Biochemical markers for cultivar identification and disease resistance evaluation in tomatoes (Lycopersicon Esculentum)

Abstract

The novelty of newly bred cultivars need to be established, since a certificate of protection is granted to breeders to protect their plant breeders' rights. Traditionally, newly bred cultivars were typified by means of unique morphological traits. Tomato cultivars, however, are morphological very similar due to breeding methods used. Such cultivars lack morphological differences and more sensitive methods are therefore needed to define novelty. Biochemical methods, such as protein and isozyme analysis and DNA based methods were investigated as alternative methods in the attempt to identify genetically uniform tomato cultivars. Protein separating methods used included SDS-PAGE of seed and leaf polypeptides, isoelectric focusing (IEF) and gradient-PAGE of leaf proteins. None of these methods, however could distinguish between the cultivars and six isozyme systems were evaluated as an alternative method. Seed isozymes alcohol dehydrogenase, acid phosphatase, phosphogluco mutase, phosphogluco isomerase, esterase and 6-phosphogluconate dehydrogenase (ADH, APS, PGM, PGI, EST and 6-PGDH, respectively) separated by means of vertical isoelectric focusing successfully distinguished between 12 of the 17 cultivars used in this study. However, closely related cultivars did not reveal any polymorphism. In an attempt to find differences between them, the study was directed to a DNA based method (RAPD's), which is more sensitive than methods detecting polymorphism at the protein level. MgC12 and DNA template concentrations were optimized to ensure reproducible banding patterns. The five 1 Omer random primers used, displayed different banding patterns, but did not display differences between different cultivars. Although the genetic diversity observed between tomato cultivars by means of isozyme analysis was a major breakthrough, this study need to be further pursued by using more isozyme systems and/or more random primers and/or restriction fragment length polymorphism to distinguish between closely related tomato cultivars. The genetic linkage between the Mi (single dominant gene for resistance to root knot nematodes) and Aps-11 (encoding Aps-1 1 isozyme) alleles were investigated to assist in identifying root knot nematode resistance in breeding lines. cultivars were firstly evaluated in the greenhouse at 30 °C for root knot nematode resistance or tolerance. Intermediate, tolerant and susceptible cultivars were identified, which support the postulation by Cap et al. (1993) that two genes for nematode resistance exist, namely a gene active at high temperatures (Mi-2) and a gene contribution to resistance at lower temperatures (Mi). The same cultivars were also analyzed for the expression of the linked Aps-11 allele by means of native-PAGE. The Aps-11 , Mi linkage were found in some resistant cultivars, while other resistant cultivars as well as the susceptible cultivars did not express the Aps-11 allele. It is postulated that an overcrossing took place between the linked Mi and Aps-11 alleles in earlier generations. This implies that tomato breeding lines whose parents underwent this overcrossing cannot be selected for root knot nematode resistance based on the presence or absence of the Aps-1 1 isozyme. The novel Aps-14 and Aps-2 isozymes of L. peruvianum and the Aps- 13 isozyme of L. esculentum were co-expressed during the second week after germination which suggested a regulatory function, since the first true leaves of a seedling appear during this period. Novel information gained from this study are: (a) Tomato cultivars also showed intermediate resistance to nematodes. (b) Isozymes, Aps-14 and some Aps-2 isozymes, were detected for the first time. (c) Aps-14 and Aps-1 3 isozymes are developmentally regulated. (d) No locally bred cultivars exhibited the Mi/Aps-1 1 linkage. (e) Aps-1+ and Aps-1 1 isozymes are dominantly inherited and (f) are homodimers.