Breeding Science 54 : 165-175 (2004)

Molecular Characterization of a 313-kb Genomic Region Containing the Self- incompatibility Locus of trifida, a Diploid Relative of

Rubens Norio Tomita§1), Go Suzuki§2), Kazuo Yoshida1), Yukihito Yano1), Tohru Tsuchiya3), Katsuyuki Kakeda1), Yasuhiko Mukai2) and Yasuo Kowyama*1)

1) Faculty of Bioresources, Mie University, 1515 Kamihama, Tsu, Mie 514-8507, Japan 2) Division of Natural Science, Osaka Kyoiku University, 4-698-1 Asahigaoka, Kashiwara, Osaka 582-8582, Japan 3) Life Science Research Center, Mie University, 1515 Kamihama, Tsu, Mie 514-8507, Japan

Diploid Ipomoea trifida is an ancestral wild species of species of the genus Ipomoea display a sporophytic type of the cultivated hexaploid sweet potato, and displays a SI that causes a complete failure of pollen germination on sporophytic self-incompatibility (SI) that is controlled the stigma surface after self-pollination. The cultivated by a single multiallelic S-locus. To characterize the ge- sweet potato, Ipomoea batatas, is a hexaploid SI species nomic region of the S-locus using a map-based cloning that is considered to be derived from the ancestral diploid SI method, a BAC library consisting of approximately species, I. trifida (Nishiyama et al. 1975). This indicates that 40,000 clones was constructed from genomic DNA of polyploidy does not necessarily lead to the breakdown of the S1-homozygote, and screened using S-linked DNA SI in the genus Ipomoea, in contrast to the gametophytic SI markers which were mapped in our previous study. We of Solanaceae, in which tetraploids are invariably self- constructed a contig covering the S-locus region with compatible (de Nettancourt 2001). To elucidate the molecular additional screening of fosmid and λ phase libraries. mechanism of SI in Ipomoea, diploid I. trifida has been RFLP analysis of recombinant using terminal end analyzed in our laboratory (Kowyama et al. 2000). SI of sequences of the BAC clones as probes indicated that the I. trifida is also controlled by a single S-locus with multiple S-locus region was delimited within a map distance of alleles and at least 49 different S-haplotypes have been iden- 0.57 cM, spanning approximately 300 kb in physical dis- tified from several natural populations of Central America tance. Remarkable suppression of genetic recombina- (Kowyama et al. 1980, 1994). tion was detected in the S-locus region. From sequence Recent molecular studies on diverse SI systems indi- analysis of the 313-kp region, 43 ORFs, many repetitive cate that the pistil and pollen components required for self sequences and 5 transposable elements were predicted. and non-self recognition of SI are encoded by two different None of the ORFs, however, showed a high homology tightly linked genes at the S-locus, and that distinct S-locus with the SI genes reported to date at the S-locus of other gene products are involved in the recognition mechanisms of families, suggesting that a unique molecular SI in several plant families (Hiscock and McInnis 2003). In mechanism is involved in the SI system of the Convol- the sporophytic SI of Brassicaceae, the female and male rec- vulaceae family. ognition determinants of SI are SRK and SP11/SCR, respec- tively (Takayama and Isogai 2003, Watanabe et al. 2003). In Key Words: Ipomoea trifida, sporophytic self-incompat- the gametophytic SI of Solanaceae, Rosaceae and Scrophu- ibility, S-locus, map-based cloning, BAC lariaceae, the female recognition determinant is an S-RNase library. (McCubbin and Kao 2000, Steinbachs and Holsinger 2002), and the candidate of the pollen determinant has recently been found to be an F-box gene (Lai et al. 2002, Ushijima et al. 2003, Entani et al. 2003). In the gametophytic SI of Introduction Papaveraceae, extracellular signaling S-proteins secreted on the stigma constitute a female factor that leads to the SI re- Self-incompatibility (SI) in flowering plants is an elab- action even under in vitro conditions (Wheeler et al. 1999, orate genetic system that prevents self-fertilization and there- Franklin-Tong and Franklin 2003). by promotes out-crossing to maintain genetic diversity within To identify the S-locus genes in Ipomoea, we used a po- a species. The majority of SI systems are regulated by a sin- sitional cloning strategy. Although we have no direct evi- gle multiallelic locus designated as S-locus (de Nettancourt dence that the male and female determinants of the SI in 2001). In the morning glory family , several Ipomoea are encoded by different genes at the S-locus, our earlier genetic study showed that dominance and co- Communicated by K. Okazaki dominance interactions between S-haplotypes were often Received November 25, 2003. Accepted December 12, 2003. different in the pollen and the stigma, suggesting the inde- §These authors contributed equally to this work pendent expression of male and female genes at the S-locus *Corresponding author (e-mail: [email protected]) of Ipomoea (Kowyama et al. 1994). If male and female 166 Tomita, Suzuki, Yoshida, Yano, Tsuchiya, Kakeda, Mukai and Kowyama components of the Ipomoea SI are encoded by different CHEF apparatus (Bio-Rad) under the following conditions: genes at the S-locus, genomic sequence analysis of the S- 2.4 V/cm with a pulse-switching interval ramped from 0.5 to locus region should lead to simultaneous cloning of the S- 1.5 sec over 20 h at 14°C. DNA of approximately 40 kb in locus genes involved in the SI system. In our previous work, size was excised from the gel and ligated with the fosmid we constructed a fine genetic map of DNA markers in the vector according to the manufacturer’s instructions. For the vicinity of the S-locus, and also obtained a DNA marker construction of the λ library, genomic DNA was partially (AAM-68) that is most tightly linked to the S-locus, with digested with MboI and ligated with a Lambda FIX II/XhoI which no recombinants were detected in an S-allele segre- partial fill-in vector (Stratagene, La Jolla, CA, USA), accord- gating population of 873 progenies examined. In that map, ing to the manufacturer’s instructions. Packaging was per- the S-locus region was delimited within 1.25 cM (Tomita et al. formed with a MaxPlax lambda packaging extract kit 2004). (Epicentre), and the λ phases were used to infect E. coli, In the present study, we identified several clones cover- XL1-blue MRA. ing the S-locus from the map-based screening of bacterial artificial chromosome (BAC), λ and fosmid libraries. These Library screening clones spanned approximately 600 kb in the genetic map. BAC clones were blotted on nylon membranes Using the terminal end sequences of these clones, the S- (Hybond N, Amersham Pharmacia, Little Chalfont, UK) using locus region was further delimited within 0.6 cM, spanning an automated workstation (Biomek 1000 HDR, Beckman approximately 300 kb. Based on sequence analysis of the Coulter, Fullerton, CA, USA) to prepare high-density mem- 313-kp region, 43 ORFs were predicted, in which there were branes (3,456 clones per 12 × 8 cm membrane) and cultured no homologies with the SI genes reported to date from other overnight at 37°C on LB containing 12.5 mg/l chloram- plants, suggesting a unique molecular mechanism of SI in phenicol. The membranes were processed as described by the Convolvulaceae family. Nakamura et al. (1997). DNA markers linked to the S-locus were used as probes to screen BAC clones. The probe DNA Materials and Methods was labeled with [α-32P] dCTP using BcaBest Labeling kit (Takara Bio, Otsu, Japan). Hybridization was performed for Construction of genomic libraries 20 h at 42°C in 5 × SSPE containing 5 × Denhardt’s solution, To construct the BAC library, high molecular-weight 0.5 mg/ml denatured salmon sperm DNA, 0.1 % SDS and 50 DNA was isolated from young leaves of an S1-homozygote % formamide. After hybridization, the filters were washed as described by Liu and Whittier (1994), embedded in agar- twice with 2 × SSC, 0.1 % SDS at room temperature for 20 ose plugs, and partially digested with HindIII (2 to 6 units/ min, washed 2 more times with 0.1 × SSC, 0.1 % SDS at 50°C ml) at 37°C for 40 min. The partially digested DNA was sub- for 20 min, and exposed to Biomax MS X-film (Eastman jected to pulsed-field gel electrophoresis (PFGE) using a Kodak, Rochester, NY, USA) at −80°C with an intensify- CHEF mapper (Bio-Rad, Hercules, CA, USA) with 1 % low ing screen. Fosmid clones were spread on 137-mm nylon melting point agarose gel (FMC, Rockland, ME, USA) in membrane disks (Hybond N, Amersham Pharmacia) and 0.5 × TBE buffer at 14°C. The PFGE conditions were 6 V/ incubated overnight on LB plates with 12.5 mg/l chloram- cm, with a constant linearly ramped pulse time of 90 sec for phenicol. The filters were processed and hybridized as de- 4 h, followed by electrophoresis at 6 V/cm, with a constant scribed above in the BAC library screening. Screening of the linearly ramped pulse time of 6 sec for 12 h. Gel slices con- λ library was performed using standard procedures. The taining DNA larger than 150 kb in size were excised, and pu- BAC, fosmid and λDNA were purified through Qiagen-tips rified by digestion with β-agarase I (New England Biolabs, (Qiagen, Hilden, Germany) for the sequencing of terminal Beverly, MA, USA), followed by ligation with a HindIII- ends. digested pBeloBAC11 vector (Kim et al. 1996) using T4 ligase (Promega, Madison, WI, USA). After the ligation, DNA sequencing DNA was electroporated into the Escherichia coli strain BAC and fosmid clones, (BAC-681, -H1, -B2, -415 and ElectroMAX DH10B (Invitrogen, Carlsbad, CA, USA) us- FOS-J1) were used for shotgun sequencing. BAC and fos- ing a Gene Pulser II (Bio-Rad) under the conditions of 1.25 mid DNAs were fragmented with an ultrasonic disrupter kV, 25 µF and 100 Ω. Recombinant clones were selected by (Tomy, Tokyo, Japan) and 2 to 5 kb fragments were cloned spreading on LB agar containing 12.5 mg/l chloramphenicol, into the pUC118 vector after end-repair treatment (Sambrook X-gal and IPTG. White colonies were picked up and cul- and Russel 2001), and transformed into JM109 competent tured in LB freezing broth in 96-well plates. After incuba- cells (Takara Bio). After transformation, recombinant clones tion at 37°C overnight, the plates were stored at −80°C. were picked up and grown in 1.8 or 3 ml of LB medium The fosmid library was constructed using the EpiFOS containing 50 mg/l of ampicillin at 37°C overnight, and fosmid library production kit (Epicentre, Madison, WI, plasmid DNA was isolated using either a QIAprep 96 turbo USA). Genomic DNA extracted from leaves was teated with miniprep or a QIAprep spin miniprep kit (Qiagen). The shot- End-Repair Enzyme kit (Epicentre), and subjected to elec- gun clones were sequenced on a CEQ 2000 DNA analysis trophoresis on 1 % low melting point agarose (FMC) with a system (Beckman Coulter). Characterization of the S-locus genomic region in Ipomoea 167

Analysis of sequence data (Fig. 1B and Fig. 2C), and among them, 6 clones (BAC-412, Sequence assembly was performed with the Sequencher -413, -416 to -419) also hybridized with the AAM-12 mark- ver 4.1 (Gene Code Corporation, Ann Arbor, MI, USA). er (Fig. 2D). Because a restriction fragment harboring both Genes were predicted using several programs: GeneMark. AF-41 and AAM-12 markers was detected in a DNA blot hmm (http://dixie.biology.gatech.edu/GeneMark), GENE- (data not shown), the physical distance between AF-41 and SCAN (http://genes.mit.edu/GENESCAN), GlimmerM (http:// AAM-12 was estimated to be approximately 70 kb. Among www.tigr.org/tdb.glimmerm/glmr) and FGENESH (http:// the 12 clones that hybridized with the AF-41 probe, the softberry.com/berry.phtml). The BLASTX program (http:// BAC-414 clone had the left most end sequence (414-T7). www.ncbi.nlm.nih.gov/BLAST) was also used to identify Using this end marker as a probe, three new BAC clones, putative coding regions. Further analysis of the putative pro- designated as BAC-B1 to BAC-B3, were identified from the teins was performed with BLASTP, and putative domains BAC library (Fig. 1B). In the largest clone, BAC-B2 (ca. 120 were also analyzed in a conserved domain database (http:// kb), both the left and right ends were sequenced, and the B2- www.ncbi.nlm.nih.gov/Structure/cdd). Repetitive sequences T7 end was located on the left side of the clone. Using the were detected and analyzed with RepeatMask (http://ftp. B2-T7 end probe, no positive clones were detected from the genome.washington) and SSRIT (http://www.gramene.org/ BAC library. gramene/searches/ssrtool). Although the BAC contigs around the AAM-68 and AF-41 markers were constructed from the screenings of the Results BAC library, a gap between the end markers 683-SP6 and B2-T7 could not be filled with any BAC clones, because no Construction and screening of BAC Library positive clones were obtained with these marker probes from To facilitate the positional cloning of the S-locus genes, the BAC library, as described above. To estimate the gap a BAC library of I. trifida was constructed using genomic size in the genomic DNA of I. trifida, the gap was directly DNA fragments prepared from S1-homozygote and partially visualized by fluorescence in situ hybridization (FISH) on digested with HindIII. The BAC library contained nearly the extended DNA fibers probed with two BAC clones 40,000 clones with an average insert size of 79.3 kb. Based (BAC-681 and BAC-B2) (Suzuki et al., unpublished data). on the haploid genome size of 533 Mb in I. trifida FISH analysis indicated that the gap size was approximately (Arumuganathan and Earle 1991), the genome coverage 44 kb in length. The gap between the contigs was small of this BAC library was estimated to be approximately 5.9 enough to be closed by λ and/or fosmid clones. We there- genome equivalents. All of the BAC clones arrayed in the fore, constructed additional λ and fosmid libraries of the high-density membranes were used for screening by colo- S1-homozygote. Screening of the λ library with the B2-T7 ny hybridization probed with four DNA markers closely marker resulted in the identification of two positive clones linked to the S-locus, SAM-23, AAM-68, AF-41 and AAM- (λ-E7 and -E8), and an additional screening with the λ-E8 12 (Fig. 1). AAM-68 was located closest to the S-locus at end marker also resulted in the identification of two clones 0 cM, and SAM-23 was located at 1.14 cM on the left side of (λ-F3 and -F7) (Fig. 1B). With probes derived from terminal the S-locus. AF-41 and AAM-12 were mapped to 0.11 cM end sequences of the λ clones, further screening of the BAC and 0.34 cM on the right side of the S-locus, respectively. library enabled to identify a BAC clone, BAC-H1, approxi- BAC library screening with the marker probes AAM- mately 30 kb in size. Using the PCR primers derived from 68 and SAM-23 enabled to obtain three and eight BAC both the right-end sequence of BAC-H1 (H1-T7) and the clones, respectively. The SAM-23 probe failed to reveal sig- left-end sequence of BAC-B2 (B2-T7), an approximately 90 nals against the three AAM-68 hybridized clones, BAC-681, bp-fragment was amplified from the genomic DNA. Thus, -682 and -683 (Fig. 2A). Both terminal ends of the largest the gap between BAC-B2 and BAC H1 was confirmed to be clone BAC-682, were sequenced, and the end sequences completely closed by the λ clones. To close the gap between were subcloned from PCR products. When the left-end se- BAC-683 and BAC-H1, approximately 200,000 clones of quence 682-T7 was used as a probe for DNA gel blot analy- the fosmid library were subjected to screening with a probe sis of the BAC clones, four of the eight BAC clones pro- from the left-end sequence of BAC-H1 (H1-SP6). Two fos- duced hybridization signals (Fig. 2B), indicating that these mid clones were identified, FOS-I1 and FOS-J1, that were four clones (BAC-232, -233, -234 and -238) overlapped 34 and 37 kb in length, respectively. The FOS-J1 clone also with the BAC-682 clones. A BAC contig was, therefore, hybridized with the 683-SP6 probe. Thus, the gap between constructed between the SAM-23 and AAM-68 markers BAC-683 and BAC-H1 was closed with the FOS-J1 clone. (Fig. 1B). Using the right most end sequence 683-SP6 as Consequently, a contig spanning approximately 600 kb cov- a probe, no positive clones were detected from the BAC ering the Ipomoea S-locus region was constructed (Fig. 1B). library. The physical distance from AAM-68 to SAM-23 was estimated to be approximately 190 kb, based on restriction Mapping of new markers and suppression of genetic recom- analysis of these BAC clones (data not shown). Screening of bination around the S-locus the BAC library probed with the marker AF-41 resulted in In our previous study (Tomita et al. 2004), the S-locus the detection of 12 positive clones, BAC-411 to BAC-4112 of I. trifida was mapped within 1.25 cM between the markers 168 Tomita, Suzuki, Yoshida, Yano, Tsuchiya, Kakeda, Mukai and Kowyama

Fig. 1. Genetic linkage map and physical map constructed around the S-locus of I. trifida. (A) The genetic linkage map of the DNA markers around the S-locus. The markers 682-T7 and 681-SP6 derived from terminal end sequences of BAC clones were newly mapped in the present study. The S-locus is delimited within the interval between markers 681-SP6 and AF-41. The numbers above and below the line indicate the genetic distance in centimorgan (cM). (B) The contigs around the S-locus constructed by screening of BAC, fosmid, and λ libraries. The five clones (BAC-681, -H1, -B2, -415 and FOS-J1) chosen for shotgun sequencing are indicated in bold charac- ters. (C) Physical map of the markers around the S-locus and the sequenced contigs (SR-1 to 7). The nucleotide sequence data of the contigs have been deposited in GenBank under accession numbers AM448010 to AM448016. Physical intervals in kb are indicated below the line. The kilobase-to-centimorgan ratios shown in parentheses were calculated from the estimated physical distances and genetic distances.

SAM-23 and AF-41. In the present study, the physical dis- cating that markers 683-T7 and AAM-68 co-segregated with tance between the two markers was estimated to be approxi- the S-locus (Fig. 1A). Based on the present RFLP analyses, mately 440 kb based on the assembling of BAC and fosmid the S-locus was further delimited within 0.57 cM between clones (Fig. 1C). To further define the location of the S- markers 681-SP6 and AF-41. The physical distance between locus, terminal end sequences derived from the BAC clones these markers was approximately 300 kb (Fig. 1C). were used as markers for RFLP analysis. A set of 20 recom- To determine whether the SI phenotype of either stigma binant plants previously detected between SAM-23 and the or pollen is altered in plants carrying recombination events S-locus from a population of 873 progenies were analyzed in the vicinity of the S-locus, as might be expected if the for RFLP using three new markers, 682-T7, 681-SP6 and recombination occurred between stigma- and pollen-part 683-T7 that corresponded to the left ends of the BAC-682, genes at the S-locus, we examined the pollination behavior BAC-681 and BAC-683 clones, respectively. We identified of eight recombinants between the 681-SP6 marker and the seven plants with recombination events between markers S-locus, and two recombinants between the AF-41 marker SAM-23 and 682-T7, five recombinants between 682-T7 and the S-locus. None of the recombinants showed any and 681-SP6, and eight recombinants between 681-SP6 and changes in the SI phenotypes of both stigma and pollen (data the S-locus. Consequently, genetic distances from the S- not shown), indicating the existence of a tight coupling of locus were estimated to be 1.14 cM for SAM-23, 0.74 cM the stigma- and pollen-part genes at the S-locus. Remarkable for 682-T7 and 0.46 cM for 681-SP6 (Fig. 1A). No recom- suppression of genetic recombination around the S-locus binants were detected between 683-T7 and the S-locus, indi- was revealed from the comparison of the genetic distances Characterization of the S-locus genomic region in Ipomoea 169

gaps were difficult to close, because of the presence of somewhat long repetitive sequences. The sizes of the se- quence gaps, however, were estimated to be rather small, that is approximately 200 bp for G2, 800 bp for G5, and less than 50 bp for the other gaps. Furthermore, Northern blot probed with shotgun DNA clones including these gap- sequences showed no detectable signals for the mRNAs iso- lated from several vegetative and reproductive tissues (data not shown), suggesting that neither the expressing genes nor the exons were present within the gap-sequences. The se- quenced contigs, designated as SR-1 to SR-7, were 82.1, 9.4, 31.6, 87.7, 7.2, 39.5 and 55.2 kb in size, respectively, added up to approximately 313 kb (Fig. 1C). The sequence data of the seven contigs have been deposited in GenBank under ac- cession numbers AY448010 to AY448016. Putative genes within the sequenced 313-kb region were predicted using GENSCAN, GeneMark.hmm, GlimmerM and FGENESH. A total of 43 putative ORFs were pre- Fig. 2. DNA gel blot analysis of the BAC clones probed with DNA markers linked to the S-locus. BAC DNAs were digested with dicted in the region and their homologs were searched in

NotI (A, B) or Sse8387I (C, D), and hybridized with a probe the BLASTX and BLASTP database (Table 1 and Fig. 3). from markers SAM-23 (A), 682-T7 (B), AF-41 (C) and AAM- Among them, 25 ORFs showed significant similarities to 12 (D). (A) Eight BAC clones (BAC-231 to -2310) and three other plant genes of known function, 7 ORFs showed simi- clones (BAC-681 to 683) were obtained from library screening larities to hypothetical genes of unknown function from with the marker probes, SAM-23 and AAM-68, respectively. Arabidopsis thaliana and Oryza sativa, and the remaining (B) Four clones (BAC-232, -233, -234 and -238) also had 11 ORFs lacked significant homology with any other genes hybridization signals for the probe 682-T7 that was derived in the database. The nucleotide sequences of the three DNA from the terminal end of BAC-682 clone, indicating the exist- markers used for the screening of the genomic libraries were ence of a physical linkage between the two markers. (C) found in ORF1 (681-SP6), ORF11 (AAM-68) and ORF41 Twelve BAC clones (BAC-411 to 4112) were obtained from (AF-41). ORF42 and ORF43, therefore, were located out- screening with the marker probe AF-41. (D) Six of the twelve clones also hybridized with the marker probe AAM-12, indi- side of the estimated S-locus region. ORF1 was located at cating the existence of a physical linkage between the two the beginning of this region and contained a partial sequence markers, AF-41 and AAM-12. DNA size markers are indicated of a disease resistance gene with a leucine-rich repeat do- on the left side in kilobases. main. ORF11showed a high homology with glycosyltrans- ferase family 2, as described in our previous paper (Tomita et al. 2004). ORF41 was similar to a histone deacetylase in- and the physical distances (Fig. 1C). The genetic distance in volved in transcriptional regulation. ORF19 and ORF20 the 250-kb region between markers AAM-68 and AF-41 showed similarities to defense-related genes encoding a was 0.11 cM , exhibiting a ratio of 2,273 kb/cM. On the other cysteine-rich protein. ORF28 was predicted to encode a hand, the flanking regions on the left side of the AAM-68 serine/arginine protein kinase and ORF9 showed a similarity marker exhibited a ratio of 108 to 214 kb/cM, and the right- to a regulatory subunit of cyclin-dependent kinase. These side interval of the AF-41 marker exhibited a ratio of 304 two kinases (ORF28 and ORF9) were structurally different kb/cM (Fig. 1C). This indicates that the genetic recombination from the Brassica SRK. Furthermore, we did not find any in the S-locus region was suppressed to approximately 1/10 receptor protein kinase in the sequenced region. Genes in- of that in the flanking regions. volved in a signal transduction pathway were predicted for ORF14 with a similarity to RelA/SpoT-like protein, which Genomic sequence of a 313-kb region surrounding the S- confers salt tolerance (Yamada et al. 2003), and for ORF25, locus which showed a high homology with non-phototropic hypo- Because the S-locus was mapped to a region between cotyl 3-like protein. markers 681-SP6 and AF-41, a set of overlapping BAC and fosmid clones covering this region (BAC-681, FOS-J1, Analysis of repetitive sequences BAC-H1, BAC-B2 and BAC-415) was chosen for shotgun To analyze the frequency and type of repetitive se- sequencing (Fig. 1B). The sequenced data assembled from quences in the genome, minimum repeats of more than 8- the shotgun clones gave an about 13-fold coverage of this re- mononucleotides, 4-dinucleotides, or 3-tri, -tetra, and -penta gion and resulted in the construction of seven contigs sepa- nucleotides were used as criteria of simple sequence repeats rated by six sequence gaps, G1 to G6 shown in Figure 3. (SSRs). A total of 915 SSRs were identified in the 313-kb Primer walking was performed to close the gaps, but these region, and the density was 29.2 SSRs per 10 kb (Table 2). 170 Tomita, Suzuki, Yoshida, Yano, Tsuchiya, Kakeda, Mukai and Kowyama

Fig. 3. Schematic representation of putative ORFs and repetitive elements in the genomic region around the S-locus. ORFs are repre- sented by black bars (exon) linked to horizontal thin lines (intron), and terminal exons are denoted by arrowheads. The designa- tion of putative ORFs is described in Table 1. Transposable elements are represented by gray dotted-bars and microsatellites (SSRs) by dots below the thick lines. The sequence gaps, G1 to G6 are denoted by inverted triangles on the thick lines. The numbers above the line indicate the physical distance in kb.

Most of the SSRs were (T/A)n-type monomer repeats, example, in the region between ORFs 25 and 31, there were (TA)n-type dimer repeats and (TAA)n-type trimer repeats. approximately 20 SSRs per 10 kb. SSRs of 8 to 11 bp in These SSRs were unequally distributed with a higher fre- length were most common (Table 2). quency in intergenic regions (Fig. 3). Around ORFs 4 and Sequence analysis also revealed the presence of five 19, there were approximately 50 SSRs per 10 kb, while, for transposable elements, including class I (retrotransposons) Characterization of the S-locus genomic region in Ipomoea 171

Table 1. Open reading frames (ORFs) predicted in the 313-kb genomic region covering the S-locus ORF Predicted homologous protein Acc. No. E-value Probable function of the predicted gene product ORF1 HcrVf3 CAC40827 4e-82 LRR disease resistance gene family ORF2 Expressed protein At2g27990 2e-25 ORF3 No homology ORF4 Zinc finger protein EPF1 S19159 1e-34 Zinc finger transcription factor-related protein ORF5 50S ribosomal protein L9 At3g44890 3e-38 Ribosomal protein L9 chloroplast precursor ORF6 Expressed protein CAE02117 3e-17 ORF7 No homology ORF8 No homology ORF9 CDK subunit 2 At2g27970 6e-30 Cyclin-dependent kinase regulatory element ORF10 Expressed protein At2g27980 e-175 PHD zinc finger protein ORF11 Glycosyltransferase family 2 At5g22740 0 Involved in cell wall biogenesis ORF12 No homology ORF13 No homology ORF14 RelA/SpoT like protein RSH1 AAL03950 6e-30 Signal transduction and transcription ORF15 No homology ORF16 No homology ORF17 No homology ORF18 No homology ORF19 γ-thionin-like protein precursor AAD21200 0.99 Defensin-like cysteine-rich protein ORF20 γ-thionin-like protein precursor S57809 0.28 Defensin-like cysteine-rich protein ORF21 Expressed protein At5g22820 e-91 ORF22 Expressed protein At1g08390 3e-28 ORF23 Phosphatidylcholine acetyltransferase At3g44830 0 Extracellular metabolism of plasma lipoproteins ORF24 Dihydroflavonol reductase At1g08390 0 Cell envelope biogenesis, and carbohydrate transport ORF25 Non-phototropic hypocoty13-like protein At3g44820 0 Signal transduction of blue light receptor ORF26 Adaptin family At5g22770 0 Involved in the formation of clathrin-coated vesicles ORF27 DNA repair protein RAD5 At5g22750 0 Involved in recombination and chromatin unwinding ORF28 Serine/arginine protein kinase At3g44820 0 Regulation of mRNA processing ORF29 Vacuolar assembly protein VPS41 P93231 0 ORF30 No homology ORF31 Expressed protein At2g27900 0 ORF32 Serine carboxipeptidase At2g27920 e-138 Amino acid transport and metabolism ORF33 Expressed protein At5g22870 e-29 ORF34 Endonuclease III protein, Ros1 At2g36490 3e-65 DNA repair, recombination and replication ORF35 No homology ORF36 Prephenate dehydratase At1g08250 e-161 Amino acid transport and metabolism ORF37 Pentatricopeptide repeat protein At4g21070 e-123 Involved in RNA stabilization ORF38 tRNA isopentenylpyrophosphatase At2g27760 e-134 Involved in translation, ribosomal structure and biogenesis ORF39 Expressed protein At3g09980 6e-29 ORF40 Hypothetical protein AAN06846 e-32 Involved in post-transcriptional gene silencing ORF41 Histone deacetylase At3g44680 0 Regulation of transcription ORF42 Neutral invertase CAD19320 0 Carbohydrate transport and metabolism ORF43 Protein kinase At2g36350 e-105 Signal transduction and class II (transposons), in the 313-kb genomic region Microsynteny between Ipomoea and Arabidopsis (Fig. 3). Between ORFs 4 and 5, a transposon En/Spm was The predicted 43 ORFs in the S-locus region of identified. A Copia-like retroelement containing an inte- Ipomoea were compared with the Arabidopsis sequences in grase domain was detected between ORFs 9 and 10. Be- GenBank using BLASTP. Thirty ORFs showed a sequence tween ORFs 12 and 13, a 122-bp nucleotide sequence similarity with an E-value of less than e-10, among which 20 showed a 74 % similarity to the helitron class of transposons could be related to 4 chromosome segments of Arabidopsis having a helicase domain. At the two positions between (Table 3 and Fig. 4). Five putative protein-encoding genes of ORFs 16–17 and 32–33, there were short nucleotide se- Ipomoea (ORFs 25, 27, 28, 32 and 33) showed similarities to quences with approximately 40 % resemblance to a retroele- more than 20 multiple homologous genes of Arabidopsis ment lacking a long terminal repeat. (Table 3). In particular, more than 50 Arabidopsis genes showed a significant similarity to ORF28. Although the 20 ORFs in the S-locus region of Ipomoea were grouped into 172 Tomita, Suzuki, Yoshida, Yano, Tsuchiya, Kakeda, Mukai and Kowyama

Table 2. Simple sequence repeats (SSRs) detected in the 313-kb Table 3. Microsynteny between the Ipomoea ORFs around the S- genomic region around the S-locus. Frequency of the locus and the genes from the Arabidopsis genome SSRs in the genomic sequence is indicated by number of Ipomoea No. of matches Syntenic BLASTP SSRs per 10 kb ORF with E < e-10 homolog1) E value

SSR type No. of SSRs SSRs/10kb ORF5 1 At3g44890 3e-38 Mononucleotide 298 9.52 ORF6 1 At5g22875 1e-16 T/A 291 9.30 ORF9 2 At2g27970 6e-30 G/C 7 0.22 ORF10 13 At2g27980 e-175 Dinucleotide 260 8.31 ORF11 14 At5g22740 0.0 TA/AT 168 5.37 ORF21 1 At5g22820 e-29 TC/GA 50 1.60 ORF22 1 At1g08390 3e-28 TG/CA 37 1.18 ORF23 3 At3g44830 0.0 GC/CA 5 0.16 ORF24 5 At1g08200 0.0 Trinucleotide 270 8.63 At2g27860 0.0 Tetranucleotide 66 2.11 ORF25 33 At3g44820 0.0 Pentanucleotide 21 0.67 ORF26 9 At5g22770 0.0 SSR length (bp) No. of SSRs SSRs/10 kb At5g22780 0.0 ORF27 39 At5g22750 0.0 8–11 603 19.26 ORF28 > 50 At3g44850 0.0 12–19 234 7.48 At5g22840 0.0 ≥ 20 78 2.49 ORF29 1 At1g08190 0.0 Total 915 29.23 ORF31 1 At2g27900 0.0 ORF32 47 At2g27920 e-138 four Arabidopsis chromosome segments, most of these ORF33 20 At5g22870 e-29 Arabidopsis genes were interspersed in a different order ORF36 6 At1g08250 e-161 At2g27820 e-158 and orientation in the Ipomoea counterparts (Fig. 4). ORF38 9 At2g27760 e-134 ORF39 6 At2g27740 7e-26 Discussion 1) Only the syntenic homology with the lowest E value is listed. In our previous study, we constructed a linkage map of DNA markers in the vicinity of the S-locus, in which the S- unit. Because recombination between these two genes would locus region was delimited within 1.25 cM (Tomita et al. result in a breakdown of the SI system, recombination events 2004). To characterize the genomic region of the S-locus at the S-locus might be suppressed (McCubbin and Kao 2000, using a map-based cloning method, a BAC library consisting Casselman et al. 2000, Silva and Goring 2001). Based on of approximately 40,000 clones was first constructed from the comparison of genetic distances and physical distances genomic DNA of the S1-homozygote, and screened using the for different intervals in the genomic region of Ipomoea, S-locus-linked markers as probes. Screening of the BAC the present study revealed a substantial suppression of ge- library alone, however, was not sufficient to cover the entire netic recombination in the S-locus region between the most S-locus region and resulted in a contig gap of approximately tightly linked markers, AAM-68 and AF-41, in which the re- 50 kb in length. When the markers SAM-23 and AF-41 combination rate per unit length was approximately 1/10 of flanking the S-locus were used as probes in the screening of that in the flanking regions of the markers. This suggests that the BAC library, more positive BAC clones were detected male and female specificities of the SI in Ipomoea are deter- than those obtained with marker AAM-68, which is tightly mined by different genes at the S-locus, at which recombi- linked to the S-locus. This could be attributed to a cloning nation events are strictly suppressed. In Solanaceae, recombi- bias of the BAC clones due to a genomic DNA specificity, nation suppression around the S-locus was reported to be such as a high frequency of repetitive sequences around the due to the centromeric localization of the locus (Entani et al. S-locus of I. trifida. By additional screening of newly con- 1999, McCubbin et al. 2000). The Ipomoea S-locus region, structed λ and fosmid libraries, the contig gap was closed however, is located at the terminal end of a chromosome ac- with their several clones. Using terminal end sequences of cording to FISH analysis (Suzuki et al., unpublished data). the BAC clones as probes for RFLP analysis of recombinant At the bz locus and a1-sh2 interval of maize and at the Mla plants, the location of the S-locus was further defined within resistance locus of barley, recombination suppressions were a narrow map distance of 0.57 cM that spanned approximate- associated with the presence of retrotransposon cluster com- ly 300 kb in physical distance. plexes (Fu et al. 2002, Yao et al. 2002, Wei et al. 2002). No The SI systems of many plant species are controlled by such large cluster of transposable elements was found in the at least two polymorphic S-locus genes, one encoding the S-locus region of I. trifida, suggesting that another mecha- male-part determinant and the other the female-part determi- nism is responsible for recombination suppression in nant, which might be transmitted as a genetically inseparable Ipomoea. The other possibility for preventing recombination Characterization of the S-locus genomic region in Ipomoea 173

Fig. 4. Microsynteny between I. trifida S-locus region and four segments of A. thaliana on chromosomes I (At1), II (At2), III (At3) and V (At5). Homologous genes between I. trifida and A. thaliana are linked by dotted lines and shown in Table 3. is heterogeneity in the genomic sequences among S- orthologs of the SRK and SP11/SCR genes are located in haplotypes (Yu et al. 1996, Boyes et al. 1999, Cui et al. 1999, an ARK3 region of chromosome IV of A. thaliana, and Shiba et al. 2003). Further comparative studies on genomic that these orthologs are nonfunctional genes encoding the organization of the S-locus region are required to reveal the truncated proteins (Kusaba et al. 2001). In our present study, structural heterogeneity among S-haplotypes of Ipomoea. 20 ORFs in the S-locus region of Ipomoea showed micro- From the sequence analysis of the 313-kb genomic re- syntenies to four segments derived from chromosomes I, II, gion covering the S-locus of I. trifida, 43 putative ORFs, III and V of A. thaliana. Of the four syntenic segments, the many repetitive sequences and 5 transposable elements were chromosome I segment was located on the short arm, on the predicted. Based on the number of ORFs, gene density was opposite arm for the colinear segment with the Brassica S- estimated to be one gene per 7.3 kb in the Ipomoea S-locus locus flanking region. None of the ORFs in the Ipomoea S- region. Comparable gene densities around the S-locus have locus region showed a significant homology with genes been reported with one gene per 5.4 to 8.3 kb in Brassica located in the chromosome IV-ARK3 region of A. thaliana. rapa (Suzuki et al. 1999, Shiba et al. 2003), one gene per This also suggests that in Ipomoea, a novel molecular SI 6.3 kb in B. napus (Cui et al. 1999), one gene per 8.0 kb in mechanism different from that of Brassica may operate, Prunus dulcis (Ushijima et al. 2003), and one gene per even though the SI system of Ipomoea is classified into the 9.0 kb in Antirrhinum majus (Lai et al. 2002). same sporophytic type as that of Brassica. Among the 43 ORFs predicted, 30 showed significant In the genomic region of approximately 60 kb from similarities to the genes related to known functions or to the ORF12 to ORF20, six of nine ORFs showed no significant hypothetical genes. None of the 30 ORFs, however, showed homology with any genes in the database. Compared with a high homology with any of the genes involved in SI of the the genome sequences of A. thaliana, no genes were homol- other plant families, suggesting that a unique SI system op- ogous with ORFs 12 to 20 except for ORF14, while in the erates in the Convolvulaceae family. Based on a compara- flanking regions outside of ORF12 and ORF20, most of the tive mapping study of the S-locus region of Brassica predicted genes were essentially homologous with the campestris (self-incompatible species) and its homeologous Arabidopsis genes (Table 1 and Fig. 4). Genomic sequence region in Arabidopsis thaliana (self-compatible species), analyses of Brassica and Prunus demonstrated that genomic Conner et al. (1998) demonstrated that the long arm segment regions covering the S-locus were highly divergent among of chromosome 1 in A. thaliana was largely colinear with S-haplotypes, while their flanking regions were rather con- the S-locus flanking region of Brassica, but differed by the served (Ushijima et al. 2003, Entani et al. 2003, Kusaba et absence of the SI specificity genes (SRK and SP11/SCR) in al. 2001, Fukai et al. 2003). The 60-kb genomic region of A. thaliana. Further study on the synteny between A. thaliana I. trifida was also found to be highly divergent among S- and a self-incompatible relative, A.lyrata, revealed that haplotypes, based on the genomic DNA gel blot probed with 174 Tomita, Suzuki, Yoshida, Yano, Tsuchiya, Kakeda, Mukai and Kowyama sequences derived from the ORFs 12 to 20 (data not shown). M.E. Nasrallah and J.B. Nasrallah (2000) Determining the phys- This circumstantial evidence suggests that the S genes in the ical limits of the Brassica S locus by recombinational analysis. S1-haplotype are located within the 60-kb region including Plant Cell 12: 23-33.

ORFs 12 to 20. In this region, only three ORFs (14, 19 and Conner,J.A., P.Conner, M.E.Nasrallah and J.B.Nasrallah (1998) Com- Brassica S 20) showed similarities to genes of known function. ORF14 parative mapping of the locus region and its homeo- log in Arabidopsis: Implications for the evolution of mating could be implicated in signal transduction for the stress re- systems in the Brassicaceae. Plant Cell 10: 801-812. sponse, because it is homologous with the plant RelA/SpoT Cui, Y., N. Brugiere, L. Jackman, Y. Bi and S.J. Rothstein (1999) Struc- gene, which regulates the synthesis of guanosine tetraphos- tural and transcriptional comparative analysis of the S locus re- phate (ppGpp) and guanosine pentaphosphate (pppGpp) and gions in two self-incompatible Brassica napus lines. Plant Cell mediates responses to pathogens and other stress factors 11: 2217-2231. (Van der Biezen et al. 2000, Yamada et al. 2003). The other de Nettancourt, D. (2001) Incompatibility and incongruity in wild and two ORFs, 19 and 20, showed a low homology with the cultivated plants, Springer-Verlag, Berlin. gamma-thionin cysteine-rich domain of Solanaceae (Lay et Entani, T., M. Iwano, H. Shiba, S. Takayama, K. Fukui and A. Isogai al. 2003). However, based on the alignment of deduced (1999) Centromeric localization of an S-RNase gene in Petunia hybrida amino acid sequences, both ORFs 19 and 20 were estimated Vilm. Theor. Appl. Genet. 99: 391-397. to be structurally different from the male determinants of Entani,T., M.Iwano, H.Shiba, F.S.Che, A.Isogai and S.Takayama (2003) Comparative analysis of the self-incompatibility (S-) lo- Brassica SI (SP11/SCR), which were also cysteine-rich pro- cus region of Prunus mume: identification of a pollen-expressed teins resembling the plant defensins (Schopfer et al. 1999, F-box gene with allelic diversity. Genes Cells 8: 203-213. Suzuki et al. 1999, Takayama et al. 2000). Franklin-Tong, V.E. and F.C.H. Franklin (2003) The different mecha- Further studies should be carried out to identify the S- nisms of gametophytic self-incompatibility. Phil. Trans. R. Soc. locus genes encoding male and female components of the SI Lond. B358: 1025-1032. in Ipomoea. First, analysis of gene expression by Northern Fu, H., Z. Zheng and H.K. Dooner (2002) Recombination rates between blot and/or RT-PCR methods may enable to screen the genes adjacent genic and retrotransposon regions in maize vary by 2 specifically expressed in either the stigma or anther/pollen. orders of magnitude. Proc. Natl. Acad. Sci. USA 99: 1082- Second, by screening cDNA libraries prepared from differ- 1087. ent S-haplotypes, highly polymorphic cDNA clones among Fukai,E., R.Fujimoto and T.Nishio (2003) Genomic organization of S S S S-haplotypes could be selected as promising candidates for the core region and the flanking regions of a class-II haplotype in Brassica rapa. Mol. Gen. Genomics 269: 361-369. the S genes. Finally, analyses of transformants conferring Hiscock, S.J. and S.M. McInnis (2003) The diversity of self-incompati- gain-of-function and loss-of-function of the SI system bility systems in flowering plants. Plant Biol. 5: 23-32. should lead to decisive evidence that the candidate genes are Kim, U.J., B.W. Birren, T. Slepak, V. Mancino, C. Boysen, H.L. Kang, involved in the SI system of Ipomoea. M.I. Simon and H. Hizuya (1996) Construction and character- ization of a human bacterial artificial chromosome library. Acknowledgements Genomics 34: 213-218. Kowyama, Y., N. Shimano and T. Kawase (1980) Genetic analysis of The authors thank Prof. Akira Isogai for his valuable incompatibility in the diploid Ipomoea species closely related comments and encouragement, and Drs. Seiji Takayama and to the sweet potato. Theor. Appl. Genet. 58: 149-155.

Hiroshi Shiba for their technical assistance in the analyses of Kowyama,Y., H.Takahasi, K.Muraoka, T.Tani, K.Hara and I.Shiotani the BAC library. We are grateful to Drs. Hidenori Sassa and (1994) The number, frequency and dominance relationships of S-alleles in diploid Ipomoea trifida. Heredity 73: 275-283. Koichiro Ushijima for their helpful comments on the con- Kowyama, Y., T. Tsuchiya and K. Kakeda (2000) Sporophytic self- struction of the fosmid library. This research was supported incompatibility in Ipomoea trifida, a close relative of sweet by a Grant-in-Aid for Scientific Research on Priority Areas potato. Ann. Bot. 85a: 191-196. (no. 11238203) and a Grant-in-Aid for Scientific Research B Kusaba, M., K. Dwyer, H. Jennifer, J. Vrebalov, J.B. Nasrallah and (no. 13460003) from the Ministry of Education, Culture, M.E. Nasrallah (2001) Self-incompatibility in the genus Sports, Science and Technology (MEXT), Japan to YK. We Arabidopsis: Characterization of the S locus in the outcrossing are also grateful to the MEXT and the Japanese Consulate in A. lyrata and its autogamous relative A. thaliana. Plant Cell 13: Sao Paulo, Brazil, for providing a fellowship to RNT. 627-643. Lai, Z., W. Ma, B. Han, L. Liang, Y. Zhang, G. Hong and Y. Xue (2002) Literature Cited An F-box gene linked to the self-incompatibility (S) locus of Antirrhinum is expressed specifically in pollen and tapetum. Arumuganathan, K. and E.D. Earle (1991) Nuclear DNA content of Plant Mol. Biol. 50: 29-42. some important plant species. Plant Mol. Biol. Reptr. 9: 208- Lay, F.T., F. Brugliera and M.A. Anderson (2003) Isolation and proper- 218. ties of floral defensins from ornamental tobacco and petunia. Boyes, D.C., M.E. Nasrallah, J. Vrebalov and J.B. Nasrallah (1999) The Plant Physiol. 131: 1283-1293. self-incompatibility (S) haplotypes of Brassica contain highly Liu, Y.G. and R.F. Whittier (1994) Rapid preparation of megabase divergent and rearranged sequences of ancient origin. Plant plant DNA from nuclei in agarose plugs and microbeds. Cell 9: 237-247. Nucleic Acids Res. 22: 2168-2169. Casselman, A.L., J. Vrebalov, J.A. Conner, A. Singhal, J. Giovannoni, McCubbin, A.G. and T.-h. Kao (2000) Molecular recognition and Characterization of the S-locus genomic region in Ipomoea 175

response in pollen and pistil interactions. Annu. Rev. Cell Dev. Takayama, S., H. Shiba, M. Iwano, H. Shimosato, F.S. Che, N. Kai, Biol. 16: 333-364. M. Watanabe, G. Suzuki, K. Hinata and A. Isogai (2000) The pol- McCubbin, A.G., C. Zuniga and T.-h. Kao (2000) Construction of a len determinant of self-incompatibility in Brassica campestris. binary bacterial artificial chromosome library of Petunia inflata Proc. Natl. Acad. Sci. USA 97: 1920-1925. and the isolation of large genomic fragments linked to the self- Tomita, R.N., K. Fukami, S. Takayama and Y. Kowyama (2004) Ge- incompatibility (S-) locus. Genome 43: 820-826. netic mapping of AFLP/AMF-derived DNA markers in the Nakamura, S., S. Asakawa, N. Ohmido, K. Fukui, N. Shimizu and vicinity of the self-incompatibility locus in Ipomoea trifida. S. Kawasaki (1997) Construction of an 800-kb contig in the Sex. Plant Reprod. 16: 265-272. near-centromeric region of the rice blast resistance gene Pi-ta2 Ushijima, K., H. Sassa, A.M. Dandekar, T.M. Gradziel, R. Tao and using highly representative BAC library. Mol. Gen. Genet. H. Hirano (2003) Structural and transcriptional analysis of the 254: 611-620. self-incompatibility locus of almond: identification of a pollen- Nishiyama, I., T. Miyazaki and S. Sakamoto (1975) Evolutionary auto- expressed F-box gene with haplotype-specific polymorphism. ploidy in the sweet potato (Ipomoea batatas (L.) Lam.) and its Plant Cell 15: 771-781. progenitors. Euphytica 24: 197-208. Van der Biezen, E.A., J. Sun, M.J. Coleman, M.J. Bibb and J.D. Jones Sambrook, J. and D.W. Russel (2001) Molecular Cloning: A Laborato- (2000) Arabidopsis RelA/SpoT homologs implicate (p)ppGpp ry Manual. 3rd Ed., Cold Spring Harbor Laboratory Press, Cold in plant signaling. Proc. Natl. Acad. Sci. USA 97: 3747-3752. Spring Harbor, New York. Watanabe, M., S. Takayama, A. Isogai and K. Hinata (2003) Recent Schopfer, C.R., M.E. Nasrallah and J.B. Nasrallah (1999) The male progresses on self-incompatibility research in Brassica species. determinant of self-incompatibility in Brassica. Science 286: Breed. Sci. 53: 199-208. 1697-1700. Wei, F., R.A. Wing and R.P. Wise (2002) Genome dynamics and evolu- Shiba, H., M. Kenmochi, M. Sugihara, M. Iwano, S. Kawasaki, G. Suzuki, tion of the Mla (powdery mildew) resistance locus in barley. M. Watanabe, A. Isogai and S. Takayama (2003) Genomic Plant Cell 14: 1903-1917. organization of the S-locus region of Brassica. Biosci. Biotech- Wheeler, M.J., A.C. Allan, N.D. Jordan, J.J. Rudd, V.E. Franklin-Tong nol. Biochem. 67: 622-623. and F.C.H. Franklin (1999) The intracellular events triggered Silva, N.F. and D.R. Goring (2001) Mechanism of self-incompatibility by the self-incompatibility response in Papaver rhoeas. Proto- in flowering plants. Cell Mol. Life Sci. 58: 1988-2007. plasma 208: 99-106. Steinbachs, J.E. and K.E. Holsinger (2002) S-RNase-mediated gameto- Yamada, A., K. Tsutsumi, S. Tanimoto and Y. Ozeki (2003) Plant RelA/ phytic self-incompatibility is ancestral in . Mol. Biol. SpoT homolog confers salt tolerance in Escherichia coli and Evol. 19: 825-829. Saccharomyces cerevisiae. Plant Cell Physiol. 44: 3-9. Suzuki, G., N. Kai, T. Hirose, K. Fukui, T. Nishio, S. Takayama, Yao, H., Q. Zhou, J. Li, H. Smith, M. Yandeau, B.J. Nikolau and A. Isogai, M. Watanabe and K. Hinata (1999) Genomic organiza- P.S. Schnable (2002) Molecular characterization of meiotic re- tion of the S locus; identification and characterization of genes combination across the 140-kb multigenic a1-sh2 interval of in SLG/SLG region of S9 haplotype of Brassica campestris maize. Proc. Natl. Acad. Sci. USA 99: 6157-6162. (syn. rapa). Genetics 153: 391-400. Yu, K., U. Schafer, T.L. Glavin, D.R. Goring and S.J. Rothstein (1996) Takayama, S. and A. Isogai (2003) Molecular mechanism of self- Molecular characterization of the S locus in two self-incompat- recognition in Brassica self-incompatibility. J. Exp. Bot. 54: ible Brassica napus lines. Plant Cell 8: 2369-2380. 149-156.