The Expression of Self-compatibility in Almond May Not Only Be Due to the Presence of the Sf Allele

in Journal of the American Society for Horticultural Science
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  • 1 Unidad de Fruticultura, Centro de Investigación y Tecnología Agroalimentaria de Aragón (CITA), Av. Montañana 930, 50059, Zaragoza, Spain
  • 2 Instituto de Tecnologia Química e Biológica (ITQB), Quinta do Marquês, 2784-505 Oeiras, Portugal
  • 3 Instituto de Tecnologia Química e Biológica (ITQB), Instituto de Biologia Experimental e Tecnológica (IBET), Quinta do Marquês, 2784-505 Oeiras, Portugal, and Universidade de Lisboa, Faculdade de Ciências, Dep. de Biologia Vegetal, Campo Grande, 1749-016 Lisbon, Portugal

The pistil (S-RNase) and the pollen [S-haplotype-specific F-box protein (SFB)] components of the Sf allele, presumably conferring self-compatibility in almond {Prunus amygdalus Batsch [syn. P. dulcis (Mill.) D.A. Webb]}, were identified and sequenced in ‘Ponç’, a local Spanish almond cultivar, confirming their identity with the published sequences of these components. Despite the presence of the Sf allele, the ‘Ponç’ phenotype was self-incompatible as confirmed by different pollination tests, including self pollen tube growth, fruit set after self-pollination, and fruit set in bagged branches. However, the pistil and the pollen of ‘Ponç’ were fully viable when pollinated by a cross-compatible pollen or used on a cross-compatible pistil. The fact that ‘Ponç’ presents two different S-proteins with RNase activity may indicate an active function of its Sf-RNase, whereas in the self-compatible almond cultivars thus far studied, the Sf-RNase has been inactive. This activation indicates that the presence of the Sf allele may not be the exclusive source of self-compatibility in almond, and other factors may also be involved in the expression of almond self-compatibility.

Abstract

The pistil (S-RNase) and the pollen [S-haplotype-specific F-box protein (SFB)] components of the Sf allele, presumably conferring self-compatibility in almond {Prunus amygdalus Batsch [syn. P. dulcis (Mill.) D.A. Webb]}, were identified and sequenced in ‘Ponç’, a local Spanish almond cultivar, confirming their identity with the published sequences of these components. Despite the presence of the Sf allele, the ‘Ponç’ phenotype was self-incompatible as confirmed by different pollination tests, including self pollen tube growth, fruit set after self-pollination, and fruit set in bagged branches. However, the pistil and the pollen of ‘Ponç’ were fully viable when pollinated by a cross-compatible pollen or used on a cross-compatible pistil. The fact that ‘Ponç’ presents two different S-proteins with RNase activity may indicate an active function of its Sf-RNase, whereas in the self-compatible almond cultivars thus far studied, the Sf-RNase has been inactive. This activation indicates that the presence of the Sf allele may not be the exclusive source of self-compatibility in almond, and other factors may also be involved in the expression of almond self-compatibility.

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.

Materials and Methods

Plant material.

Nine almond genotypes were studied. Four of these, ‘Ponç’, ‘Desmayo Largueta’ [S1S10 (Ortega et al., 2006)], ‘Marcona’ [S11S12 (Bošković et al., 1999)], and ‘Bertina’ [S6S11 (Bošković et al., 1999)] are local Spanish cultivars, and three, ‘Soleta’ (SfS23), ‘Cambra’ (SfS3), and selection G-2–22 [SfS6 (Kodad and Socias i Company, 2008; Kodad et al., 2008)], are releases from the Centro de Investigación y Tecnología Agroalimentaria de Aragón (CITA) breeding program. ‘Tuono’ [SfS1 (Crossa-Raynaud and Grasselly, 1985)] is the most used SC cultivar in breeding programs, and ‘Ferragnès’ [S1S3 (Crossa-Raynaud and Grasselly, 1985)] is a release from the Institut National de la Recherche Agronomique, France (INRA) breeding program. All plant samples were obtained from the Spanish almond germplasm collection located at CITA, maintained as living plants grafted on the almond × peach [Prunus persica (L.) Batsch] hybrid clonal rootstock INRA GF-677 using the standard management practices (Espiau et al., 2002).

DNA extraction.

Genomic DNA was extracted from leaves following the CTAB extraction method based on Doyle and Doyle (1987). For PCR amplification, DNA was diluted to 20 ng·μL−1 in water.

S-RNase and SFB alleles PCR amplification.

To confirm the identity of the S alleles of these cultivars, the S alleles were amplified according to Tamura et al. (2000), using the primers AS1II and AmyC5R designed for the C1 and C5 conserved regions of almond S-RNase alleles. The regions between the signal peptide and the conserved region C5 of the Sf allele of ‘Cambra’ and the putative Sf allele of ‘Ponç’ were amplified according to Ortega et al. (2006) using the forward primer PaConsI-F (Sonneveld et al., 2003) and the reverse primer EM_PC5consRD (Sutherland et al., 2004). PCR reactions were performed in a volume of 30 μL containing 1× PCR buffer (Promega, Madrid, Spain), 1.5 mm MgCl2, 0.2 mm of each dNTPs, 0.5 μm of each primer, one unit of Taq DNA polymerase, and 75 ng of genomic DNA. The PCR program for S-RNase allele identification consisted of an initial denaturation of 2 min at 94 °C, followed by 34 cycles of 1 min at 95 °C, 2 min at 50 °C, and 4 min at 72 °C followed by a final extension of 10 min at 72 °C. A fragment of the SFB locus was PCR-amplified with primers SFB-C1F and SFB-FB3 (Yamane et al., 2003), SFB-C2F (Ikeda et al., 2004), and F-BOX3A′ (Vaughan et al., 2006). About 50 ng of genomic DNA was used for PCR amplification in 50 μL of reaction mixture containing 1× PCR buffer, 1.5 mm of MgCl2, 200 μm each of dNTPs, 400 mm of each primer, and one unit of Taq DNA polymerase. For the SFB haplotype amplification, the PCR reactions were run with a program of 35 cycles at 94 °C for 1 min, 54 °C for 1 min, and 72 °C for 1 min 30 s, with initial denaturing at 94 °C for 3 min and a final extension of 72 °C for 10 min.

The PCR products were separated on 1% agarose gels containing 1× TAE buffer and were stained with ethidium bromide (0.4 μg·mL−1). DNA bands were visualized under ultraviolet light and the images were registered using Gel Doc 2000 (Bio-Rad, Hercules, CA) with Quantity One software (version 4.0.1; Bio-Rad). GeneRuler™ DNA Ladder Mix (Fermentas, Porto, Portugal) was used as the molecular size standard.

Cloning and gDNA sequencing.

For the SFB haplotype, the cloning of the target sequences was done directly from the PCR product after checking the presence of the amplification on agarose gel. For the S-alleles, before cloning, the bands corresponding to the target S alleles were purified using the Wizard Plus Miniprep DNA Purification System (Promega) and were quantified on a 1.5% agarose gel using a standard 1-kb DNA ladder (Invitrogen, Madrid, Spain). The purified PCR products were cloned into the vector pCR2.1 using a TA Cloning Kit (Invitrogen). Insertion was confirmed by restriction enzyme digestion with EcoRI. Plasmids were isolated using the QIA prep Spin Miniprep Kit (Qiagen, Hilden, Germany). For each allele, at least three plasmids from different PCR reactions were sequenced from both ends.

Stylar S-RNase analysis.

S-genotype of ‘Ponç’ was also determined by separation of stylar RNases linked to SI alleles by NEpHGE as described by Bošković and Tobutt (1996), using as reference the other cultivars included in this study.

Pollination tests.

Physiological SI in ‘Ponç’ and ‘Cambra’ was studied with three different methods: pollen tube growth, fruit set after artificial hand pollination, and fruit set after bagging. During two consecutives years (2006–07), 50 flowers at stage D (Felipe, 1977) were collected from trees growing in the field and taken in plastic bags to the laboratory, emasculated, and placed in trays with tap water. Their pistils were self- or cross-pollinated with ‘Marcona’ pollen as a control. Pollen was collected and its viability was checked before pollination (Kodad and Socias i Company, 2006). After pollination, trays were kept at room temperature and samples of 10 pistils were collected every 24 h after pollination, completing 120 h since pollination. The pistils were autoclaved in a 5% solution of Na2SO3 for 12 min at 1.2 kg·cm−2. Self- and cross-compatibility was assessed by pollen tube growth after observation in a Leitz Ortholux II microscope (Leitz, Wetzlar, Germany) with ultraviolet illumination of a mercury lamp (Osram HBO 200 W/4; Torrejón de Arsoz, Madrid, Spain) by fluorescence of the callose deposits of the pollen tubes by aniline blue staining after squashing.

Eight branches per cultivar were selected in the field and at least 100 flowers were emasculated per branch and were self-pollinated. In other branches, the flowers were cross-pollinated with ‘Marcona’ and ‘Desmayo Largueta’ pollen. Another group of branches were bagged to evaluate the level of self-fertilization of ‘Ponç’. Fruit set was recorded in June for all treatments.

Results

‘Ponç’ S-RNase allele identity.

The screening of the S-RNase allele diversity in the almond germplasm bank of Zaragoza using allele-specific and conserved primers has allowed the identification of the Sf allele in ‘Ponç’ (Kodad et al., 2008). This allele, using the AS1II and AmyC5R primers, corresponds to a band of size ≈1200 bp. The deduced amino acid sequence of the Sf-allele in ‘Ponç’ is identical to that of the first Sf allele (AY291117) amplified in almond from two SC genotypes, ‘Lauranne’ and Institut de Recerca i Tecnologia Agroalimentària (IRTA) selection 12–2 (Channuntapipat et al., 2001). However, it is only 98% identical to the Sf allele sequenced in ‘Tuono’ (AF157009) by Ma and Oliveira (2001) and 99.3% to that by Bošković et al. (2007; AM690356), who gave different sequences for the same allele. The other allele with a band of size ≈1700 bp is identical to S27 (AM231675; Ortega et al., 2006).

Using the forward primer PaConsI-F and the reverse primer EM_PC5consRD, a band of 1600 bp was amplified in ‘Ponç’ and ‘Cambra’. The sequencing of this Sf showed the typical feature of the Prunus T2-type RNase with five conserved domains (C1, C2, C3, RC4, and C5) and one hypervariable region (Fig. 1). The position of the two introns was also identical to that of other almond S-RNases reported by Ortega et al. (2006). The comparison of the sequences as the Sf allele of ‘Ponç’ at the g-DNA and at the deduced amino acid sequence levels showed the same sequence and position of the signal peptide and the first intron of the Sf allele identified in ‘Cambra’, a SC cultivar obtained from the cross of ‘Tuono’ × ‘Ferragnès’ (Socias i Company and Felipe, 1999). Thus, the two alleles are identical and no alteration was observed in the primary structure of the encoded almond Sf-RNase.

Fig. 1.
Fig. 1.

Multiple alignment of the deduced amino acid sequence of the almond Sf allele identified in the present study and those deposited in the database. The conserved regions (C1–C5) and the hypervariable region (RHV) described in Rosaceae (Ushijima et al., 1998) are boxed. Conserved cysteine and histidine residues are shadowed. European Molecular Biology Laboratory Nucleotide Sequence database (EMBL/GenBank) accession numbers are as follows: Sf of ‘Tuono’ a (AM690356; Bošković et al., 2007); Sf of ‘Tuono’ b (AF157009; Ma and Oliveira, 2001); Sf of ‘Cambra’ (EU684318); Sf of IRTA selection 12–2 (AY291117; Channuntapipat et al., 2001); Sfa of ‘Ponç’ (EU293146); and S30 of ‘Fra Giulio Grande’ (AM690361; Bošković et al., 2007).

Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 134, 2; 10.21273/JASHS.134.2.221

The application of NEpHGE in ‘Ponç’ revealed two different S-RNase bands instead of one, as expected in SC genotypes (Bošković et al., 1999), corresponding to S27 and Sf (Fig. 2). The presence of two bands suggests that the two S-RNases of ‘Ponç’ are functional, including its Sf-RNase.

Fig. 2.
Fig. 2.

Zymogram of almond S alleles after NEpHGE showing two bands for ‘Ponç’ compared to a single band for the self-compatible ‘Tuono’ and selection G-2–22.

Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 134, 2; 10.21273/JASHS.134.2.221

SFB identity in ‘Ponç’.

When the SFB-C1F and SFB-FB3 primers were tested in ‘Cambra’ (SfS3) and ‘Ferragnès’ (S1S3), DNA sequencing revealed two different kinds of clones; however, just one kind of clone was found in ‘Ponç’. The deduced amino acid sequence from the partial genomic DNA of the SFB1 and SFB3 identified in ‘Ferragnès’, SFB11 and SFB12 in ‘Marcona’, SFB3 and SFBf in ‘Cambra’, and SFBf in ‘Ponç’ contained a partial sequence of the F-Box motif determined in other SFB S-haplotypes (Ushijima et al., 2003), and two variable regions V1 and V2 (Ikeda et al., 2004).

For ‘Ponç’ and ‘Cambra’, the use of SFB-C2F and F-BOX3A′ primers allowed the sequencing of the hypervariable regions HVa and HVb (Ikeda et al., 2004), located downstream to the V2 region (Fig. 3). The deduced amino acid of the SFBf sequence identified in ‘Ponç’ was identical to that sequenced in ‘Tuono’ (AM711126; Bošković et al., 2007) and in ‘Lauranne’ (AB361036; Hanada et al., 2009).

Fig. 3.
Fig. 3.

Alignment of the deduced almond SFBf amino acid sequences. The F-box motif, the variable regions V1 and V2, and the hypervariable regions HVa and HVb are boxed (Ikeda et al., 2004). European Molecular Biology Laboratory Nucleotide Sequence database (EMBL/GenBank) accession numbers are as follows: SFB30 of ‘Fra Giulio Grande’ (AM711127; Bošković et al., 2007); SFBf of ‘Tuono’ (AM711126; Bošković et al., 2007); SFBf of ‘Lauranne’ (AB361036); and SFBf of ‘Ponç’ (EU310402).

Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 134, 2; 10.21273/JASHS.134.2.221

The partial sequences of SFB3, SFB12, and SFBf have been deposited in the database with accession numbers EU293149, EU310402, and EU310402, respectively.

Pollination tests.

After confirming the identity of the Sf-allele RNase and of the SFB S-haplotype of ‘Ponç’ with those of other SC cultivars, pollination tests were conducted to confirm ‘Ponç’ SC. Pollen viability of ‘Ponç’ was tested before pollination, showing a high percentage of germination in vitro (≈92%). Pollen germination was also high in self-pollinated pistils in the laboratory, with similar numbers of pollen tubes in the upper third of the style compared with cross-pollinated pistils, with an average of 47 pollen tubes. Pollen tube growth was arrested in the middle third of the style in the self-pollinated pistils, and no pollen tubes were observed at the style base or in the ovary. Conversely, in the cross-pollinated pistils with pollen of ‘Marcona’ and ‘Desmayo Largueta’, more than three pollen tubes were observed at the style base. These results were consistent during the 2 years of study. In the field tests, no fruit set was obtained after hand self-pollination, but normal sets, according to the criteria of Kester and Griggs (1959), were obtained after cross-pollination with ‘Marcona’ and ‘Desmayo Largueta’ pollen, as well as after selfing ‘Cambra’ (Table 1). Furthermore, no fruit set was obtained in bagged branches of ‘Ponç’ during the 2 years of study. The field pollinations by hand or in bagged branches were unaffected by adverse weather conditions in both years, as confirmed by the normal sets in the compatible crosses.

Table 1.

Fruit set after artificial hand-pollination of almond cultivars in the field (100–150 flowers per treatment).

Table 1.

Discussion

S-RNase and SFB identity and self-(in)compatibility expression in ‘Ponç’.

The cloning and sequencing of the Sf-RNase allele in ‘Ponç’ (EU310402) showed that it was found to be identical to that of ‘Cambra’ (EU684318). As well, the deduced amino acid sequence (C1-C5) of the ‘Ponç’ Sf–RNase allele was identical to that determined here in ‘Cambra’ and to that of ‘Lauranne’ and the IRTA selection 12–2 (AY291117; Channuntapipat et al., 2001), reported to be derived from selfing ‘Lauranne’ (Bošković et al., 1999). Hanada et al. (2009) have also identified the partial sequence of the Sf-RNase allele in ‘Lauranne’ and found it to be identical to the AY291117 sequence. However, all of these sequences are different from that of ‘Tuono’ published by Ma and Oliveira (2001; AF157009) or by Bošković et al. (2007; AM690356). The former (AF157009) has valine instead of isoleucine and histidine instead of arginine in the C2 region (Fig. 1). The latter has histidine instead of arginine (Fig. 1). Taking into account that ‘Cambra’ and ‘Lauranne’ are both seedlings from ‘Tuono’, these cultivars must have inherited the identical Sf–RNase allele from ‘Tuono’, as well as the IRTA selection 12–2 from ‘Lauranne’.

Separately, Bošković et al. (2007) have identified in the SI ‘Fra Giulio Grande’, also from the same Italian region of Apulia as ‘Tuono’, an S–RNase identical to our Sf–RNase of ‘Ponç’ and ‘Cambra’ and to Sf–RNase of ‘Lauranne’ and IRTA 12–2 (Channuntapipat et al., 2001). However, Bošković et al. (2007) reported for ‘Tuono’ a different sequence (AM690356), with a substitution of histidine by arginine in the C2 region from the consensus Sf sequence (Fig. 1). Thus, they suggested a new allele, S30, (AM690361) present in ‘Fra Giulio Grande’, but not differing from the consensus Sf sequence, concluding that this S30 is the wild-type allele from which Sf was a stylar mutation called S30° conferring SC in almond. This terminology is confusing because they attribute a new number (S30) for an existing allele (Sf) and establish a new allele (S30°) as a result of a possible missequencing. Consequently, the S30–RNase allele identified by Bošković et al. (2007) and the Sf–RNase allele of ‘Ponç’ correspond to the Sf–RNase allele already sequenced in SC almond cultivars and selections (Channuntapipat et al., 2001).

The pollination tests showed that ‘Ponç’ is physiologically SI. However, although the growth of the self-pollen was arrested in ‘Ponç’ pistils, the good growth of a cross-compatible pollen showed that these pistils are able to sustain the tube growth of foreign pollen. In addition, the good germination of ‘Ponç’ pollen indicates its high viability, as well as the fruit set of ‘Marcona’ after hand pollination by ‘Ponç’ pollen. There were no available cultivars sharing the same S genotype as ‘Ponç’, therefore the functionality and ability of the pollen harboring the same S-genotype than ‘Ponç’ in reaching the style base could not be tested.

The ribonuclease activity of the S-RNases is shown to be essential for the SI reaction (Huang et al., 1994; Sassa et al., 1997). In almond, SC has been associated with the absence of a stylar RNase band (Bošković et al., 1999), as well as in japanese pear (Pyrus serotina Rehd.) (Sassa et al., 1992). The fact that RNase activity is required for the function of the S-RNases, and that S-RNases are taken up by self- and nonself-pollen tubes, suggests that the Sf-RNase of ‘Ponç’ is functional and degrades the RNA of self-pollen. Bošković et al. (2007) have also identified three Italian SI almond cultivars, ‘Cinquanta Vignali’, ‘Fra Giulio Grande’, and ‘Santoro’, with the S-RNase allele identical to that found in ‘Ponç’ that showed ribonuclease activity. The ribonuclease activity of the Sf–RNase allele of ‘Ponç’ explains the results of pollen tube growth and fruit set when ‘Ponç’ was self-pollinated.

SC in ‘Cambra’, IRTA 12–2, and ‘Lauranne’ could be the result of a mutation or alteration in the coding region of the Sf-RNase gene, as mentioned in other species. In Lycopersicon peruvianum (L.) Mill., some spontaneous SC accessions have an amino acid substitution at one of the essential histidines in the catalytic domain of the S-RNase that leads to a complete loss of enzymatic activity (Kowyama et al., 1994; Royo et al., 1994). In Petunia inflata R. Fries, the mutant S3 allele mutagenized by replacing the codon for His-93 with a codon for aspargine, producing a mutant protein that does not exhibit any detectable ribonuclease activity (Huang et al., 1994). However, in the present study, no mutation of histidine to other amino acids was detected in the Sf allele in SC almond cultivars.

Recently, Tao et al. (2007) reported an inactive S2m–RNase in several cultivars of peach and attributed the reduction of RNase stability to the replacement of a structurally important cysteine residue in the C5 region by tyrosine. In the present study, we have not completely sequenced the SfRNase clone, but the complete deduced amino acid sequence of SfRNase of IRTA selection 12–2 (AY291117) contains a cysteine residue.

The SC observed in some cultivars of several Prunus species has been reported to be due to a defective function of the pollen, as a result of a mutation, deletion, or insertion in the coding region of the SFB S-haplotype, such as in P. avium and P. mume Sieb. et Zucc. (Ushijima et al., 2004), P. armeniaca L. (Vilanova et al., 2006), and P. persica (Tao et al., 2007). However, the identity of the partial sequence of ‘Ponç’ SFBf and that of ‘Lauranne’ (AB361036; Hanada et al., 2009) and ‘Tuono’ (AM711126; Bošković et al., 2007) indicates that there is no alteration in any of the two components of the Sf allele in ‘Ponç’, either in the pistil or the pollen parts. Moreover, the identity of SFBf from ‘Lauranne’ and ‘Tuono’, and of SFB30 (AM711127) from ‘Fra Giulio Grande’, a SI Italian cultivar (Bošković et al., 2007), indicates that the origin of SC in almond is not related to any alteration or mutation in the SFB gene in almond.

These results confirm that ‘Ponç’ and ‘Cambra’ in the present study, as well as ‘Lauranne’ (Hanada et al., 2009), share the same SFBf allele but present a different phenotypic expression. As far as we know, this is the first time that an almond cultivar has been identified as possessing the Sf allele, associated with SC expression, but being phenotypically SI. Alonso and Socias i Company (2005) reported that some inbred SC genotypes, having ‘Tuono’ as a donor for the SC trait, in spite of the presence of the Sf allele, showed a SI phenotype, indicated by a very low number of pollen tubes at the base of low number of pistils. However, this was the result of the inbreeding depression observed in that population, whereas in the present work, all self-pollinated pistils showed the arrest of the pollen tubes in the middle part of the style, which is a different phenomenon. This Sf allele, showing ribonuclease activity and being expressed in ‘Ponç’, may be called Sfa (active Sf-allele).

Hypothesis on the origin of self-compatibility in almond.

It has been suggested that SC in almond could be explained by quantifying the transcript expression of the S-RNases in the style. Watari et al. (2007) reported that the low transcriptional level of the Se-RNase resulted in an insufficient Se-RNase accumulation in the pistils, thus conferring SC in japanese plum (Prunus salicina Lindl.). Qin et al. (2006) have estimated the threshold amount of S12-RNase required for pollen rejection in Solanum chacoesense Bitt. and reported that an insufficient accumulation confers SC. In almond, SC has already been attributed to the lack of ribonuclease activity of the Sf allele in the pistil (Bošković et al., 1999). However, it is not known thus far whether the change affecting Sf-RNase expression in almond is at the transcriptional or translational level.

The absence of any alteration in the coding region of the Sf-allele in the SI and SC cultivars indicates that the appearance of SC in almond could be due to a change outside the coding region or to the presence of a modifier gene. Yamane et al. (2003) reported that the inactivation of the S6m-RNase in Prunus cerasus L. is due to an insert of 2600 bp located upstream of the S-RNase coding sequence. Further analysis should be undertaken to ascertain if there is any insertion in the Sf allele from SC and SI almond cultivars that could explain the inactivation of the Sf allele in SC almond cultivars. In Prunus pseudocerasus Lindl., it has been suggested that SC could be caused by mutations in other genes critical for the SI reaction (Huang et al., 2008).

In conclusion, we have isolated and sequenced, for the first time, the coding sequence for Sf–RNase and partial SFBf from two cultivars originated from two different geographic regions and showing a different phenotypic expression. The present results show that the coding region of the Sf gene may not be the exclusive origin of SC in almond. Further experiments involving the complete sequence of the Sf–RNase gene in SC and SI cultivars to confirm its identity and the expression analysis of the Sfa–RNase in ‘Ponç’ is required to determine the characteristics of the transcribed proteins of the expressed Sfa allele in almond.

Literature Cited

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    • Export Citation
  • Bošković, R. & Tobutt, K.R. 1996 Correlation of stylar ribonuclease zymograms with incompatibility alleles in sweet cherry Euphytica 90 245 250

  • Bošković, R., Tobutt, K.R., Ortega, E., Sutherland, B.C. & Godini, A. 2007 Self-(in)compatibility of the almond P. dulcis and P. webbii: Detection and cloning of ‘wild-type Sf’ and new self-compatibility alleles encoding inactive S-RNases Mol. Genet. Genomics 278 665 676

    • Search Google Scholar
    • Export Citation
  • Bošković, R., Tobutt, K.R., Duval, H., Batlle, I., Dicenta, F. & Vargas, J.F. 1999 A stylar ribonucleases assay to detect self-compatible seedlings in almond progenies Theor. Appl. Genet. 99 800 810

    • Search Google Scholar
    • Export Citation
  • Bošković, R., Tobutt, K.R., Batlle, I., Duval, H., Martínez-Gómez, P. & Gradziel, T.M. 2003 Stylar ribonuclease in almond: Correlation with and prediction of incompatibility genotypes Plant Breed. 122 70 76

    • Search Google Scholar
    • Export Citation
  • Channuntapipat, C., Sedgley, M. & Collins, G. 2001 Sequences of cDNAs and genomic DNAs encoding the S1, S7, S8 and Sf alleles from almond, Prunus dulcis Theor. Appl. Genet. 103 1115 1122

    • Search Google Scholar
    • Export Citation
  • Crane, M.B. & Lawrence, W.J.C. 1929 Genetical and cytological aspects of incompatibility and sterility in cultivated fruits J. Pomol. Hort. Sci. 7 276 301

    • Search Google Scholar
    • Export Citation
  • Crossa-Raynaud, P. & Grasselly, C. 1985 Éxistence de groupes d'interstérilite chez l'amandier Options Méditerranéennes CIHEAM/IAMZ 85 1 43 45

  • Doyle, J.J. & Doyle, J.L. 1987 A rapid DNA isolation procedure for small quantities of fresh tissue Phytochem. Bul. 19 11 15

  • Entani, T., Iwano, M., Shiba, H., Che, F.S., Isogai, A. & Takayama, S. 2003 Comparative analysis of the self-incompatibility (S-) locus region of Prunus mume: Identification of a pollen-expressed F-box gene with allelic diversity Genes Cells 8 203 213

    • Search Google Scholar
    • Export Citation
  • Espiau, M.T., Ansón, J.M. & Socias i Company, R. 2002 The almond germplasm bank of Zaragoza Acta Hort. 591 275 278

  • Felipe, A.J. 1977 Almendro. Estados fenológicos Información Técnica Económica Agraria 27 8 9

  • Hanada, T., Fukuta, K., Yamane, H., Tao, R., Alonso, J.M. & Socias i Company, R. 2009 Cloning of self-compatible Sf locus in almond Acta Hort. in press

  • Huang, S., Lee, H.S., Karunanandaa, B. & Kao, T.H. 1994 Ribonuclease activity of Petunia inflata S proteins is essential for rejection of self-pollen Plant Cell 6 1021 1028

    • Search Google Scholar
    • Export Citation
  • Huang, S.X., Wu, H.Q., Li, Y.R., Wu, J., Zhang, S.J., Heng, W. & Zhang, S.L. 2008 Competitive interaction between two functional S-haplotypes confer self-compatibility on tetraploid Chinese cherry (Prunus pseudocerasus Lindl. cv. Nanjing Chuisi) Plant Cell Rep. 27 1075 1085

    • Search Google Scholar
    • Export Citation
  • Ikeda, K., Igic, B., Ushijima, K., Yamane, H., Hauck, N.R., Nakano, R., Sassa, H., Iezzoni, A.F., Kohn, J.R. & Tao, R. 2004 Primary structural features of the S haplotype-specific F-box protein, SFB, in Prunus Sex. Plant Reprod. 16 235 243

    • Search Google Scholar
    • Export Citation
  • Ikeda, K., Ushijima, K., Yamane, H., Tao, R., Hauck, N.R., Sebolt, A.M. & Iezzoni, A.F. 2005 Linkage and physical distances between the S-haplotype S-RNase and SFB genes in sweet cherry Sex. Plant Reprod. 17 289 296

    • Search Google Scholar
    • Export Citation
  • Kao, T.-H. & Tsukamato, T. 2004 The molecular and genetic bases of S-RNase-based self-incompatibility Plant Cell 16 72 83

  • Kester, D.E. & Griggs, W.H. 1959 Fruit setting in the almond: The effect of cross-pollinating various percentages of flowers Proc. Amer. Soc. Hort. Sci. 74 214 219

    • Search Google Scholar
    • Export Citation
  • Kodad, O. & Socias i Company, R. 2006 Pollen source effect on pollen tube growth in advanced self-compatible almond selections (Prunus amygdalus Batsch) Adv. Hort. Sci. 20 256 261

    • Search Google Scholar
    • Export Citation
  • Kodad, O. & Socias i Company, R. 2008 Fruit set evaluation for self-compatibility selection in almond Scientia Hort. 118 260 265

  • Kodad, O., Alonso, J.M., Sánchez, A., Oliveira, M.M. & Socias i Company, R. 2008 Evaluation of genetic diversity of S-alleles in an almond germplasm collection J. Hort. Sci. Biotechnol. 83 603 608

    • Search Google Scholar
    • Export Citation
  • Kowyama, Y., Kunz, C., Lewis, I., Newbigin, E., Clarke, A.E. & Anderson, M.A. 1994 Self-compatibility in a Lycopersicon peruvianum variant (LA2157) is associated with a lack of style S-RNase activity Theor. Appl. Genet. 88 859 864

    • Search Google Scholar
    • Export Citation
  • Ma, R.C. & Oliveira, M.M. 2001 Molecular cloning of the self-incompatibility genes S1 and S3 from almond (Prunus dulcis) cv. Ferragnès Sex. Plant Reprod. 14 163 167

    • Search Google Scholar
    • Export Citation
  • McClure, B.A., Haring, V., Ebert, P.R., Anderson, M.A., Simpson, R.J., Sakiyama, F. & Clarke, A.E. 1989 Style self-incompatibility gene products of Nicotiana alata are ribonucleases Nature 342 955 957

    • Search Google Scholar
    • Export Citation
  • Ortega, E., Bošković, R., Sargent, D.J. & Tobutt, K.R. 2006 Analysis of S-RNase alleles of almond (Prunus dulcis): Characterization of new sequences, resolution of synonyms and evidence of intragenic recombination Mol. Genet. Genomics 276 413 426

    • Search Google Scholar
    • Export Citation
  • Qin, X., Liu, B., Soulard, J., Morse, D. & Cappadocia, M. 2006 Style by style analysis of two sporadic self-compatible Solanum chacoense lines supports a primary role for S-RNase in determining pollen rejection thresholds J. Expt. Bot. 57 2001 2013

    • Search Google Scholar
    • Export Citation
  • Royo, J., Kunz, C., Kowyama, Y., Anderson, M., Clarke, A.E. & Newbigin, E. 1994 Loss of a histidine residue at the active site of S-locus ribonuclease is associated with self-compatibility in Lycopersicon peruvianum Proc. Natl. Acad. Sci. USA 91 6511 6514

    • Search Google Scholar
    • Export Citation
  • Rubí, V. 1980 El almendro Delegación de Baleares, Ministerio de Agricultura Palma de Mallorca, Spain

    • Export Citation
  • Sassa, H., Hirano, H. & Ikehashi, H. 1992 Self-incompatibility-related RNases in styles of japanese pear (Pyrus serotina Rehd.) Plant Cell Physiol. 33 811 814

    • Search Google Scholar
    • Export Citation
  • Sassa, H., Hirano, H., Nishio, T. & Koba, T. 1997 Style-specific self-incompatibility mutation caused by deletion of the S-RNase gene in japanese pear (Pyrus serotina) Plant J. 12 223 227

    • Search Google Scholar
    • Export Citation
  • Socias i Company, R. 1984 Genetic approach to the transmission of self-compatibility in almond (Prunus amygdalus Batsch) Options Méditerranéennes CIHEAM/IAMZ 84 1 123 127

    • Search Google Scholar
    • Export Citation
  • Socias i Company, R. 1990 Breeding self-compatible almonds Plant Breed. Rev. 8 313 318

  • Socias i Company, R. 2002 Latest advances in almond self-compatibility Acta Hort. 591 205 212

  • Socias i Company, R. 2004 The contribution of Prunus webbii to almond evolution Plant Genet. Resour. Newsl. 14 9 13

  • Socias i Company, R. & Felipe, A.J. 1999 ‘Blanquerna’, ‘Cambra’, y ‘Felisia’: Tres nuevos cultivares autógamos de almendro. Información Técnica Económica Agraria 95 5 111 117

    • Search Google Scholar
    • Export Citation
  • Socias i Company, R., Kester, D.E. & Bradley, M.V. 1976 Effects of temperature and genotype on pollen tube growth of some self-compatible and self-incompatible almond cultivars J. Amer. Soc. Hort. Sci. 101 490 493

    • Search Google Scholar
    • Export Citation
  • Sonneveld, T., Tobutt, K.R. & Robbins, T.P. 2003 Allele-specific PCR detection of sweet cherry self-incompatibility (S) alleles S1 to S16 using consensus and allele-specific primers Theor. Appl. Genet. 107 1059 1070

    • Search Google Scholar
    • Export Citation
  • Sutherland, B.G., Robbins, T.P. & Tobutt, K.R. 2004 Primers amplifying a range of Prunus S-alleles Plant Breed. 123 582 584

  • Tamura, M., Ushijima, K., Sassa, H., Hirano, H., Tao, R., Gradziel, T.M. & Dandekar, A.M. 2000 Identification of self-incompatibility genotype of almond by allele-specific PCR analysis Theor. Appl. Genet. 101 344 349

    • Search Google Scholar
    • Export Citation
  • Tao, R., Watari, A., Hanada, T., Habu, T., Yaegaki, H., Yamaguchi, M. & Yamane, H. 2007 Self-compatible peach (Prunus persica) has mutant versions of the S haplotypes found in self-incompatible Prunus species Plant Mol. Biol. 63 109 123

    • Search Google Scholar
    • Export Citation
  • Ushijima, K., Sassa, H., Dandekar, A.M., Gradziel, T.M., Tao, R. & Hirano, H. 2003 Structural and transcriptional analysis of the self-incompatibility locus of almond: Identification of a pollen-expressed F-Box gene with haplotype-specific polymorphism Plant Cell 15 771 781

    • Search Google Scholar
    • Export Citation
  • Ushijima, K., Yamane, Y., Watari, A., Kakehi, E., Ikeda, K., Huak, R.H., Iezzoni, A.F. & Tao, R. 2004 The S-haplotype-specific F-Box protein gene, SFB, is defective in self-compatible haplotypes of Prunus avium and P. mume Plant J. 39 573 586

    • Search Google Scholar
    • Export Citation
  • Vaughan, S.P., Russell, K., Sargent, D.J. & Tobutt, K.R. 2006 Isolation of S-locus F-box alleles in Prunus avium and their application in a novel method to determine self-incompatibility genotypes Theor. Appl. Genet. 112 856 866

    • Search Google Scholar
    • Export Citation
  • Vilanova, S., Badenes, M.L., Burgos, L., Martínez-Calvo, J., Llácer, G. & Romero, C. 2006 Self-compatibility of two apricot selections is associated with two pollen part mutations of different nature Plant Physiol. 142 629 641

    • Search Google Scholar
    • Export Citation
  • Watari, A., Hanada, T., Yamane, H., Esumi, T., Tao, R., Yaegaki, H., Yamaguchi, M., Beppu, B. & Kataoka, I. 2007 A low transcriptional level of Se-RNase in the Se-haplotype confers self-compatibility in japanese plum J. Amer. Soc. Hort. Sci. 132 396 406

    • Search Google Scholar
    • Export Citation
  • Yamane, H., Ikeda, K., Hauck, N.R., Iezzoni, A.F. & Tao, R. 2003 Self-incompatibility (S) locus region of the mutated S6-haplotype of sour cherry (Prunus cerasus) contains a functional pollen S allele and non-functional pistil allele J. Expt. Bot. 54 2431 2437

    • Search Google Scholar
    • Export Citation

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Contributor Notes

This work was supported by the Spanish Interministerial Commission for Science and Technology (CICYT; grant no. AGL2007-65853-C02-02) and by research line L38 of the Instituto de Tecnologia Química e Biológica (Portugal). O. Kodad gratefully acknowledges a scholarship from the Spanish National Institute for Agricultural and Food Research and Technology (INIA) and from the Consolidated Research Group A12 of Aragón.

We thank O. Frontera and J. Búbal for their technical assistance.

Corresponding author. E-mail: rsocias@aragon.es.

  • View in gallery

    Multiple alignment of the deduced amino acid sequence of the almond Sf allele identified in the present study and those deposited in the database. The conserved regions (C1–C5) and the hypervariable region (RHV) described in Rosaceae (Ushijima et al., 1998) are boxed. Conserved cysteine and histidine residues are shadowed. European Molecular Biology Laboratory Nucleotide Sequence database (EMBL/GenBank) accession numbers are as follows: Sf of ‘Tuono’ a (AM690356; Bošković et al., 2007); Sf of ‘Tuono’ b (AF157009; Ma and Oliveira, 2001); Sf of ‘Cambra’ (EU684318); Sf of IRTA selection 12–2 (AY291117; Channuntapipat et al., 2001); Sfa of ‘Ponç’ (EU293146); and S30 of ‘Fra Giulio Grande’ (AM690361; Bošković et al., 2007).

  • View in gallery

    Zymogram of almond S alleles after NEpHGE showing two bands for ‘Ponç’ compared to a single band for the self-compatible ‘Tuono’ and selection G-2–22.

  • View in gallery

    Alignment of the deduced almond SFBf amino acid sequences. The F-box motif, the variable regions V1 and V2, and the hypervariable regions HVa and HVb are boxed (Ikeda et al., 2004). European Molecular Biology Laboratory Nucleotide Sequence database (EMBL/GenBank) accession numbers are as follows: SFB30 of ‘Fra Giulio Grande’ (AM711127; Bošković et al., 2007); SFBf of ‘Tuono’ (AM711126; Bošković et al., 2007); SFBf of ‘Lauranne’ (AB361036); and SFBf of ‘Ponç’ (EU310402).

  • Alonso, J.M. & Socias i Company, R. 2005 Self-compatibility expression in self-compatible almond genotypes may be due to inbreeding J. Amer. Soc. Hort. Sci. 130 868 869

    • Search Google Scholar
    • Export Citation
  • Alonso, J.M. & Socias i Company, R. 2007 Negative inbreeding effects in tree fruit breeding: Self-compatibility transmission in almond Theor. Appl. Genet. 115 151 158

    • Search Google Scholar
    • Export Citation
  • Bošković, R. & Tobutt, K.R. 1996 Correlation of stylar ribonuclease zymograms with incompatibility alleles in sweet cherry Euphytica 90 245 250

  • Bošković, R., Tobutt, K.R., Ortega, E., Sutherland, B.C. & Godini, A. 2007 Self-(in)compatibility of the almond P. dulcis and P. webbii: Detection and cloning of ‘wild-type Sf’ and new self-compatibility alleles encoding inactive S-RNases Mol. Genet. Genomics 278 665 676

    • Search Google Scholar
    • Export Citation
  • Bošković, R., Tobutt, K.R., Duval, H., Batlle, I., Dicenta, F. & Vargas, J.F. 1999 A stylar ribonucleases assay to detect self-compatible seedlings in almond progenies Theor. Appl. Genet. 99 800 810

    • Search Google Scholar
    • Export Citation
  • Bošković, R., Tobutt, K.R., Batlle, I., Duval, H., Martínez-Gómez, P. & Gradziel, T.M. 2003 Stylar ribonuclease in almond: Correlation with and prediction of incompatibility genotypes Plant Breed. 122 70 76

    • Search Google Scholar
    • Export Citation
  • Channuntapipat, C., Sedgley, M. & Collins, G. 2001 Sequences of cDNAs and genomic DNAs encoding the S1, S7, S8 and Sf alleles from almond, Prunus dulcis Theor. Appl. Genet. 103 1115 1122

    • Search Google Scholar
    • Export Citation
  • Crane, M.B. & Lawrence, W.J.C. 1929 Genetical and cytological aspects of incompatibility and sterility in cultivated fruits J. Pomol. Hort. Sci. 7 276 301

    • Search Google Scholar
    • Export Citation
  • Crossa-Raynaud, P. & Grasselly, C. 1985 Éxistence de groupes d'interstérilite chez l'amandier Options Méditerranéennes CIHEAM/IAMZ 85 1 43 45

  • Doyle, J.J. & Doyle, J.L. 1987 A rapid DNA isolation procedure for small quantities of fresh tissue Phytochem. Bul. 19 11 15

  • Entani, T., Iwano, M., Shiba, H., Che, F.S., Isogai, A. & Takayama, S. 2003 Comparative analysis of the self-incompatibility (S-) locus region of Prunus mume: Identification of a pollen-expressed F-box gene with allelic diversity Genes Cells 8 203 213

    • Search Google Scholar
    • Export Citation
  • Espiau, M.T., Ansón, J.M. & Socias i Company, R. 2002 The almond germplasm bank of Zaragoza Acta Hort. 591 275 278

  • Felipe, A.J. 1977 Almendro. Estados fenológicos Información Técnica Económica Agraria 27 8 9

  • Hanada, T., Fukuta, K., Yamane, H., Tao, R., Alonso, J.M. & Socias i Company, R. 2009 Cloning of self-compatible Sf locus in almond Acta Hort. in press

  • Huang, S., Lee, H.S., Karunanandaa, B. & Kao, T.H. 1994 Ribonuclease activity of Petunia inflata S proteins is essential for rejection of self-pollen Plant Cell 6 1021 1028

    • Search Google Scholar
    • Export Citation
  • Huang, S.X., Wu, H.Q., Li, Y.R., Wu, J., Zhang, S.J., Heng, W. & Zhang, S.L. 2008 Competitive interaction between two functional S-haplotypes confer self-compatibility on tetraploid Chinese cherry (Prunus pseudocerasus Lindl. cv. Nanjing Chuisi) Plant Cell Rep. 27 1075 1085

    • Search Google Scholar
    • Export Citation
  • Ikeda, K., Igic, B., Ushijima, K., Yamane, H., Hauck, N.R., Nakano, R., Sassa, H., Iezzoni, A.F., Kohn, J.R. & Tao, R. 2004 Primary structural features of the S haplotype-specific F-box protein, SFB, in Prunus Sex. Plant Reprod. 16 235 243

    • Search Google Scholar
    • Export Citation
  • Ikeda, K., Ushijima, K., Yamane, H., Tao, R., Hauck, N.R., Sebolt, A.M. & Iezzoni, A.F. 2005 Linkage and physical distances between the S-haplotype S-RNase and SFB genes in sweet cherry Sex. Plant Reprod. 17 289 296

    • Search Google Scholar
    • Export Citation
  • Kao, T.-H. & Tsukamato, T. 2004 The molecular and genetic bases of S-RNase-based self-incompatibility Plant Cell 16 72 83

  • Kester, D.E. & Griggs, W.H. 1959 Fruit setting in the almond: The effect of cross-pollinating various percentages of flowers Proc. Amer. Soc. Hort. Sci. 74 214 219

    • Search Google Scholar
    • Export Citation
  • Kodad, O. & Socias i Company, R. 2006 Pollen source effect on pollen tube growth in advanced self-compatible almond selections (Prunus amygdalus Batsch) Adv. Hort. Sci. 20 256 261

    • Search Google Scholar
    • Export Citation
  • Kodad, O. & Socias i Company, R. 2008 Fruit set evaluation for self-compatibility selection in almond Scientia Hort. 118 260 265

  • Kodad, O., Alonso, J.M., Sánchez, A., Oliveira, M.M. & Socias i Company, R. 2008 Evaluation of genetic diversity of S-alleles in an almond germplasm collection J. Hort. Sci. Biotechnol. 83 603 608

    • Search Google Scholar
    • Export Citation
  • Kowyama, Y., Kunz, C., Lewis, I., Newbigin, E., Clarke, A.E. & Anderson, M.A. 1994 Self-compatibility in a Lycopersicon peruvianum variant (LA2157) is associated with a lack of style S-RNase activity Theor. Appl. Genet. 88 859 864

    • Search Google Scholar
    • Export Citation
  • Ma, R.C. & Oliveira, M.M. 2001 Molecular cloning of the self-incompatibility genes S1 and S3 from almond (Prunus dulcis) cv. Ferragnès Sex. Plant Reprod. 14 163 167

    • Search Google Scholar
    • Export Citation
  • McClure, B.A., Haring, V., Ebert, P.R., Anderson, M.A., Simpson, R.J., Sakiyama, F. & Clarke, A.E. 1989 Style self-incompatibility gene products of Nicotiana alata are ribonucleases Nature 342 955 957

    • Search Google Scholar
    • Export Citation
  • Ortega, E., Bošković, R., Sargent, D.J. & Tobutt, K.R. 2006 Analysis of S-RNase alleles of almond (Prunus dulcis): Characterization of new sequences, resolution of synonyms and evidence of intragenic recombination Mol. Genet. Genomics 276 413 426

    • Search Google Scholar
    • Export Citation
  • Qin, X., Liu, B., Soulard, J., Morse, D. & Cappadocia, M. 2006 Style by style analysis of two sporadic self-compatible Solanum chacoense lines supports a primary role for S-RNase in determining pollen rejection thresholds J. Expt. Bot. 57 2001 2013

    • Search Google Scholar
    • Export Citation
  • Royo, J., Kunz, C., Kowyama, Y., Anderson, M., Clarke, A.E. & Newbigin, E. 1994 Loss of a histidine residue at the active site of S-locus ribonuclease is associated with self-compatibility in Lycopersicon peruvianum Proc. Natl. Acad. Sci. USA 91 6511 6514

    • Search Google Scholar
    • Export Citation
  • Rubí, V. 1980 El almendro Delegación de Baleares, Ministerio de Agricultura Palma de Mallorca, Spain

    • Export Citation
  • Sassa, H., Hirano, H. & Ikehashi, H. 1992 Self-incompatibility-related RNases in styles of japanese pear (Pyrus serotina Rehd.) Plant Cell Physiol. 33 811 814

    • Search Google Scholar
    • Export Citation
  • Sassa, H., Hirano, H., Nishio, T. & Koba, T. 1997 Style-specific self-incompatibility mutation caused by deletion of the S-RNase gene in japanese pear (Pyrus serotina) Plant J. 12 223 227

    • Search Google Scholar
    • Export Citation
  • Socias i Company, R. 1984 Genetic approach to the transmission of self-compatibility in almond (Prunus amygdalus Batsch) Options Méditerranéennes CIHEAM/IAMZ 84 1 123 127

    • Search Google Scholar
    • Export Citation
  • Socias i Company, R. 1990 Breeding self-compatible almonds Plant Breed. Rev. 8 313 318

  • Socias i Company, R. 2002 Latest advances in almond self-compatibility Acta Hort. 591 205 212

  • Socias i Company, R. 2004 The contribution of Prunus webbii to almond evolution Plant Genet. Resour. Newsl. 14 9 13

  • Socias i Company, R. & Felipe, A.J. 1999 ‘Blanquerna’, ‘Cambra’, y ‘Felisia’: Tres nuevos cultivares autógamos de almendro. Información Técnica Económica Agraria 95 5 111 117

    • Search Google Scholar
    • Export Citation
  • Socias i Company, R., Kester, D.E. & Bradley, M.V. 1976 Effects of temperature and genotype on pollen tube growth of some self-compatible and self-incompatible almond cultivars J. Amer. Soc. Hort. Sci. 101 490 493

    • Search Google Scholar
    • Export Citation
  • Sonneveld, T., Tobutt, K.R. & Robbins, T.P. 2003 Allele-specific PCR detection of sweet cherry self-incompatibility (S) alleles S1 to S16 using consensus and allele-specific primers Theor. Appl. Genet. 107 1059 1070

    • Search Google Scholar
    • Export Citation
  • Sutherland, B.G., Robbins, T.P. & Tobutt, K.R. 2004 Primers amplifying a range of Prunus S-alleles Plant Breed. 123 582 584

  • Tamura, M., Ushijima, K., Sassa, H., Hirano, H., Tao, R., Gradziel, T.M. & Dandekar, A.M. 2000 Identification of self-incompatibility genotype of almond by allele-specific PCR analysis Theor. Appl. Genet. 101 344 349

    • Search Google Scholar
    • Export Citation
  • Tao, R., Watari, A., Hanada, T., Habu, T., Yaegaki, H., Yamaguchi, M. & Yamane, H. 2007 Self-compatible peach (Prunus persica) has mutant versions of the S haplotypes found in self-incompatible Prunus species Plant Mol. Biol. 63 109 123

    • Search Google Scholar
    • Export Citation
  • Ushijima, K., Sassa, H., Dandekar, A.M., Gradziel, T.M., Tao, R. & Hirano, H. 2003 Structural and transcriptional analysis of the self-incompatibility locus of almond: Identification of a pollen-expressed F-Box gene with haplotype-specific polymorphism Plant Cell 15 771 781

    • Search Google Scholar
    • Export Citation
  • Ushijima, K., Yamane, Y., Watari, A., Kakehi, E., Ikeda, K., Huak, R.H., Iezzoni, A.F. & Tao, R. 2004 The S-haplotype-specific F-Box protein gene, SFB, is defective in self-compatible haplotypes of Prunus avium and P. mume Plant J. 39 573 586

    • Search Google Scholar
    • Export Citation
  • Vaughan, S.P., Russell, K., Sargent, D.J. & Tobutt, K.R. 2006 Isolation of S-locus F-box alleles in Prunus avium and their application in a novel method to determine self-incompatibility genotypes Theor. Appl. Genet. 112 856 866

    • Search Google Scholar
    • Export Citation
  • Vilanova, S., Badenes, M.L., Burgos, L., Martínez-Calvo, J., Llácer, G. & Romero, C. 2006 Self-compatibility of two apricot selections is associated with two pollen part mutations of different nature Plant Physiol. 142 629 641

    • Search Google Scholar
    • Export Citation
  • Watari, A., Hanada, T., Yamane, H., Esumi, T., Tao, R., Yaegaki, H., Yamaguchi, M., Beppu, B. & Kataoka, I. 2007 A low transcriptional level of Se-RNase in the Se-haplotype confers self-compatibility in japanese plum J. Amer. Soc. Hort. Sci. 132 396 406

    • Search Google Scholar
    • Export Citation
  • Yamane, H., Ikeda, K., Hauck, N.R., Iezzoni, A.F. & Tao, R. 2003 Self-incompatibility (S) locus region of the mutated S6-haplotype of sour cherry (Prunus cerasus) contains a functional pollen S allele and non-functional pistil allele J. Expt. Bot. 54 2431 2437

    • Search Google Scholar
    • Export Citation
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