly polymorphic. We identified seven alleles of TNF receptor type 2 and five alleles of TNF receptor type 1. Using these polymorphic markers in two sets of recombinant inbred strains of mice, the chromosomal localization of Tnfr-1 was mapped to mouse chromosome 6 and Tnfr-2 was located to the distal portion of mouse chromosome 4.     

PCR-analyzed microsatellites: Data concerning laboratory and wild-derived mouse inbred strains

We have investigated 67 primers designed by Dr. J. Todd and co-workers to amplify microsatellites sequences in the mouse. We report on additional polymorphisms concerning seven laboratory inbred strains, complementary to those already published. We include the survey of three independently derived strains of Mus spretus: SPE/Pas, SEG/Pas and SPR/Smh. SPE/Pas and SEG/Pas are very close (3% polymorphism), whereas the third one, (SPR/Smh), is very different from the other two strains (33% polymorphism). Seventy-four to 84% of the microsatellites analyzed in this study are polymorphic between C57BL/6Pas and Mus spretus strains. By comparison, 36–46% are polymorphic between laboratory inbred strains involved in established sets of recombinant inbred strains. A strain derived from Mus musculus musculus (PWK/Pas) was found to be very different from both C57BL/6Pas (70% polymorphism) and SPE/Pas (82% polymorphism). These results emphasize the interest of using Mus musculus musculus inbred strains to establish interspecific crosses, particularly when considering their breeding performances.     

Phylogenetic relationships and genetic polymorphisms in wild Indian mice

Randomly amplified polymorphic DNA (RAPD) analysis was used to examine the extent of variability in 11 Indian wild derived commensal house mice (Mus musculus) populations and compared with inbred strains of musculus and domesticus subspecies as well as commonly used laboratory inbred strains C57BL/6J and DBA/2J. Arbitrary designed 10 mer oligonucleotide primers with 60-70% (G+C) content were used to amplify DNA template. Out of 52 primers screened initially on the laboratory strains, 20 were selected for analysis on the basis of amplification product in the size range of 200-1400 bp. Among 353 total polymorphic bands, 220 bands (64%) were found to be polymorphic in Indian wild mice, 85 bands (25%) in wild derived inbred strains and 37 bands (11%) in laboratory mice strains. The amplification patterns produced by primers were statistically analysed by Jaccard's similarity coefficient the value of which ranged from 0.56 to 0.80. High level of genetic diversity was seen in the Indian wild mice populations as compared to the controls. The UPGMA phenogram grouped mice population into two major clusters except Bikaner [BIK], Bilaspur [BIL] and Ranikhet [RK] populations which were placed outside the close-knit clusters. Inspite of low values of bootstrap estimates obtained by Wagner and Dollo parsimony analysis, the results were comparable with UPGMA phenogram when constitution of the populations in the major cluster was considered. Indian mice populations appeared to be diverse from laboratory inbred mice strains.     

Rat Gene Mapping Using Pcr-Analyzed Microsatellites

One hundred and seventy-four rat loci which contain short tandem repeat sequences were extracted from the GenBank or EMBL data bases and used to define primers for amplification by the polymerase chain reaction (PCR) of the microsatellite regions, creating PCR-formatted sequence-tagged microsatellite sites (STMSs). One hundred and thirty-four STMSs for 118 loci, including 6 randomly cloned STMSs, were characterized: (i) PCR-analyzed loci were assigned to specific chromosomes using a panel of rat x mouse somatic cell hybrid clones. (ii) Length variation of the STMSs among 8 inbred rat strains could be visualized at 85 of 107 loci examined (79.4%). (iii) A genetic map, integrating biochemical, coat color, mutant and restriction fragment length polymorphism loci, was constructed based on the segregation of 125 polymorphic markers in seven rat backcrosses and in two F2 crosses. Twenty four linkage groups were identified, all of which were assigned to a defined chromosome. As a reflection of the bias for coding sequences in the public data bases, the STMSs described herein are often associated with genes. Hence, the genetic map we report coincides with a gene map. The corresponding map locations of the homologous mouse and human genes are also listed for comparative mapping purposes.     

Amplification fragment length polymorphism in Brucella strains by use of polymerase chain reaction with arbitrary primers.

DNA heterogeneity among members of the genus Brucella was demonstrated with the arbitrarily primed polymerase chain reaction (AP-PCR). Simple, reproducible genomic fingerprints from DNA of 25 different Brucella strains were generated with five arbitrarily chosen primers, alone and in pairs, with the PCR. Reaction conditions were optimized for each primer. Several DNA segments were amplified in each sample with all of the primers. PCR products that are not shared among all strains act as polymorphic markers. Polymorphism was apparent for each primer. The Brucella strains can be distinguished according to the banding patterns of their amplified DNA on agarose gels, and the differences can be diagnostic of specific strains. To determine genetic relatedness among the Brucella strains, similarity coefficients were calculated. Statistical analysis of the similarity coefficients revealed the degrees of relatedness among strains of the genus Brucella.     

Identification of polymorphic microsatellite markers from RAPD product in turbot (Scophthalmus maximus) and a test of cross‐species amplification

Five polymorphic microsatellite loci have been isolated and characterized from random amplified polymorphic DNA product in turbot, Scophthalmus maximus. Twelve microsatellites were selected for designing microsatellite primers, of which five gave working primer pairs. They had between four and nine alleles. Observed and expected heterozygosities varied from 0.76 to 0.90 and from 0.63 to 0.83, respectively. Five additional fish species assessed for cross‐species amplification revealed between one and three positive amplifications and between zero and two polymorphic loci per species.     

随机扩增多态性DNA技术在鲍氏层孔菌菌株鉴别中的应用

用20个随机引物对7个不同来源的鲍氏层孔菌菌株进行了RAPD分析．结果表明120个随机引物中,有17个引物的扩增产物DNA条带表现出明显的多态性,不同引物对供试菌株扩增出现的DNA条带数目少则10条,多达33条．DNA片段从250bp到2000bp;采用17个引物对7个鲍氏层孔菌菌株共扩增出DNA片段带377条,不同引物扩增出的DNA片段谱带存在较大差异．采用UPGMA系统聚类法,将7个菌株聚类为两大类,能直观准确地揭示菌株间的差异并加以鉴别．     

The development and mapping of functional markers in Fragaria and their transferability and potential for mapping in other genera

We have developed 46 primer pairs from exon sequences flanking polymorphic introns of 23 Fragaria gene sequences and one Malus sequence deposited in the EMBL database. Sequencing of a set of the PCR products amplified with the novel primer pairs in diploid Fragaria showed the products to be homologous to the sequences from which the primers were originally designed. By scoring the segregation of the 24 genes in two diploid Fragaria progenies FV × FN (F. vesca × F. nubicola F₂) and 815 × 903BC (F. vesca × F. viridis BC₁) 29 genetic loci at discrete positions on the seven linkage groups previously characterised could be mapped, bringing to 35 the total number of known function genes mapped in Fragaria. Twenty primer pairs, representing 14 genes, amplified a product of the expected size in both Malus and Prunus. To demonstrate the applicability of these gene-specific loci to comparative mapping in Rosaceae, five markers that displayed clear polymorphism between the parents of a Malus and a Prunus mapping population were selected. The markers were then scored and mapped in at least one of the two additional progenies.     

The development of eight microsatellite loci in the wild daffodil Narcissus triandrus (Amaryllidaceae)

We report microsatellite primer pairs for the wild tristylous daffodil, Narcissus triandrus (Amaryllidaceae). From enriched libraries, we identified 58 unique microsatellite loci. We designed primer pairs for 27 of these loci and screened genomic DNA from 38 to 40 adults from a single population. For eight polymorphic loci, the number of alleles per locus ranged from five to 17. As six primers also amplified loci in three other Narcissus species, including two horticultural varieties, we expect that some of these markers will be transferable to other Narcissus species.     

Development of a real-time PCR assay for the detection of Cladosporium fulvum in tomato leaves

Aims: The aim of this study was to develop a sensitive real-time polymerase chain reaction (PCR) assay for the rapid detection of Cladosporium fulvum in tomato leaves. Methods and Results: Three PCR primer pairs were designed based on the nucleotide sequences of: (i) the internal transcribed spacer regions of ribosomal RNA; (ii) a microsatellite region amplified by the microsatellite primer M13; and (iii) the β-tubulin gene of C. fulvum. Each primer pair amplified the expected target DNA fragment from geographically diverse isolates of C. fulvum. No PCR products were amplified with these primer pairs from DNA of other fungal species. Among the three pairs of primers, the primer pair CfF1/CfR1 developed based on the microsatellite region was the most sensitive. Using this sensitive primer pair, a real-time PCR assay was developed to detect early infection of C. fulvum in tomato leaves. Significance and Impact of the Study: DNA regions amplified by the microsatellite primer M13 have a high potential for developing highly sensitive species-specific PCR primers for the detection of phytopathogenic fungi. The real-time PCR assay developed in this study is useful in monitoring early infection of C. fulvum, and can help growers make timely decisions on fungicide application.     

Detection of Intra-Clonal Genetic Variability in Vegetatively Propagated Tea Using RAPD Markers

The role of random amplified polymorphic DNA (RAPD) markers in detecting intra-clonal genetic variability in vegetatively propagated UPASI-9 clone of tea (Camellia sinensis) was studied. Twenty five decamer primers were used, of which three did not amplify, three gave single bands and the rest of nineteen primers generated upto twelve bands (an average of 6.3 bands per primer). Twenty one primers exhibiting amplified products gave monomorphic banding patterns. Only one primer (OPE-17) gave a unique extra band of similar size in four plants.     

Characterization of polymorphic microsatellite markers in the water rat (Hydromys chrysogaster)

The water rat (Hydromys chrysogaster) is well adapted to a semiaquatic life and is endemic to dispersed regions of Australia and New Guinea. To analyse the genetic diversity of water rat populations, polymorphic microsatellite markers were developed. A partial genomic library was screened for microsatellite sequences. Following isolation of the microsatellite sequences, primers were designed to amplify seven loci and of these loci, five were polymorphic. The sample tested for polymorphisms came from areas across Australia and New Guinea. Between three and 13 alleles were detected for each locus. In addition the primers amplified two loci in Mus musculus and Rattus rattus.     

Isolation and characterization of microsatellite DNA markers for spinner dolphin (<Emphasis Type="Italic">Stenella longirostris</Emphasis>)

Practically no studies on the population genetics of the spinner dolphin (Stenella longirostris) exist. Seventeen pairs of DNA primers, cloned from an Mbo I digestion of S. longirostris liver DNA, were selected from a total of 288 sequences. Eight polymorphic microsatellite DNA markers were selected from the 17 primer pairs following amplification of DNA from skin samples of 65 spinner dolphins. Characterization of the polymorphisms revealed between three and nine alleles per loci. The observed heterozygosity ranged from 0 to 0.6032, while the expected heterozygosity ranged from 0.5834 to 0.73. Seven of the eight designed primer pairs amplified DNA from three other delphinid species. There was a marked low observed heterozygosity in the spinner dolphin suggesting a high level of inbreeding within this species in the southern Atlantic.