Journal of Heredity Advance Access published online on August 14, 2008
Journal of Heredity, doi:10.1093/jhered/esn063
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Brief Communication |
The Prunus Self-Incompatibility Locus (S locus) Is Seldom Rearranged
Instituto de Biologia Molecular e Celular (IBMC), University of Porto, Rua do Campo Alegre 823, 4150-180 Porto, Portugal (Vieira, Santos, and Vieira); Experimental Farm, Graduate School of Agriculture, Kyoto University, Takatsuki 569-0096, Japan (Habu); Laboratory of Pomology, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan (Tao)
Address correspondence to Cristina P. Vieira, Molecular Evolution Group, IBMC, University of Porto, Rua do Campo Alegre 823, 4150-180 Porto, Portugal, or e-mail: cgvieira{at}ibmc.up.pt.
Self-incompatibility enables flowering plants to discriminate between self- and non-selfpollen. In Prunus, the 2 genes determining specificity are the S-RNase (the female determinant that is a glycoprotein with ribonuclease activity) and the SFB (the male determinant, a protein with an F-box motif). In all Prunus S haplotypes characterized so far, with the exception of Prunus armeniaca S2 haplotype, the 2 genes have opposite transcription orientations. Nevertheless, the relative transcription orientation observed in P. armeniaca S2 haplotype has been postulated to be the one present in all S haplotypes from this species. We show that this is not the case by demonstrating that that the relative transcription orientation of the pollen and pistil genes of the P. armeniaca S17 haplotype is that which is commonly found in Prunus. Using a phylogenetic approach, we show that the relative transcription orientation of the S-RNase and SFB genes is seldom changed (less than once every 380 million years). This contrasts with the Brassica sporophytic S locus where chromosomal rearrangements are often observed in the region between the pollen and pistil genes.
Key Words: Prunus • self-incompatibility • SFB • S-RNase • transcription orientation
Self-incompatibility (SI) is a genetic mechanism that prevents inbreeding and promotes outcrossing. This mechanism comprises recognition of self- or self-related pollen, by cells of the pistil, followed by rejection of the incompatible pollen, through aborted development, either immediately after pollination on the stigma surface or at a subsequent stage during pollen tube growth into the stigma or style (de Nettancourt 1977). In gametophytic SI (GSI), pollen specificity is determined by the S-locus genotype of the haploid gametophyte. At least 2 linked genes, one determining the pistil specificity and another one determining pollen specificity, must be linked and coevolve as a genetic unit for GSI to work (see for instance, Roalson and McCubbin 2003). In Prunus, the pistil and pollen GSI components are the S-RNase and SFB gene, respectively. In Prunus, the S-RNase and SFB genes of the characterized S haplotypes are less than 70 kb apart (Ushijima et al. 2001). This implies that using long template PCR systems, both genes can be amplified without the need for cosmid contigs.
All speciation events giving rise to extant Prunus species happened in the last 5 million years (Vieira et al. 2008). Nevertheless, in GSI, specificities are maintained by frequency-dependent selection. Therefore, GSI specificities can be much older than speciation events. The age of the oldest Prunus specificity has been estimated to be in between 15 and 20 million years old (Vieira et al. 2008). Although, in extant Prunus species, most species sampled exhibit less than 34 specificities, the ancestral to extant Prunus species harbored at least 102 specificities (Vieira et al. 2008). Nevertheless, for 32 Prunus haplotypes, only the relative orientation of the 2 genes is known (Table 1). In all except the Prunus armeniaca S2 haplotype (Romero et al. 2004), the S-RNase and the SFB genes have opposite transcription orientations (Entani et al. 2003; Ushijima et al. 2003, 2004; Yamane et al. 2003; Nunes et al. 2006; Sonneveld et al. 2005; Surbanovski et al. 2007; Tao et al. 2007; Watari et al. 2007). The S-RNase–SFB intergenic region has been studied in detail in 7 Prunus spinosa haplotypes and only one 18-bp conserved element, that is a typical polyA signal, is found, that is located in the first 20 bp of the 3' noncoding region of the S-RNase gene (Nunes et al. 2006). Therefore, in principle, the S-locus region could have been rearranged, although the small size of the region encompassing the S genes could impose some constraint.
|
It has been assumed that within P. armeniaca, the orientation of the S-RNase and SFB genes is conserved (see Figure 1 in Romero et al. 2004, where the transcription orientation of the S-RNase and SFB genes from the S1 and S4 P. armeniaca haplotypes is shown as being the same as that observed in S2 haplotype), despite the observation that in the phylogenetic tree specificities from different species are mingled (see for instance Figure 1 in Vieira et al. 2008). The S17 P. armeniaca S-RNase and P. spinosa S3.1 and S3.2 S-RNase cluster together with high bootstrap support (Vieira et al. 2008). At the nucleotide level, they are 98% identical. The same pattern is also observed for the SFB gene (Vieira et al. 2008) as expected because the 2 genes show correlated evolutionary histories (Nunes et al. 2006). In P. spinosa, both the S3.1 and S3.2 haplotypes have the commonest orientation and the intergenic region is small (less than 1.7 kb; Nunes et al. 2006). Therefore, in this work, we determined the relative transcription orientation of the 2 genes in the S17 P. armeniaca haplotype. Using a plylogenetic approach, we show that in contrast to what is observed in Brassica, in Prunus the relative transcription orientation of the pollen and pistil genes is highly conserved.
 |
Material and Methods |
|---|
 Top Material and Methods Results and DiscussionFundingReferences |
|---|
Genomic DNA was extracted from young leaves of the P. armeniaca, cv. Satsuki, for which the S17-RNase and SFB17 were sequenced, using the cetyl trimethyl ammonium bromide (CTAB) method as described by Doyle JI and Doyle JL (1987). The Expand long template PCR system (Roche, Germany) was used to amplify P. armeniaca S17 haplotype using the 42fmix (TTGCTTTMTTCTTKTGTT) and 370f (TCCAACGAGCACCAACAT) primers for the S-RNase and SFB genes, respectively, according to manufacturer's instructions for system 3 and 48 °C as annealing temperature. The resulting amplification product of about 2.8 kb was excised from the gel and the DNA extracted using the QIAEX II Gel Extraction Kit (Qiagen, Hilden, Germany). This band was cloned using the pCR-XL-TOPO vector (Invitrogen, Carslabad, CA, USA). Fourteen colonies were screened for the presence of inserts using the primer pair 370f and 1010r for the SFB gene (5' TCCAACGAGCACCAACAT 3' and 5' CTTGCTTGGWYTCGTAAT 3'; Nunes et al. 2006). Plasmidic DNA was purified from 3 different colonies using the QIAprep Miniprep kit (Qiagen). This DNA was then used as template for the sequencing reaction that was performed using the ABI PRISM Big Dye cycle-sequencing kit (Perkin-Elmer, Foster City, CA, USA) and specific primers (see below) or the primers for the M13 forward and reverse priming sites of the pCR2.1 vector. The specific primers used to obtain the complete S3-haplotype sequence are as follows: forward primers—5' TATTTTCARTTTGTGCAA 3'; 5' ATTTTCATACTCTTTTGT 3'; 5' TTTGGGAAGGSGAATGGA 3'; and 5' AACAAAATACCACTTCAT 3' and reverse primers—5' TCCCATACTCAAAAAGAAG 3'; 5' AATTTTAYKGAAACRAGATG 3'; 5' GGGGGTTTTGTTTTTGTG 3'; and 5' TTGAGAAAGGTCCCATCT 3'. Sequencing runs were performed by Stabvida Inc. (Lisboa, Portugal).
Analyses of Sequences
The intergenic DNA sequence was deposited in GenBank (accession number EU516388). The nucleotide sequences were aligned using ClustalX v. 1.64b (Thompson et al. 1997), and minor manual adjustments were performed using Proseq version 2.43 (http://helios.bto.ed.ac.uk/evolgen/filatov/proseq.html). Analyses of DNA polymorphism were performed using DnaSP 4.1 (Rozas et al. 2003). The Prunus S-RNase tree was built using the 30 S haplotypes for which an S-RNase sequence longer than 180 amino acids is available. The minimum evolution method for inferring the phylogeny using distance matrices with pairwise deletion obtained with the Kimura 2 parameter model, as implemented in the MEGA software (Kumar et al. 2004), was used. A close-neighbor-interchange heuristic search (level 2) was performed. For the heuristic search, the initial tree was obtained using the distance based Neighbor-Joining method. In all, 1000 bootstrap replicates were used.
|
Results and Discussion |
|---|
|
TopMaterial and MethodsResults and DiscussionFundingReferences |
|---|
In the P. armeniaca S17 haplotype, the S-RNase and SFB gene present opposite transcription directions, as in P. spinosa. The size of the intergenic region is smaller (974 bp) than that for the P. spinosa S3.1 and S3.2 haplotypes (1343 and 1694, respectively). The divergence rate found for the intergenic region is low (0.056 and 0.058 for the P. spinosa S3.1–P. armeniaca S17 and the P. spinosa S3.2–P. armeniaca S17, respectively). This value is similar to that found for S-RNase silent sites (synonymous and intron sites; 0.058 and 0.061 for the P. spinosa S3.1–P. armeniaca S17 and the P. spinosa S3.2–P. armeniaca S17, respectively).
In P. armeniaca, 2 relative orientations are present for the S-RNase and SFB genes. Based on the observation that Prunus alleles from different species are found mingled in the phylogenetic tree (Figure 1; see also Vieira et al. 2008) and that most Prunus haplotypes show the opposite transcription orientation, we predict that most P. armeniaca haplotypes will also have this orientation. Therefore, it cannot be assumed that in Prunus, all haplotypes within species will have identical gene orientations.
|
In Prunus, changes in the relative transcription orientation of the pollen and pistil genes are a rarity. The minimum evolution tree shown in Figure 1 indicates that a single change in orientation happened in the external branch leading to S2 P. armeniaca sequence. In order to calculate the rate (per million years) for the change in the relative transcription orientation of the pollen and pistil genes, we added the estimated length (the units are percentage of amino acid changes; see Figure 1) of all branches except that of the S2 P. armeniaca. The data presented in Vieira et al. (2008) suggest that, for the S-RNase gene, the rate of amino acid change is about 1% per million years. Using this information to convert the units, it is estimated that a change in the relative transcription orientation of the pollen and pistil genes happens once every 380 million years. This result contrasts with that of Brassica species, where the 2 genes determining pistil and pollen specificity at the sporophytic S locus (SRK, the pistil protein that is a receptor kinase and SP11, a cysteine-rich pollen coat protein located a few kilobases away from the SRK) are frequently found in various relative orientations and different orders in the S-locus genomic region (Cui et al. 1999; Watanabe et al. 2000; Shiba et al. 2003). The lengths of the SP11–SRK region also varies (from about 1 to 23 kb, see Figure 1 in Shiba et al. 2003; although larger regions (60–104 kb; Watanabe et al. 2000) are also observed due to transposable element insertions. No such elements have been found in the intergenic region of the S-RNase–SFB gene, although they are frequent in the 5' and 3' region of the S-RNase–SFB genes (Entani et al. 2003; Ushijima et al. 2003). It should also be noted that the age of the extant S-allele lineage in the sporophytic SI system in Brassica is twice that of estimated in Prunus (about 40 millions years; Uyenoyama 1995). Therefore, the age of the 2 systems seems not to be the only cause for the difference observed.
|
Funding |
|---|
|
TopMaterial and MethodsResults and DiscussionFundingReferences |
|---|
Fundação para a Ciência e Tecnologia (research project POCI/BIA-BDE/59887/2004 funded by POCI 2010, cofunded by Fundo Europeu de Desenvolvimento Regional funds).
|
Footnotes |
|---|
Corresponding Editor: Jim Hamrick
Received January 15, 2008
Accepted July 22, 2008
|
References |
|---|
|
TopMaterial and MethodsResults and DiscussionFundingReferences |
|---|
-
Cui Y, Brugière N, Jackman L, Bi YM, Rothstein SJ. Structural and transcriptional comparative analysis of the S locus regions in two self-incompatible Brassica napus lines. Plant Cell (1999) 11:2217–2231.
de Nettancourt D. Incompatibility in angiosperms (1977) Berlin (Germany): Springer.
Doyle JI, Doyle JL. A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem Bull (1987) 19:11–15.
Entani T, Iwano M, Shiba H, Che FS, Isogai A, Takayama S. Comparative analysis of the self-incompatibility (S-) locus region of Prunus mume: identification of a pollen-expressed F-box gene with allelic diversity. Genes Cells (2003) 8:203–213.[Abstract]
Ikeda K, Ushijima K, Yamane H, Tao R, Hauck NR, Sebolt AM, Iezzoni AM. Linkage and physical distances between the S-haplotype S-RNase and SFB genes in sweet cherry. Sex Plant Reprod (2005) 17:289–296.[CrossRef]
Kumar S, Tamura K, Nei M. MEGA3: Integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinform (2004) 5:150–163.
Nunes MDS, Santos RAM, Ferreira SM, Vieira J, Vieira CP. Variability patterns and positively selected sites at the gametophytic self-incompatibility pollen SFB gene in a wild self-incompatible Prunus spinosa (Rosaceae) population. New Phytol (2006) 172:577–587.[CrossRef][Web of Science][Medline]
Roalson EH, McCubbin AG. S-RNases and sexual incompatibility: structure, functions, and evolutionary perspectives. Mol Phylogenet Evol (2003) 29:490–4506.[CrossRef][Web of Science][Medline]
Romero C, Vilanova S, Burgos L, Martínez-Calvo J, Vicente M, Llácer G, Badenes ML. Analysis of the S-locus structure in Prunus armeniaca L. Identification of S-haplotype specific S-RNase and F-box genes. Plant Mol Biol (2004) 56:145–157.[CrossRef][Web of Science][Medline]
Rozas J, Sanchez-DelBarrio JC, Messeguer X, Rozas R. DnaSP, DNA polymorphism analyses by the coalescent and other methods. Bioinformatics (2003) 19:2496–2497.
Shiba H, Kenmochi M, Sugihara M, Iwano M, Kawasaki S, Suzuki G, Watanabe M, Isogai A, Takayama S. Genomic organization of the S-locus region of Brassica. Biosci Biotechnol Biochem (2003) 67:622–626.[CrossRef][Medline]
Sonneveld T, Tobutt KR, Vaughan SP, Robbins TP. Loss of pollen-S function in two self-compatible selections of Prunus avium is associated with deletion/mutation of an S haplotype-specific F-box gene. Plant Cell (2005) 17:37–51.
Surbanovski N, Tobutt KR, Konstantinovi
M, Maksimovi V, Sargent DJ, Stevanovi V, Boskovi RI. Self-incompatibility of Prunus tenella and evidence that reproductively isolated species of Prunus have different SFB alleles coupled with an identical S-RNase allele. Plant J (2007) 50:723–734.[CrossRef][Web of Science][Medline]
Tao R, Watari A, Hanada T, Habu T, Yaegaki H, Yamaguchi M, Yamane H. Self-compatible peach (Prunus persica) has mutant versions of the S haplotypes found in self-incompatible Prunus species. Plant Mol Biol (2007) 63:109–123.[CrossRef][Web of Science][Medline]
Thompson J, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG. The ClustalX window interface: Flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res (1997) 25:4876–4882.
Ushijima K, Sassa H, Dandekar AM, Gradziel TM, Tao R, Hirano H. Structural and transcriptional analysis of the self-incompatibility locus of almond: Identification of a pollen-expressed F-box gene with haplotype-specific polymorphism. Plant Cell (2003) 15:771–781.
Ushijima K, Sassa H, Tamura M, Kusaba M, Tao R, Gradziel TM, Dandekar AM, Hirano H. Characterization of the S-locus region of almond (Prunus dulcis): Analysis of a somaclonal mutant and a cosmid contig for an S haplotype. Genetics (2001) 158:379–386.
Ushijima K, Yamane H, Watari A, Kakehi E, Ikeda K, Hauck NR, Iezzoni AF, Tao R. The S haplotype-specific F-box protein gene, SFB, is defective in self-compatible haplotypes of Prunus avium and P. mume. Plant J (2004) 39:573–586.[CrossRef][Web of Science][Medline]
Uyenoyama MK. A generalized least-squares estimate for the origin of sporophytic self-incompatibility. Genetics (1995) 139:975–992.[Abstract]
Vieira J, Fonseca NA, Santos RAM, Habu T, Tao R, Vieira CP. The number, age, sharing and relatedness of S-locus specificities in Prunus. Genet Res (2008) 90:17–26.[Web of Science][Medline]
Watanabe M, Ito A, Takada Y, Ninomiya C, Kakizaki T, Takahata Y, Hatakeyama K, Hinata K, Suzuki G, Takasaki T, et al. Highly divergent sequences of the pollen self-incompatibility (S) gene in class-I S haplotypes of Brassica campestris (syn. rapa) L. FEBS Lett (2000) 473:139–144.[CrossRef][Web of Science][Medline]
Watari A, Hanada T, Yamane H, Esumi T, Tao R, Yaegaki H, Yamaguchi M, Beppu K, Kataoka I. A low transcriptional level of Se-RNase in the Se-haplotype confers self-compatibility in Japanese plum. J Am Soc Hort Sci (2007) 132:396–406.
Yamane H, Ushijima K, Sassa H, Tao R. The use of the S haplotype-specific F-box protein gene, SFB, as a molecular marker for S-haplotypes and self-compatibility in Japanese apricot (Prunus mume). Theor Appl Genet (2003) 107:1357–1361.[CrossRef][Web of Science][Medline]
Zhang S-L, Huang S-X, Kitashiba H, Nishio T. Identification of S-haplotype-specific F-box gene in Japanese plum (Prunus salicina Lindl.). Sex Plant Reprod (2007) 20:1–8.[CrossRef]
CiteULike 
Connotea 
Del.icio.us What's this?
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||