Isolation of RNA from blackcurrant (Ribes nigrum L.) fruit.

Extraction of high-quality RNA from blackcurrant fruit has hitherto proved difficult, probably owing to high levels of phenolic and polysaccharide components in the berries. The procedure described here is a modification of one described for grape berries, and yields RNA suitable for in vitro translations, RNA blot analysis, and cDNA library construction.

The isolation of RNA from raspberry (Rubus idaeus) fruit.

Previous attempts to extract high-quality, total RNA from raspberry (Rubus idaeus) fruits using published protocols have proven to be unsuccessful. Even the use of protocols developed for the extraction of RNA from other fruit tissue has resulted in low yields (1) or the isolation of degraded RNA (2). Here, we report on the development of a quick and simple method of extracting total RNA from raspberry fruit. Using this method, high yields of good quality, undegraded RNA were obtained from fruit at all stages of ripening. The RNA is of sufficient quality for northern analysis and cDNA library construction.

Responses of cells and protoplasts of Coffea arabica genotypes to partially purified culture filtrates produced by Colletotrichum kahawae.

Hypocotyl-derived calli of genotypes and segregating populations of Coffea arabica, differing in susceptibility to Colletotrichum kahawae, were used to produce cell suspensions and protoplasts which were exposed to partially purified culture filtrates (PPCFs) prepared from the pathogen. The growth and viability of PPCF-treated cells and protoplasts were measured using packed cell volume, fluorescein diacetate staining and a colorimetric assay involving the tetrazolium salt MTT. Differential responses of cells and protoplasts were influenced by genotype, time of exposure and PPCF concentration. Protoplasts of resistant genotypes responded differentially from more susceptible genotypes as early as 4 h after challenge with the phytotoxin, suggesting that they were more sensitive than cell suspensions to the treatments. Protoplasts exposed to PPCFs from C. kahawae may therefore be used to screen and select genotypes resistant to, or tolerant of, coffee berry disease.

An examination of the ability of RAPD markers to determine the relationships within and between Rubus species.

RAPD markers were generated from 13 different Rubus species in order to assess the degree of similarity between species from the important subgenera. All ten primers revealed scorable polymorphisms within both the closely related and the genetically diverse individuals. Three hundred and seventy-two markers were generated and scored from the material analysed. Estimates of similarity, dendrograms and principle co-ordinate analysis were calculated, with the results generally being in agreemen with previous classifications of the species studied, confirming the validity and usefulness of the RAPD method. However, amongst the species studied, R. macraei of the Idaeobats proved more diverse and grouped in with both the Idaeobats and Eubats at only 26% similarity.

Gene flow from cultivated to wild raspberries in Scotland: developing a basis for risk assessment for testing and deployment of transgenic cultivars.

In order to help establish a basis for assessing the risk associated with the testing and large-scale deploymen of transgenic raspberries, wild and feral raspberry populations in Scotland were surveyed for evidence of the escape of genes introduced into raspberry cultivars by traditional breeding. The genes concerned were introduced into cultivars using traditional breeding techniques and were deployed at known times 20 to 30 years prior to the present survey. Escape of the semidominant L 1 gene, affecting fruit size and plant morphology, could not be detected after 30 years in test plots at the Scottish Crop Research Institute near Dundee. The recessive gene s, conferring spinelessness, was detected at very low frequencies (estimated at 0.004) in wild populations within the commercial production locales where cultivars carrying this gene had been introduced on a large scale beginning 21-years prior to this survey. This gene was not, however, found in any areas remote from the commercial production locales. The results of the survey indicate that escape does occur following large-scale deployment but that gene flow events are probably infrequent and spread is localized for genes having probable neutral selective value.

A comparison of methods for the estimation of genetic diversity in strawberry cultivars.

RAPD markers were used to examine the genetic relatedness of eight strawberry cultivars released from four breeding programmes around the world. Ten random primers successfully amplified DNA fragments from each cultivar and specific fingerprints were generated from the molecular marker data. The cultivars were traced back to founding clones and the relationships between the cultivars were examined from both the molecular and the pedigree data.

Microsatellites as DNA markers in Sitka spruce.

Nine microsatellite loci were found by screening a genomic DNA library of Sitka spruce (Picea sitchensis) with the four oligonucleotide probes (TG), (CAC), (GATA) and (AT). Pairs of flanking primers were generated for seven microsatellites. Five primer pairs were used to screen up to 58 Sitka spruce clones. The five loci SStg3a, SStg4, SStg4a, SStg4c and SSgataS were found to have 15, 13, 4, 3 and 6 different length alleles respectively, and in using a combination of them almost all 58 Sitka spruce genotypes could be identified. The five primer pairs were successful in amplifying DNA from two other spruce species (Picea albutilia and Picea smithiana), while only one primer pair could amplify DNA from the pine species, Pinus sylvestris and Pinus latifolia. The inheritance of microsatellites in Sitka spruce was co-dominant Mendelian.

RAPD fingerprinting of blackcurrant (Ribes nigrum L.) cultivars.

Ribes nigrum germplasm was screened for random amplified polymorphic DNA (RAPD) markers. Fiftyfour markers were identified which generated individual fingerprints for each of 21 cultivars. Genetic variation within R. nigrum germplasm, as detected by RAPDs, demonstrated that the genetic basis for improvement of blackcurrant is narrower than would be expected by the analysis of parentage.