Identification of RAPD markers linked to the Tm-2 locus in tomato

Tm-2 and Tm-2a are genes conferring resistance to tomato mosaic virus in Lycopersicon esculentum. They are allelic and originated from different lines of L. peruvianum, a wild relative of tomato. In this study, random amplified polymorphic DNA (RAPD) markers linked to these genes were screened in nearly isogenic lines (NILs). To detect RAPDs differentiating NILs, 220 different 10-base oligonucleotide primers were examined by the polymerase chain reaction (PCR), and 43 of them generated 53 consistent polymorphic fragments among the NILs. Out of these 53 fragments, 13 were arbitrarily chosen and examined in respect of whether they were linked to the netted virescent (nv) gene, since nv is tightly linked to the Tm-2 locus and its phenotype is more easily distinguishable. As a result, all 13 markers were shown to be linked to nv, and hence to the Tm-2 locus. Among them, two fragments specific to the NIL carrying Tm-2 three specific to the NIL carrying Tm-2a, and four specific to both of these NILs were closely linked to nv.     

Gynogenic lines of onion (Allium cepa L.): evidence of their homozygosity

Haploid induction via gynogenesis offers the possibility of using doubled haploid (DH) inbred lines in onion breeding. A first DH line that originated from the open-pollinated (OP) cultivar Dorata di Parma was obtained after overcoming difficulties associated with the haploidy of the regenerants. Spontaneous chromosome doubling occurs seldom in onion. The first DH line obtained was cloned and selfed to produce sufficient seeds for genetic studies. The homozygosity of the DH gynogenic line was revealed on the basis of the low standard deviations of the bulb traits polar diameter, shape index and weight with respect to those of the S₁ line or the OP cultivar. In the DH line, moreover, segregation of RAPD and alpha esterase markers was not noted. Out of four primers revealing polymorphism at 16 ge-netic loci in the OP cultivar Dorata di Parma, none produced polymorphism in the DH gynogenic line. The Est-1 locus, homozygous in 22 plants (Est-1 ^1/1 in 3 and Est-1 ^2/2 in 19) and heterozygous (Est-1 ^1/2) in 11 plants of the OP cultivar, always carried the same alleles in the DH line. We also tested genetic stability during micropropagation of a second halpoid line obtained via gynogenesis from var. Senshyu Yellow. Seventeen plants of this line were tested to detect changes occurring during the tissue culture process. Again no polymorphism was observed. The high genetic homogeneity observed in the two gynogenic lines of onion could be related to the absence of the callus phase during the gynogenic process.     

Identification of a RAPD marker linked to the pendula gene in Norway spruce (Picea abies (L.) Karst. f. pendula)

The pendula phenotype of Norway spruce [Picea abies (L.) Karst f. pendula] is characterized by narrow crowns and strong apical dominance and is controlled by a single dominant gene (P). This defined genetic control presents one of the few opportunities to map a single gene controlling a morphological trait in a forest tree. We used random amplified polymorphic DNA (RAPD) markers and bulked segregant analysis to identify one locus OPH10_720, linked to the pendula gene. The estimated recombination frequency (r) between OPH10_720 and P was 0.046 (SE _r =0.032). Mapping of the pendula gene is an important first step towards the ultimate identification and cloning of this gene.     

DNA Isolation and AFLP™ Genetic Fingerprinting of shape Theobroma cacao (L.)

Accurate identification of parental plants and their hybrids is essential for an effective breeding programme. Traditional classification of cocoa varieties relies on the characterisation of agricultural traits at plant maturity. A rapid and reliable method is described, based on genotypic analysis. An efficient DNA isolation procedure was developed, yielding unsheared DNA of high purity. Two genetic fingerprinting techniques, RAPD and AFLP, were evaluated for their suitability in distinguishing cocoa varieties. RAPD analysis was unsatisfactory due to the low frequency of polymorphisms and poor reproducibility. AFLP was reliable in distinguishing phenotypically identical, known varieties of cocoa. Importantly, AFLP also revealed intra- and inter-varietal variation.Abbreviations: AFLP, amplified fragment length polymorphism; APS, ammonium persulphate; CTAB, hexadecyltrimethylammonium bromide; DEB, DNA extraction buffer; f.wt., fresh weight; NEB, nuclei extraction buffer; PMSF, phenylmethanesulphonyl fluoride; RAPD, random amplified polymorphic DNA; T4 PNK, Bacteriophage T4 polynucleotide kinase; Taq, Thermus aquaticus; TBE, tris-borate-EDTA; TEMED, NNNN tetramethylethylenediamine.     

A Simple and Rapid Procedure for Isolation of Total DNA Suitable for Fingerprint Analysis from shape Amaranthus

A simple, efficient and reliable method is described for isolation of total DNA from young leaves of Amaranthus species. This procedure yields a high amount (600–800 µg DNA/g fresh leaf tissue) of good quality DNA free from contaminating proteins, polysaccharides, and coloured pigments. The DNA is suitable for digestion with several restriction endonucleases, preparation of Southern blots, and PCR amplification. The DNA has been successfully used for generating DNA fingerprint profiles and RAPD banding patterns in two species of Amaranthus. The procedure is suitable for processing of a large number of samples simultaneously.     

Detection of a specifically amplified DNAfragment in Brucella abortus by arbitrarilyprimed polymerase chain reaction

Randomly amplified polymorphic DNA (RAPD) profiles of Brucella and non-Brucella DNA were established after polymerase chain reaction (PCR) amplification. Five arbitrary oligonucleotide primers were screened to generate Brucella-specific DNA fingerprints. The arbitrary primer OPB-01 (5-GTTTCGCTCC-3) produced DNA bands specific to Brucella. Amplification conditions must be optimized for reproductibility. Accordingly, we optimized and established the conditions, which included Mg²⁺, enzyme (DNA polymerase), primer, template and deoxyribonucleoside triphosphate (dNTP) concentrations as well as the optimum number of thermal cycles to produce OPB-01 directed Brucella DNA fingerprints.The optimized RAPD method can produce a 1.3 kb DNA fragment specific to Brucella. This DNA fragment was common to eight biovars of B. abortus and one biovar of B. melitensis. The fragment was not detected in genetically related species such as Ochrobactrum anthropi and other non-Brucella organisms associated with farm animals. We anticipate the use of this fragment as a possible probe for the detection of Brucella organisms.     

Host-based genetic differentiation in the aphid Aphis gossypii Glover, evidenced from RAPD fingerprints

Samples of the aphid Aphis gossypii (Glover) were collected from different host plants at 18 locations in southern France, La Réunion, Portugal and Laos. RAPD (random amplified polymorphic DNA) patterns of the 480 aphids were obtained using three random primers. A large number of RAPD bands were shared by all aphids of the 18 populations, but some RAPD bands appeared to be population specific. Over all aphids, a total of 37 polymorphic bands were identified and defined 142 RAPD phenotypes. A cluster analysis based on genetic distance revealed that the 18 aphid populations were divided into two groups, depending on whether they were collected on a cucurbit host plant. An analysis of molecular variance ( AMOVA ) was also performed and confirmed the differentiation into two groups. Several RAPD bands that were obtained using random primer A11 could be considered as diagnostic loci as they were fixed in populations collected on cucurbits and were always absent in those collected on noncucurbit host plants. These results represent the first evidence for genetic structuring within the species A. gossypii , according to host-plant type.     

RAPD markers of mitochondrial origin exhibit lower population diversity and higher differentiation than RAPDs of nuclear origin in Douglas fir

We developed a method of screening RAPD markers for the presence of organelle DNA products using enriched organelle DNA probes, then used these markers to compare the structure of nuclear and mitochondrial RAPD diversity in Douglas fir. Of 237 screened RAPD fragments from 25 primers, 16% were identified as originating in the mitochondrial genome and 3% in the chloroplast genome. The mitochondrial DNA probe correctly distinguished fragments with known maternal inheritance (which is exclusive for the mitochondrial genome in the Pinaceae), and neither of the organelle probes hybridized to biparentally inherited fragments. Mitochondrial RAPD markers exhibited low diversity within populations compared to nuclear RAPD diversity ( H _S = 0.03 and 0.22, respectively), but were much more highly differentiated than were fragments of nuclear origin at both the population ( G _ST = 0.18 and 0.05, respectively) and racial levels ( G _ST = 0.72 and 0.25, respectively). Both nuclear and mitochondrial DNA based phylogenetic analyses identified the varieties as monophyletic groups; the nuclear RAPD markers further separated the north and south interior races.     

Characterization of two morphological groups of isolates ofPythium ultimum var.ultimum in a vegetable field

Comparisons were made between two morphological groups ofPythium ultimum var.ultimum strains isolated in a vegetable field in Japan. The groups were distinguished as having smaller or larger sexual organs by the sizes of their antheridia and oogonia. Morphological study indicated that the two groups comprised a single taxon,P. ultimum var.ultimum, by the current taxonomical keys. The smaller group grew faster in the lower temperature range of 4–15°C, whereas the larger group grew faster in the higher temperature range of 25–37°C. Random amplified polymorphic DNA (RAPD) and isozyme analyses revealed genetic dissimilarity between the two groups. Cluster analysis of the isozyme banding patterns with four otherPythium spp. demonstrated that the genetic dissimilarity between the two groups was equivalent to species level. In the field survey, the smaller group was frequently detected in February, May and September but not in July, while the larger group was detected mainly in July and September. The two groups were not distinguishable by their pathogenicity to spinach seedlings.