Molecular Characterization of a 313-Kb Genomic Region Containing the Self- Incompatibility Locus of Ipomoea Trifida, a Diploid Relative of Sweet Potato

Breeding Science 54 : 165-175 (2004) Molecular Characterization of a 313-kb Genomic Region Containing the Self- incompatibility Locus of Ipomoea trifida, a Diploid Relative of Sweet Potato 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 plants 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 plant 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 Convolvulaceae, 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).

Load more