Cloning and Characterization of a Self-compatible Sf Haplotype in Almond [Prunus dulcis (Mill.) D.A. Webb. syn. P. amygdalus Batsch] to Resolve Previous Confusion in Its Sf-RNase Sequence

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  • 1 Graduate School of Agriculture, Kyoto University, Oiwake-cho, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan
  • | 2 Department of Plant Science, University of California, Davis, CA 95616
  • | 3 Unidad de Fruticultura, CITA de Aragón, Av. Montañana 930, 50059 Zaragoza, Spain

Most of the self-compatible (SC) cultivars of almond [Prunus dulcis (Mill.) D.A. Webb. syn. P. amygdalus Batsch] have the Sf haplotype. In this study, we cloned and characterized the S locus region of the Sf haplotype of SC ‘Lauranne’. The relative transcriptional orientation of SFBf and Sf-RNase and the physical distance between them are similar to those of other functional self-incompatible (SI) S haplotypes of Prunus, indicating that the genomic structure of the SC Sf haplotype appears to be intact. Although there is no apparent mutation in the coding sequence of SFBf, the Sf-RNase sequence in this study and previously reported Sf-RNase sequences show discrepancies. First, as opposed to previous indications, the ‘Lauranne’ Sf-RNase sequence encodes a histidine residue in place of a previously reported arginine residue in the conserved C2 region of Prunus S-RNase. Direct sequencing of the polymerase chain reaction products from the Sf-RNase of ‘Tuono’ confirmed that ‘Tuono’ Sf-RNase also encodes the histidine residue. We found another difference in the ‘Lauranne’ Sf-RNase sequence and other reported Sf-RNase sequences. Namely, ‘Lauranne’ Sf-RNase encodes a phenylalanine residue in place of a previously reported leucine residue in the conserved C5 region of Prunus S-RNase. This is also the case for ‘Tuono’ Sf-RNase. Expression analysis of Sf-RNase and SFBf by reverse transcriptase–polymerase chain reaction showed that Sf-RNase transcripts were barely detectable in pistil, whereas SFBf transcripts were accumulated at a similar level to the level that was observed with SFB of other functional SI S haplotypes of almond. We discuss the possible molecular mechanisms of SC observed with the Sf haplotype with special references to the expression of Sf-RNase.

Abstract

Most of the self-compatible (SC) cultivars of almond [Prunus dulcis (Mill.) D.A. Webb. syn. P. amygdalus Batsch] have the Sf haplotype. In this study, we cloned and characterized the S locus region of the Sf haplotype of SC ‘Lauranne’. The relative transcriptional orientation of SFBf and Sf-RNase and the physical distance between them are similar to those of other functional self-incompatible (SI) S haplotypes of Prunus, indicating that the genomic structure of the SC Sf haplotype appears to be intact. Although there is no apparent mutation in the coding sequence of SFBf, the Sf-RNase sequence in this study and previously reported Sf-RNase sequences show discrepancies. First, as opposed to previous indications, the ‘Lauranne’ Sf-RNase sequence encodes a histidine residue in place of a previously reported arginine residue in the conserved C2 region of Prunus S-RNase. Direct sequencing of the polymerase chain reaction products from the Sf-RNase of ‘Tuono’ confirmed that ‘Tuono’ Sf-RNase also encodes the histidine residue. We found another difference in the ‘Lauranne’ Sf-RNase sequence and other reported Sf-RNase sequences. Namely, ‘Lauranne’ Sf-RNase encodes a phenylalanine residue in place of a previously reported leucine residue in the conserved C5 region of Prunus S-RNase. This is also the case for ‘Tuono’ Sf-RNase. Expression analysis of Sf-RNase and SFBf by reverse transcriptase–polymerase chain reaction showed that Sf-RNase transcripts were barely detectable in pistil, whereas SFBf transcripts were accumulated at a similar level to the level that was observed with SFB of other functional SI S haplotypes of almond. We discuss the possible molecular mechanisms of SC observed with the Sf haplotype with special references to the expression of Sf-RNase.

Most of the fruit tree species in the genus Prunus (Rosaceae), including almond [Prunus dulcis (Mill.) D.A. Webb.], exhibit the S-RNase-based gametophytic self-incompatibility (GSI) system (de Nettancourt, 2001; Yamane and Tao, 2009). The GSI reaction in Prunus is controlled by the S locus, which contains the style-specific ribonuclease gene (S-RNase) (Tao et al., 1997; Ushijima et al., 1998) and the pollen-specific F-box protein gene (SFB) (Ushijima et al., 2003; Yamane et al., 2003) for the pistil and pollen specificities, respectively. Because the pistil and pollen determinant genes have been identified, the variants of the S-RNase and SFB combinations are called S haplotypes and the variants of a given S locus gene are called pistil and pollen S alleles.

Almond cultivars are largely self-incompatible (SI) (Tufts and Philip, 1922), although self-compatible (SC) cultivars exist (Socias i Company, 1990). Grasselly and Olivier (1976) reported that several SC cultivars such as Tuono, Filippo Ceo, Occhiorosso, and Genco were found among the almond population of the Italian region of Puglia, where P. webbii (Spach) Vierh. grows wild. Thus, most of the SC almond selections were considered to be derived from interspecific hybridization with SC P. webbii (Socias i Company, 2004). As a consequence, the SC Sf haplotype of ‘Tuono’ has been long thought to be derived from P. webbii. However, a recent finding of the almond SI S30 haplotype, a putative wild-type Sf haplotype, poses a question about this hypothesis (Bošković et al., 2007). The origin and molecular basis of SC in Sf haplotype is intriguing for future SC breeding programs in almond.

We previously sequenced the partial Sf-RNase sequence that was flanked by the Pru-C2 and Pru-C5 primer sequences that were designed from the conserved C2 and C5 regions, respectively, of rosaceous S-RNase (Tao et al., 1999; Ushijima et al., 1998) and the full coding sequence for SFBf (Hanada et al., 2009). No substantial differences or defects in the deduced amino acid sequence were found in the partial Sf-RNase and full-length SFBf sequences. Although our full-length SFBf sequence completely matches the partial SFBf sequences that were reported by other research groups, partial Sf-RNase sequences that were posted on the public database by several research groups, including our group, show differences (Barckley et al., 2006; Bošković et al., 2007; Channuntapipat et al., 2001; Ma and Oliveira, 2002). Although it appeared later that the Sf-RNase sequence reported by Barckley et al. (2006) could be from misannotated S1-RNase (synonymous to Sb-RNase) because their Sf-RNase sequence shows 100% match to S1-RNase of Tuono (S1Sf), there are still some minor differences among the Sf-RNase sequences reported by the different research groups. This led us to thoroughly reinvestigate the S locus of the Sf haplotype in almond. We, therefore, isolated fosmid clones that contained the Sf locus of the SC ‘Lauranne’ to determine the full coding sequence of the Sf-RNase and the structure of the S locus region of the Sf haplotype. Furthermore, we conducted expression analysis of S locus genes by reverse transcriptase–polymerase chain reaction (RT-PCR) and discuss the possible molecular mechanisms of SC observed with the Sf haplotype with special references to the expression of Sf-RNase.

Materials and Methods

Plant materials.

Young leaves, pistils, and pollen grains were collected from three almond cultivars, Lauranne (S3Sf), Ferragnès (S1S3), and Tuono (S1Sf), that were grown at the CITA de Aragón in Zaragoza, Spain. ‘Lauranne’ is from ‘Ferragnès’ × ‘Tuono’, and the Sf and S3 haplotypes in ‘Lauranne’ are estimated to be from ‘Tuono’ and ‘Ferragnès’, respectively. We also used young leaf samples from ‘Tuono’ that was grown at the University of California at Davis, CA. Leaf samples were collected, frozen in liquid nitrogen, lyophilized, and stored at –20 °C with desiccant until used.

Isolation of DNA.

Genomic DNA was isolated from young leaves using the Nucleon PhytoPure® plant and fungal DNA extraction kit (GE Healthcare, Piscataway, NJ) with some modifications as described previously (Watari et al., 2007). In brief, 0.5 g of freeze-dried leaves was ground to a powder with MULTI-BEADS SHOCKER® (YASUI KIKAI, Osaka, Japan), suspended in washing buffer [0.1 M HEPES (pH 8.0), 0.1% (w/v) PVP, 2% (v/v) 2-mercaptoethanol], and mixed thoroughly. The mixture was centrifuged (6500 × g at 4 °C for 15 min) to collect the pellet; the pellet was resuspended in washing buffer and centrifuged again. This partially purified nuclear fraction was used to isolate DNA with the Nucleon PhytoPure® plant and fungal DNA extraction kit.

Cloning and characterization of the Sf haplotype.

A fosmid library was constructed from the genomic DNA of ‘Lauranne’ (S3Sf) using the CopyControl Fosmid Library Production Kit (Epicentre, Madison, WI) as previously described (Ikeda et al., 2004; Ushijima et al., 2001). The library was screened with a DIG-dUTP-labeled (Roche Diagnostics, Basel, Switzerland) cDNA probe corresponding to the coding sequence between the conserved C2 and C4 regions of rosaceous S-RNase (Tao et al., 1999; Ushijima et al., 1998). A genomic clone that contained the S locus of the Sf haplotype was selected and used for further studies. DNA sequences of both strands of the Sf-RNase were determined by primer walking using the BigDye Terminator Version 3.1 Cycle Sequencing Kit and a 3730xl DNA analyzer (Applied Biosystems, Foster City, CA).

The transcriptional orientation and physical distance between Sf-RNase and SFBf were determined by long PCR using an Expand Long Template PCR system (Roche Applied Science, Mannheim, Germany) as previously described (Watari et al., 2007). Each reaction consisted of 1× Expand Long Template buffer 3.5 mm of each dNTP, 2.75 mm MgCl2, 300 nM of each primer, and 25 ng of fosmid DNA as a template, and 1 U of Expand Long Template Enzyme mix in a 15-μL reaction volume. Long PCR was performed using a program with an initial denaturing step at 94 °C for 2 min, 10 cycles of 94 °C for 10 s, 58 °C for 30 s, 68 °C for 15 min, 20 cycles of 94 °C for 10 s, 58 °C for 30 s, 68 °C for 15 min with an extension of an extra 20 s after each cycle, and final extension at 68 °C for 7 min. Three different primer combinations were used: Pru-C2 (5′-CTA TGG CCA AGT AAT TAT TCA AAC C-3′) and SFB C5F (5′-TAC CAY WTM ATT GAG AAA GGT CC-3′), Pru-C2 and rtPd_SFBf_F (5′-TGA TTA CGA CTC CAA GCC AAC TC-3′), and rtPd_Sf_F (5′-CCT CCT TCG TTG CAA AGG AA-3′) and SFB C5F. Primer positions are shown in Figure 1. Amplified fragments were separated by 0.5% agarose gel electrophoresis and visualized with ethidium bromide under ultraviolet light. Amplified fragments were also purified using ExoSAP-IT (USB, Cleveland, OH) and sequenced by the DTCS Quick Start kit and CEQ 8000 Genetic Analysis System (Beckman Coulter, Fullerton, CA).

Fig. 1.
Fig. 1.

Schematic diagrams illustrating Sf-RNase and SFBf gene organization in the S locus region of the Sf haplotype. The nucleotide “A” of the start codons of Sf-RNase and SFBf are at +1. Gray boxes represent exons of the S-RNase and SFB. Oligonucleotide primers used to amplify the regions A, B, and C that include the intergeneric region between the Sf-RNase and SFBf are indicated by open triangles with the primer names. The agarose gel image of the amplified products is shown below. Lanes A, B, and C show the polymerase chain reaction products from the regions A, B, and C, respectively.

Citation: HortScience horts 44, 3; 10.21273/HORTSCI.44.3.609

DNA sequences encoding the region around the conserved C2 and C5 regions (Fig. 2) of the Sf-RNase of ‘Tuono’ were amplified by PCR using two primer sets, AS1 II (5′-TAT TTT CAA TTT GTG CAA CAA TGG-3′) and PdSf_2intR (5′-TAA ATG CCA AAT AGT A-3′), and PdSf_F1 (5′-AAA GCA GCA ACT CAA AGA ATA C-3′) and PdSf_R2 (5′-AAA AAG ATA AGA AAC AAA AGA C-3′), respectively (Fig. 3). All PCR reactions contained 1× ExTaq buffer, 200 μM of each dNTP, 400 nM of each primer, 50 ng of template genomic DNA, and 0.5 U of TaKaRa ExTaq polymerase (TaKaRa Bio, Ohtsu, Japan) in a 25-μL reaction volume. PCR was performed using a program with an initial denaturing step at 94 °C for 1 min, 35 cycles of 94 °C for 1 min, 58 °C for 1 min, 72 °C for 2 min, and a final extension at 72 °C for 7 min. Amplified fragments were purified by ExoSAP-IT and sequenced using DTCS Quick Start kit and CEQ 8000 genetic Analysis System.

Fig. 2.
Fig. 2.

Deduced amino acid sequence alignment of the reported SC Sf-RNases and the SI S30-RNase from P. dulcis and P. webbii. The deduced amino acid sequences were aligned using Clustal W. Conserved regions C1, C2, C3, RC4, and C5 and the rosaceous hypervariable region are boxed (Ushijima et al., 1998). The positions of the conserved histidine residues essential for catalytic activity are marked with open triangles. An open circle shows the phenylalanine residue that is different from the previously reported Sf-RNase sequences. Gaps are marked by dashes. Asterisks indicate positions that have a single, fully conserved residue. Abbreviations and EMBL accession numbers are as shown in Table 2.

Citation: HortScience horts 44, 3; 10.21273/HORTSCI.44.3.609

Fig. 3.
Fig. 3.

Direct polymerase chain reaction (PCR) sequencing of the C2 (I) and C5 (II) regions of the Sf-RNase of ‘Tuono’. The determined ‘Tuono’ sequences are identical to the sequences of the respective portions of the Sf-RNase of ‘Lauranne’. Coding region and intron sequences are indicated by bold type and italics, respectively. Deduced amino acid sequences are shown under the nucleotide sequences. Boxed sequences indicate conserved regions of Prunus S-RNase. PCR primer name and directions are indicated by arrows.

Citation: HortScience horts 44, 3; 10.21273/HORTSCI.44.3.609

Reverse transcriptase–polymerase chain reaction.

Total RNA was isolated from pistils and pollen grains from ‘Lauranne’, ‘Tuono’, and ‘Ferragnès’ by cold phenol method and first-strand cDNAs were synthesized by SuperScript III RT (Invitrogen, Carlsbad, CA) with adapter-dT primer as described (Watari et al., 2007). Gene-specific primers for S1-RNase, Sf-RNase, SFB1, and SFBf were designed from Sf-RNase (AB433984), SFBf (AB361036), Sb-RNase (AB011469), and SFBb (AB092967) sequences. Almond Sb-RNase and SFBb are synonyms of almond S1-RNase and SFB1, respectively (Channuntapipat et al., 2001). The primer combinations used to amplify the RT-PCR products from S1-RNase, Sf-RNase, SFB1, and SFBf were Pru-T2 and rtPd_SbR, Pru-T2 and rtPd_SfR, Pd_SFBb_F1 and Pd_SFBb_R1, and Pd_SFBf_F1 and Pd_SFBf_R1, respectively (Table 1). As references, RT-PCR for actin gene was also conducted using the ActF1 and ActR1 primers. The PCR mixture contained 1× ExTaq buffer, 200 μM each of dNTPs, 400 nM each of primers, 20 ng of template cDNA, and 0.5 U of TaKaRa ExTaq polymerase (TaKaRa Bio). PCR for S-RNase was performed using a program of 30 cycles of 94 °C for 30 s, 56 °C for 30 s, and 72 °C for 1 min 30 s with an initial denaturation step at 94 °C for 3 min and a final extension at 72 °C for 7 min. PCR for SFB was performed using a program of 30 cycles of 94 °C for 30 s, 53 °C for 30 s, and 72 °C for 1 min 30 s with an initial denaturation step at 94 °C for 3 min and a final extension at 72 °C for 7 min. PCR condition for actin is exactly the same as that described in Yamane et al. (2003). Amplified PCR products were separated by electrophoresis on 1.5% agarose gels, stained with ethidium bromide, and visualized under ultraviolet light.

Table 1.

DNA sequences of oligonucleotide primers used for reverse transcriptase–polymerase chain reaction analysis.

Table 1.

Results

Several positive clones were obtained from the fosmid library constructed from ‘Lauranne’ (S3Sf). With Prunus S-RNase-specific (Tao et al., 1999) and SFB-specific (Yamane et al., 2003) PCRs, we selected several clones that contained full-length Sf-RNase and SFBf sequences. Long PCR conducted using one of the fosmid clones yielded three different length of fragments (A:7.6k, B:7.3k, C:6.3k) with three primer pairs, respectively (Fig. 1). Based on the length of amplified products, the physical distance between Sf-RNase and SFBf was estimated to be ≈6 kb with opposite relative transcriptional orientations. The coding region of Sf-RNase was completely sequenced, and the deduced amino acid sequence was aligned with other reported Sf-RNase and S30-RNase sequences (Table 2; Fig. 2). The five conserved regions (C1, C2, C3, RC4, and C5) and a rosaceous hypervariable region (Ushijima et al., 1998) were present in the Sf-RNase sequence predicted from the genomic clone for Sf-RNase. To date, several different sequences of Sf-RNase from different almond cultivars and P. webbii accessions have been reported (Bošković et al., 2007; Channuntapipat et al., 2001; Ma and Oliveira, 2002). Furthermore, S30-RNase was recently identified in the SI S30 haplotype, and its sequence indicated that it was the wild-type Sf-RNase (Bošković et al., 2007) (Fig. 2). Compared with these sequences, the ‘Lauranne’ Sf-RNase sequence showed several differences. First, the ‘Lauranne’ Sf-RNase sequence had a conserved histidine residue in the C2 region and there was no indication of replacement of the histidine residue with an arginine residue. Direct sequencing of the PCR products from the Sf-RNase of ‘Tuono’ genomic DNA also indicated no replacement of the conserved histidine residue in the C2 region of the two ‘Tuono’ clones from CITA in Spain and the University of California at Davis in the United States (Fig. 3). We found another difference in the ‘Lauranne’ Sf-RNase sequence and other reported Sf-RNase sequences. Namely, the ‘Lauranne’ Sf-RNase encodes a phenylalanine residue in place of a previously reported leucine residue in the C5 region. DNA sequencing of the PCR fragment amplified from ‘Tuono’ genomic DNA revealed that the two clones of ‘Tuono’ Sf-RNase also encoded a phenylalanine residue in place of a leuicine residue (Fig. 3).

Table 2.

Reported Sf and S30 RNases from P. dulcis and P. webbii

Table 2.

Gene-specific primers for SFBf, Sf-RNase, SFB1, and S1-RNase that were developed in this study (Table 1) could specifically amplify the respective genes among the three target S haplotypes, S1, S3, and Sf of ‘Lauranne’ (S3Sf), ‘Tuono’ (S1Sf), and ‘Ferragnès’ (S1S3) (Fig. 4). In the expression analysis for SFB, SFB1, fragments were specifically amplified from the genomic DNA and pollen cDNA of ‘Ferragnès’ and ‘Tuono’, whereas no amplification was observed either with the genomic DNA or pollen cDNA of ‘Lauranne’. Similarly, SFBf fragment was amplified from the genomic DNA and pollen cDNA of ‘Tuono’ and ‘Lauranne’, whereas no amplification was observed either with the genomic DNA or pollen cDNA of ‘Ferragnès’. As expected, SFB showed pollen-specific gene expression and no amplification was observed with stylar cDNA of all three cultivars tested. In terms of the size of PCR products, there is no defect in SFBf. In the expression analysis for S-RNase, S1-RNase fragments were amplified from the genomic DNA and style cDNA of ‘Ferragnès’ and ‘Tuono’, whereas no amplification was observed either from the genomic DNA or style cDNA of ‘Lauranne’. As expected, S-RNase shows pistil-specific expression, and no amplification was observed from pollen cDNA. Sf-RNase fragment was amplified from the genomic DNA of ‘Tuono’ and ‘Lauranne’, whereas no amplification was observed from stylar cDNA of ‘Tuono’ and ‘Lauranne’ under our PCR condition. It appeared that Sf-RNase transcripts were not accumulated at the detectable level by our RT-PCR conditions in the styles of ‘Lauranne’ and ‘Tuono’.

Fig. 4.
Fig. 4.

Expression analysis of SFB1, SFBf, S1-RNase, Sf-RNase, and actin gene of almond. Polymerase chain reaction (PCR) was performed with cDNA from pollen (P), style (S), and genomic DNA (G) of ‘Ferragnès’ (FER), ‘Tuono’ (TUO), and ‘Lauranne’ (LAU). Because there is no intron sequence in the coding region of SFB, PCR products from SFB from genomic DNA show the same size as those from cDNA. Because PCR for S-RNase was designed to amplify the region containing an intron, amplified products from cDNA and genomic DNA showed different sizes. Arrow indicates the position where PCR products from cDNA for Sf-RNase should be located.

Citation: HortScience horts 44, 3; 10.21273/HORTSCI.44.3.609

Discussion

SC in SI Prunus species is usually conferred by mutated dysfunctional S haplotypes (Hauck et al., 2006; Tao et al., 2007; Tsukamono et al., 2006; Ushijima et al., 2004), although SC associated with the pollen-part general factor has been reported in sweet cherry (Wünsch and Hormaza, 2004) and apricot (Vilanova et al., 2006). In almond, the Sf haplotype is thought to be a mutated SC S haplotype because cosegregation of the SC and the Sf haplotype has been described (Bošković et al., 1999). The Sf haplotype could be a stylar part mutant, because RNase activity gel assays indicated that the Sf-RNase of the Sf haplotype encoded no RNase activity. This conjecture is further confirmed by a pollination study conducted by Bošković et al. (2007) showing that Sf pollen tube growth was rejected in the style of the S30 haplotype, a putative wild type of the Sf haplotype.

Bošković et al. (2007) compared almond Sf-RNase and S30-RNase sequences and found a replacement of the conserved histidine residue in the C2 region of Sf-RNase with an arginine residue to which they ascribed the SC observed with Sf-RNase. Although this argument is quite reasonable because the replaced histidine residue is one of the two histidine residues essential for T2/S-type RNase activity, our sequencing results pose a question regarding this argument. As opposed to the previous studies, we found a conserved histidine residue in the Sf-RNase sequences from ‘Lauranne’ and the two clones of ‘Tuono’. Although it is unknown why this kind of discrepancy occurred, our finding calls into question the previously reported molecular basis of SC in the Sf haplotype (Bošković et al., 2007).

There is another discrepancy in the C5 region between the previously reported Sf-RNase and our Sf-RNase sequences. Namely, our Sf-RNase sequences encode a phenylalanine residue in place of the previously reported leucine residue in the C5 region. This replacement in the C5 region was not described in the previous studies, probably because this region is used as a primer to clone the Sf-RNase fragment and the sequence was mistakenly reported. Although most Prunus S-RNases have a leuicine residue at this position in the C5 region, we propose that this replacement does not affect the functionality of the Sf-RNase because the fully functional S2-RNase of the S2 haplotype of apricot have a phenylalanine residue at that position (Romero et al., 2004).

Expression analysis by RT-PCR indicated that Sf-RNase transcripts were not accumulated at the detectable level in style in our PCR condition. To confirm there were no Sf-RNase transcripts in the style, we isolated total RNA and conducted RT-PCR several times. On one occasion, a trace level of transcripts was detected from ‘Tuono’ style when the PCR cycles were increased to 35 cycles (data not shown). It may be possible, therefore, that transcription of Sf-RNase was not completely shut down but the transcripts are present at a very low level in the style. Because the transcriptional level of Prunus S-RNase is very high and usually more amplification products were obtained from stylar cDNA than genomic DNA, we conclude that the transcription level of Sf-RNase is not enough for a SI reaction. This could be supported by the fact that no Sf-RNase activity was found in the RNase activity gel (Bošković et al., 1999), although our data do not support Bošković et al. (2007), in which no Sf-RNase activity was ascribed to the replacement of the histidine residue with the arginine residue in the C2 region. Although it is unlikely that the replacement found in the coding region for the C5 region in Sf-RNase affects its transcription, it is intriguing to investigate if S30-RNase has this replacement in the C5 region.

Further comparisons of the Sf haplotype and the original SI S30 haplotype could clarify the molecular basis of SC with the Sf haplotype and could lead to the development of DNA markers to distinguish them for future SC breeding in almond. However, because it has been suggested that the possession of the Sf haplotype does not necessarily result in SC, possibly because of inbreeding depression (Alonso and Socias i Company, 2005), early detection of SC seedlings should take this inbreeding effect into consideration.

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  • Ushijima, K., Sassa, H., Tao, R., Yamane, H., Dandekar, A.M., Gradziel, T.M. & Hirano, H. 1998 Cloning and characterization of cDNAs encoding S-RNases from almond (Prunus dulcis): Primary structural features and sequence diversity of the S-RNases in Rosaceae Mol. Gen. Genet. 260 261 268

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  • Ushijima, K., Yamane, H., Watari, A., Kakehi, E., Ikeda, K., Hauck, N.R., 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

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  • 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

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  • Watari, A., Hanada, T., Yamane, H., Esumi, T., Tao, R., Yaegaki, H., Yamaguchi, M., Beppu, K. & 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

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  • Wünsch, A. & Hormaza, J.I. 2004 Genetic and molecular analysis in Cristobalina sweet cherry, a spontaneous self-compatible mutant Sex. Plant Reprod. 17 203 210

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  • Yamane, H., Ikeda, K., Ushijima, K., Sassa, H. & Tao, R. 2003 A pollen-expressed gene for a novel protein with an F-box motif that is very tightly linked to a gene for S-RNase in two species of cherry, Prunus cerasus and P. avium Plant Cell Physiol. 44 764 769

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  • Yamane, H. & Tao, R. 2009 Molecular basis of self-(in)compatibility and current status of S-genotyping in rosaceous fruit trees J. Jpn. Soc. Hort. Sci. (submitted).

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Contributor Notes

This work was supported by a Grant-in-Aid (no. 20248004) for Scientific Research (A) to R.T.

To whom reprint requests should be addressed; e-mail rtao@kais.kyoto-u.ac.jp.

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    Schematic diagrams illustrating Sf-RNase and SFBf gene organization in the S locus region of the Sf haplotype. The nucleotide “A” of the start codons of Sf-RNase and SFBf are at +1. Gray boxes represent exons of the S-RNase and SFB. Oligonucleotide primers used to amplify the regions A, B, and C that include the intergeneric region between the Sf-RNase and SFBf are indicated by open triangles with the primer names. The agarose gel image of the amplified products is shown below. Lanes A, B, and C show the polymerase chain reaction products from the regions A, B, and C, respectively.

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    Deduced amino acid sequence alignment of the reported SC Sf-RNases and the SI S30-RNase from P. dulcis and P. webbii. The deduced amino acid sequences were aligned using Clustal W. Conserved regions C1, C2, C3, RC4, and C5 and the rosaceous hypervariable region are boxed (Ushijima et al., 1998). The positions of the conserved histidine residues essential for catalytic activity are marked with open triangles. An open circle shows the phenylalanine residue that is different from the previously reported Sf-RNase sequences. Gaps are marked by dashes. Asterisks indicate positions that have a single, fully conserved residue. Abbreviations and EMBL accession numbers are as shown in Table 2.

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    Direct polymerase chain reaction (PCR) sequencing of the C2 (I) and C5 (II) regions of the Sf-RNase of ‘Tuono’. The determined ‘Tuono’ sequences are identical to the sequences of the respective portions of the Sf-RNase of ‘Lauranne’. Coding region and intron sequences are indicated by bold type and italics, respectively. Deduced amino acid sequences are shown under the nucleotide sequences. Boxed sequences indicate conserved regions of Prunus S-RNase. PCR primer name and directions are indicated by arrows.

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    Expression analysis of SFB1, SFBf, S1-RNase, Sf-RNase, and actin gene of almond. Polymerase chain reaction (PCR) was performed with cDNA from pollen (P), style (S), and genomic DNA (G) of ‘Ferragnès’ (FER), ‘Tuono’ (TUO), and ‘Lauranne’ (LAU). Because there is no intron sequence in the coding region of SFB, PCR products from SFB from genomic DNA show the same size as those from cDNA. Because PCR for S-RNase was designed to amplify the region containing an intron, amplified products from cDNA and genomic DNA showed different sizes. Arrow indicates the position where PCR products from cDNA for Sf-RNase should be located.

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    • Search Google Scholar
    • Export Citation
  • Ushijima, K., Yamane, H., Watari, A., Kakehi, E., Ikeda, K., Hauck, N.R., 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
  • 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, K. & 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
  • Wünsch, A. & Hormaza, J.I. 2004 Genetic and molecular analysis in Cristobalina sweet cherry, a spontaneous self-compatible mutant Sex. Plant Reprod. 17 203 210

    • Search Google Scholar
    • Export Citation
  • Yamane, H., Ikeda, K., Ushijima, K., Sassa, H. & Tao, R. 2003 A pollen-expressed gene for a novel protein with an F-box motif that is very tightly linked to a gene for S-RNase in two species of cherry, Prunus cerasus and P. avium Plant Cell Physiol. 44 764 769

    • Search Google Scholar
    • Export Citation
  • Yamane, H. & Tao, R. 2009 Molecular basis of self-(in)compatibility and current status of S-genotyping in rosaceous fruit trees J. Jpn. Soc. Hort. Sci. (submitted).

    • Search Google Scholar
    • Export Citation
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