Self-incompatibility (SI) is the ability of a fertile hermaphrodite flowering plant to prevent self-fertilization by discriminating between self and nonself pollen. In almond, as well as in other Prunus L. species, the SI system is of the gametophytic type (Socias i Company et al., 1976) and is controlled by a single S locus with multiple alleles (Crane and Lawrence, 1929). To ensure that flowers may be efficiently pollinated to reach an economically acceptable fruit set (Kester and Griggs, 1959), insect-dependent cross-pollination of intercompatible and simultaneously blooming cultivars is required. As an alternative to SI cultivars, self-compatible (SC) ones may be used to expand monovarietal orchards (Socias i Company, 1990).
To breed SC cultivars, it is important to understand the molecular mechanisms underlying this behavior. The S locus produces in the pistil a basic glycoprotein with ribonuclease (S-RNase) activity (McClure et al., 1989). This protein is taken up by the growing pollen tubes, causing their subsequent arrest in the style and thus preventing self-fertilization by its own pollen (Kao and Tsukamato, 2004). The expression of SC in almond is attributed to the presence of the Sf allele, whose expression is dominant over the other alleles of the S series (Socias i Company, 1984). Bošković and Tobutt (1996) reported that in cherry (Prunus avium L.), the S alleles code for stylar ribonucleases that can be detected by electrophoretic separation of stylar proteins and subsequent staining for activity. In almond, Bošković et al. (1999, 2003) applied the analysis of stylar S-RNases by nonequilibrium pH gradient electrofocusing (NePHGE) to detect SC seedlings in almond progenies from crosses in which one parent was SC. In these analyses, SC cultivars only showed one band with RNase activity. Additional studies carried out at the genetic level allowed the partial sequence of the Sf allele gene associated with S-RNase of SC in almond to be obtained (Channuntapipat et al., 2001; Ma and Oliveira, 2001).
The origin and the mechanism of almond SC are still unknown. Two hypotheses have been put forward to explain how SC appeared. The first hypothesis suggests a natural mutation in the SI system (Crossa-Raynaud and Grasselly, 1985), and the second suggests a gene transfer through spontaneous interspecific hybridization between Prunus amygdalus and P. webbii (Vierh.) Spach (Socias i Company, 2004). Independent of its origin, it is accepted that the loss of activity of an S-RNase in the style is the possible reason for SC in almond (Bošković et al., 1999). Supporting the previous suggestions from Bošković et al. (1999), Hanada et al. (2009) have reported that the possible origin of SC in almond may be due to the lack of or to the very low level of the transcription of the S-RNase in the pistil. However, Bošković et al. (2007) have suggested another possible origin for ‘Tuono’ SC—a mutation in the C2 region from histidine to arginine. Both hypotheses could explain the absence of RNase activity repeatedly observed in SC almond genotypes (Alonso and Socias i Company 2005; Bošković et al., 1999).
In other studies, Ushijima et al. (2003) sequenced, for the first time, the pollen S haplotype termed F-Box (SFB), finding that this could be a good candidate for the pollen S product because it was confirmed to be specifically expressed in the pollen tube and to be physically linked to the S-RNase gene (Entani et al., 2003, Ikeda et al., 2005). SFB features such as pollen-specific expression, tight linkage with the S-RNase gene, a high level of allelic polymorphism, and the presence of regions under positive selection are consistent with this being the pollen determinant in Prunus (Entani et al., 2003; Ikeda et al., 2004, 2005; Yamane et al., 2003). Hanada et al. (2009) have sequenced the SFBf in SC ‘Lauranne’ and found the same sequence as did Bošković et al. (2007) in SC ‘Tuono’ and in SI ‘Cinquanta Vignali’ and ‘Fra Giulio Grande’.
Recently, Kodad et al. (2008) identified the S genotype of 39 cultivars in the almond germplasm bank of Zaragoza, Spain, using the PCR approach, combining different primers. Several Spanish local cultivars from the island of Majorca, including Ponç, were found to posses the consensus Sf allele as sequenced by Channuntapipat et al. (2001), considered to confer the SC trait in almond. This cultivar presents high productivity, good nut quality, medium blooming time, and good behavior under drought conditions (Rubí, 1980). The confirmation of SC in ‘Ponç’ would also open new horizons in almond breeding programs as an alternative source of SC other than ‘Tuono’ and closely related cultivars from the Italian region of Puglia (Socias i Company, 2002). This alternative would avoid the problems related to inbreeding in the expression and transmission of SC in almond, which presumably results from lethal and deleterious genes that also affect some horticultural traits, including vigor, bud density, and plant size (Alonso and Socias i Company, 2007).
The aim of this work was to confirm the molecular identity of the Sf-RNase and SFBf of ‘Ponç’ by genomic DNA sequencing and by comparison with the S genotype of other SC cultivars. This study was complemented by assessing pollen tube growth and fruit set, after controlled artificial self-pollination and bagging branches at bloom, as an indicator of phenotypic expression of SC/SI.
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