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Most commercial cultivars of japanese plum (Prunus salicina Lindl.) exhibit S-RNase-based gametophytic self-incompatibility (GSI), although some self-compatible (SC) cultivars exist. In this study, we characterized S-RNase and SFB, the pistil and pollen S determinants of the specificity of the GSI reaction, respectively, from four S-haplotypes, including a SC (S) and three SI (S, S, and S) S-haplotypes of japanese plum. The genomic organization and structure of the SC S-haplotype appear intact, because the relative transcriptional orientation of its S-RNase and SFB and their intergenetic distance are similar to those of the other three SI S-haplotypes of japanese plum and other Prunus L. species. Furthermore, there is no apparent defect in the DNA sequences of S-RNase and SFB. However, a series of transcriptional analyses, including real-time reverse transcriptase–polymerase chain reaction, showed that the S-RNase transcript levels in the pistil are significantly lower than those of the S-, S-, and S-RNases, although transcripts of SFB, SFB, SFB, and SFB are present at similar levels in pollen. Furthermore, no S-RNase spot was detected in two-dimensional polyacrylamide gel electrophoresis profiles of stylar extracts of the cultivars with the S-haplotype. We discuss the possible molecular basis of SC observed with the S-haplotype with special reference to the insufficient S-RNase accumulation incited by the very low transcriptional level of S-RNase in pistils. Self-incompatibility (SI) is a widespread mechanism in flowering plants that prevents inbreeding and promotes outcrossing (de Nettancourt, 2001). In the Rosaceae, Scrophulariacae, and Solanaceae plant families, SI is gametophytically controlled by the S locus, which contains the genes that control the pollen and pistil specificities. The pistil determinant of the three families is S-RNase, which encodes a basic glycoprotein with ribonuclease activity that is present in the extracellular matrix of the style-transmitting tract. The F-box genes named SFB or SLF were found to be good candidates for the pollen S component in Prunus (Rosaceae) (Entani et al., 2003; Ushijima et al., 2003; Yamane et al., 2003b) and Antirrhinum L. (Scrophulariacae) (Lai et al., 2002). A subsequent transformation experiment demonstrated that SLF is the pollen S component in Petunia inflata R.E. Fries (Solanaceae) (Sijacic et al., 2004). SI in Prunus interests pomologists and has been extensively studied because most Prunus fruit tree species are SI and unable to bear fruit parthenocarpically. The pistil S gene, S-RNase, has been characterized at the molecular level in most SI Prunus fruit tree species, including almond [P. dulcis (Mill.) D.A. Webb. (Ushijima et al., 1998)], sweet cherry [P. avium L. (Tao et al., 1999)], sour cherry [P. cerasus L. (Yamane et al., 2001)], japanese apricot [P. mume Siebold & Zucc. (Yaegaki et al., 2001)], and apricot [P. armeniaca L. (Romero et al., 2004)]. Recently, pollen S component SFB (S haplotype-specific F-box protein gene) has also been characterized in almond (Ushijima et al., 2003), sweet and sour cherries (Yamane et al., 2003b), japanese apricot (Entani et al., 2003; Yamane et al., 2003c), and apricot (Romero et al., 2004). The results from these studies have led to the development of not only molecular typing methods for S-haplotypes (Tao et al., 1999), but also molecular markers for self-compatibility (SC) in Prunus fruit tree species (Ikeda et al., 2004b; Tao et al., 2000, 2002a, 2002b). There have been several reports on the characterization of japanese plum S-RNase and SFB. Yamane et al. (1999) first isolated a japanese plum S-RNase from the SI cultivar ‘Sordum’. Based on the S-RNase DNA sequence, Beppu et al. (2002, 2003) developed a polymerase chain reaction (PCR)-based Shaplotype typing system and identified 14 different japanese plum S-RNases corresponding to the Sto S-haplotypes. Later, Beppu et al. (2005) reported that one of the 14 S-haplotypes, the S-haplotype, could be responsible for SC in some of the cultivars based on pollination and genetic studies. Tao et al. (2007) and Zhang et al. (2007) isolated SFB from several S-haplotypes. Because only partial sequences are available for S-RNase and SFB from all of the S-haplotypes of japanese plum, except for the S-haplotype (Tao et al., 2007), the nature of the SC conferred by the S-haplotype is unclear. In the present study, we thoroughly characterized the pistil and pollen S determinants of the specificity of the gametophytic self-incompatibility (GSI) reaction, S-RNase Received for publication 31 Oct. 2006. Accepted for publication 12 Mar. 2007. Ms. Watari and Mr. Hanada contributed equally. Corresponding author. E-mail: rtao@kais.kyoto-u.ac.jp. 396 J. AMER. SOC. HORT. SCI. 132(3):396–406. 2007. and SFB, respectively, from four S-haplotypes, including a SC (S) and three SI (S, S, and S) S-haplotypes of japanese plum, to clarify the molecular basis of the SC observed in the S-haplotype. Elucidation of the molecular basis of SC observed with S-haplotype could lead to the development of not only the molecular markers for SC, but also the novel SC breeding strategies in japanese plum. A series of molecular analyses that were conducted in this study suggest that the SC in the S-haplotype is incited by the accumulation of insufficient levels of S-RNase incited by a very low S-RNase transcription level in the pistil. Materials and Methods PLANT MATERIAL. Twelve japanese plum cultivars were used in this study (Table 1). Young leaves, pollen grains, and styles with stigmas at the balloon stage of development were collected and stored at –80 C until use. ISOLATION OF DNA. Total DNA was isolated from young leaves as described previously (Yamane et al., 1999). In brief, leaves were ground to a powder using a mortar and pestle and homogenized in washing buffer [0.1 M HEPES (pH 8.0), 0.1% PVP, 2% mercaptoethanol]. The homogenate was centrifuged (6500 gn at 4 C for 20 min) to collect the pellet, and the pellet was resuspended in washing buffer and centrifuged again. Next, the pellet was suspended in extraction buffer [0.2 M Tris-HCl (pH 8.0), 0.25 M NaCl, 25 mM ethylenediaminetetraacetic acid, 6% PVP], and one-half volume of 7.5 M ammonium acetate was added. The mixture was incubated on ice for 30 min, after which one volume of chloroform was added. After centrifugation (2500 gn at 4 C for 15 min), the supernatant was transferred to a new tube and one volume of isopropanol was added. The mixture was centrifuged (8000 gn at 4 C for 20 min) to collect the pellet. The resulting DNA pellet was dissolved in 10 mM Tris-HCl + 1 mM EDTA buffer (pH 8.0) (TE). After RNase treatment, the DNA solution was further purified by phenol/chloroform extraction and the DNA was precipitated with ethanol, rinsed with 75% ethanol, and dissolved in TE. CONSTRUCTION AND SCREENING OF A GENOMIC LIBRARY. A fosmid library was constructed from genomic DNA of ‘Sordum’ (SS) and ‘Santa Rosa’ (SS) using the CopyControl Fosmid Library Production Kit (Epicentre, Madison, Wis.). The library was screened with a DIG-dUTP-labeled (Roche, Basel, Switzerland) probe corresponding to a partial fragment of the coding region of almond S-RNase or SFB (Tao et al., 2007). Plasmid was extracted from positive colonies, and DNA sequences were determined with the BigDye Terminator version 3.1 Cycle Sequencing Kit and an ABI PRISM 310 Genetic Analyzer (Applied Biosystems, Foster City, Calif.). The deduced amino acid sequences of the SFBs and S-RNases were aligned using Clustal X (Thompson et al., 1997). ANALYSIS OF THE PHYSICAL DISTANCES BETWEEN S-RNASE AND SFB. PCR reactions were performed to determine the physical distance between S-RNase and SFB in the Table 1. S-haplotypes of 16 japanese plum cultivars. S-haplotype Cultivar Reference SS Sordum Yamane et al., 1999 Burmosa Beppu et al., 2003 SS Rio Beppu et al., 2002 SS Terada Beppu et al., 2002 SS Botan Beppu et al., 2003 SS Oishinakate Beppu et al., 2002 Taiyo Beppu et al., 2002 SS Santa Rosa Beppu et al., 2002 Beauty Beppu et al., 2002 SS Formasa Beppu et al., 2002 SS Frontier Beppu et al., 2003 SS Honey Rosa Beppu et al., 2003 SS Yonemomo Beppu et al., 2002 SS Bakemonosumomo Beppu et al., 2002 SS Harypickstone Beppu et al., 2003 SS Lantz Beppu et al., 2003 Table 2. DNA sequences of oligonucleotide primers.

A Low Transcriptional Level of Se-RNase in the Se -haplotype Confers Self-compatibility in Japanese Plum