Leaf explants were taken from mature leaves of two almond [Prunus dulcis (Miller) D.A. Webb] cultivars, Ne Plus Ultra and Nonpareil selection 15-1, and maintained in vitro to grow shoot tips. Shoot tips were grown also from a pre-existing in vitro culture of an almond-peach rootstock, P. dulcis `Titan' × P. persica `Nemaguard'. The shoot tips were harvested, cryopreserved, and tested for survival after 3 days and then at intervals of 3 months up to two years. The mean survival was 80% for `Ne Plus Ultra', 54% for `Nonpareil', and 78% for the hybrid rootstock, and there were no significant differences in survival between 3 days and 24 months. The effects of in vitro culture and cryopreservation on DNA integrity were examined by both RAPD-PCR, and restriction enzyme digestion followed by RAPD-PCR, using DNA from the original trees from which the explants were derived, from leaves regrown from cultures that had undergone several passages of in vitro culture, and from leaves regrown from cryopreserved shoot tips. No detectable differences were found between the DNA fingerprints of each DNA sample using RAPD-PCR with seven different 10-mer primers. However, differences were detected when the DNA was first digested with the isoschizomeric pairs, Hpa II/Msp I and Bsp 143 I/Mbo I and then subjected to RAPD-PCR with six different 10-mer primers. Changes in the structure and methylation of DNA were found that were probably related to the process of in vitro culture, and in addition, methylation changes were detected that were probably associated with the cryopreservation process. These changes did not appear to be caused by the vitrification solution used before immersion of shoot tips in liquid nitrogen. While cryopreservation appears to be an ideal method for the long-term storage of almond germplasm, the significance of the alterations to both methylation and structure of DNA needs to monitored in regenerated plants, especially as they relate to agronomic performance when the regenerants become reproductively mature.
Olive leaf spot is a disease of olive (Olea europaea L.) caused by the fungal pathogen, Spilocea oleaginea Cast. Progeny derived from crosses among susceptible, resistant, and semiresistant parental lines were assessed in the field for 8 years and classified as either resistant or susceptible. DNA from some of the progeny of this segregating population was used to identify molecular markers linked to olive leaf spot disease using the randomly amplified polymorphic DNA (RAPD) technique and bulked segregant analysis (BSA). Two DNA bulks were constructed, each containing 13 progeny showing either resistance or susceptibility for the disease, and screened for polymorphisms using 100 primers. One primer produced two polymorphic bands, one of ≈700 base pairs (bp) from the susceptible bulk and the other of ≈780 bp from the resistant bulk. The 780 bp marker appeared in 70.6% of the segregating progeny and 100% of parents showing resistance to leaf spot disease, while the 700 bp marker appeared in 47.1% of the segregating progeny and 100% of the parents showing susceptibility. These markers can be used as screening tools in olive improvement programs.
A sequence-tagged site (STS) was developed to identify a genetic marker linked to resistance to olive leaf spot caused by the pathogen, Spilocea oleaginea (Cast) (syn. Cycloconium oleaginum Cast.). The STS was based on a randomly amplified polymorphic DNA (RAPD) marker of about 780 base pairs (bp) linked to olive leaf spot resistance. Several primer pairs were developed to flank the sequence, and one pair produced the expected polymorphism between resistant and susceptible individuals tested, and was used as an STS marker. This primer pair was tested against parents and 34 individuals from a population segregating for resistance to olive leaf spot, and 12 commercial olive (Olea europaea L.) cultivars showing various levels of resistance to the disease. The STS marker was present in 71.4% of the parents and progeny that were designated as resistant, and was absent in 87% of the parents and progeny showing susceptibility. These primers were also able to distinguish cultivars such as `Koroneiki' and `Leccino', that are reported to show resistance to olive leaf spot, from `Barouni' and `Mission', that are reported to be susceptible. This is the first report of a STS marker for olive, and its use will assist greatly in screening olive progeny for resistance to leaf spot in breeding programs.
The potential for hybridization among three species of Eucalyptus L'Hér in the Series Macrocarpae, E. macrocarpa Hook (Mottlecah), E. pyriformis Turcz. (pear-fruited mallee), and E. youngiana F. Muell. (large-fruited mallee), was investigated using molecular data generated by randomly amplified polymorphic DNA (RAPD)-polymerase chain reaction (PCR) analysis. Samples of DNA from seedlings derived from controlled pollinations, and from different individuals from each species, were amplified with six different 10-mer primers. The presence or absence of RAPD fragments was used to generate a dendrogram based on genetic similarity, an ordination derived by multidimensional scaling (MDS), and a minimum spanning tree (MST) to show the relative links and dissimilarities between the individuals tested. Two clusters were identified on the unweighted pair-group method arithmetric average dendrogram. The first included all of the E. macrocarpa genotypes and all but one of the E. macrocarpa hybrids. The second included all of the E. youngiana and E. pyriformis genotypes and their hybrids. The MDS ordinations placed the hybrid seedlings between the parent species. From the 30 progeny investigated, 28 were assessed from the molecular data to be hybrids from controlled pollinations. The remaining two seedlings appeared to be derived from self-pollination. The parentage of two mature trees, thought to be natural hybrids involving the three species, was also investigated. One was confirmed as a cross between E. youngiana and E. pyriformis, but the second was less certain because of its low genetic similarity to all other individuals, and may be a hybrid involving species not included in this study.
The National Olive Variety Assessment (NOVA) collection, established at the Roseworthy Campus of the University of Adelaide, contains six replicate trees of 100 olive (Olea europaea L.) accessions grown in the same environment. The DNA fingerprints of these accessions were compared, using randomly amplified polymorphic DNA (RAPD), to those of a number of cultivars obtained from international collections. A total of 86 uniquely named accessions in the NOVA collection resulted in 58 different genotypes. Different names were synonyms for the same genotype, and homonyms were also found where accessions with the same name had different DNA fingerprints. A rapid and efficient protocol was developed to identify unknown olive genotypes using a two-stage process. Data from DNA fingerprints were added to a matrix already containing binary data from recognized standard cultivars. The estimated probability of any given RAPD profile randomly occurring at this stage ranged between 6 × 10-4 and 2 × 10-8. In the second stage, the approximate identity of the unknown genotype, revealed by the resulting dendrogram, was confirmed by comparing it with appropriate standards under identical conditions of DNA amplification and band separation. The data collected in this report form the basis of a genetic database that can be used for the identification of olive samples.