Reveals S Locus Duplications in the Ancestral Rosaceae ...

Evolutionary Analysis of Genes for S-RNase-based Self-incompatibilityReveals S Locus Duplications in the Ancestral Rosaceae

Takuya Morimoto, Takashi Akagi and Ryutaro Tao*

Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan

Flowering plants have developed a genetically determined self-incompatibility (SI) system to maintain geneticdiversity within a species. The Solanaceae, the Rosaceae, and the Plantaginaceae have the S-RNase-basedgametophytic SI (GSI) system, which uses S-RNase and F-box proteins as the pistil S and pollen Sdeterminants, respectively. SI is associated with culture and breeding difficulties in rosaceous fruit trees, suchas apple, pear, and stone fruit species; therefore, researchers in the pomology field have long studied themechanism and genetics of SI in order to obtain clues to overcome these difficulties. Here, we investigated theevolutionary paths of the S-RNase genes by tracking their duplication patterns. Phylogenetic analysis andestimation of proxy ages for the establishment of S-RNase and its homologs in several rosaceous speciesshowed that the divergence of S-RNase in the subtribe Malinae and the genus Prunus predated the gene inmost recent common ancestors of Rosaceae species. Furthermore, the duplicated S-RNase-like genes wereaccompanied by duplicated pollen S-like F-box genes, suggesting segmental duplications of the S locus.Analysis of the expression patterns and evolutionary speeds of duplicated S-RNase-like genes in Prunussuggested that these genes have lost the SI recognition function, resulting in a single S locus. Furthermore, theS loci in the current Rosaceae species might have evolved independently from the duplicated S loci, whichcould explain the presence of genus-specific SI recognition mechanisms in the Rosaceae. The results of thepresent study should be valuable for the future development of artificial SI control and for self-compatiblebreeding in rosaceous horticultural plant species.

Key Words: F-box gene, gene duplication, Malinae, Prunus.

Introduction

Self-incompatibility (SI) is one of the most importantreproductive mechanisms for maintaining genetic diver-sity within a species (de Nettancourt, 2001). It is widelyused in angiosperms, with at least 19 orders, 71 fami-lies, and 250 genera—comprising approximately 60%of angiosperm species—showing this behavior (Allenand Hiscock, 2008). Most herbaceous annual crops lostthe SI characteristic to become self-compatible (SC)during domestication because SI hinders effective cropproduction. In contrast, many fruit tree species and cul-tivars still retain SI, probably because of breeding diffi-culties associated with their long generation time and

Received; February 1, 2015. Accepted; March 3, 2015.First Published Online in J-STAGE on April 4, 2015.This work was supported by Grants-in-Aid (nos. 20248004,24248007, and 15H02431) for Scientific Research (A) from the JapanSociety for the Promotion of Science (JSPS) to R.T.* Corresponding author (E-mail: [email protected]).

large size. SI is associated with difficulties in effectivebreeding and culture practices; therefore, its mechanismand genetics have long interested researchers in thefields of agriculture and horticulture.

In most cases, SI is genetically controlled by a singlepolymorphic S locus that harbors pistil S determinantand pollen S determinant genes. The mechanism can beeither gametophytic (GSI) or sporophytic (SSI), de-pending on the genetic control of the pollen SI pheno-type (Tao and Iezzoni, 2010). The Solanaceae, theRosaceae, and the Plantaginaceae recruit the secretedcytotoxic ribonuclease (S-RNase) as pistil S determi-nant for their SI system, which is referred to as theS‑RNase-based GSI (McClure, 2009). The pollen Sdeterminant(s) encodes an F-box protein called SLF (S-locus F-box) in the Solanaceae and the Plantaginaceae,SFBB (S-locus F-box brothers) in the subtribe Malinae(Rosaceae), and SFB (S haplotype-specific F-boxprotein) in Prunus (Rosaceae) (Meng et al., 2011; Sassaet al., 2010; Tao and Iezzoni, 2010; Ushijima et al.,2003). The most recent common ancestor (MRCA) of

The Horticulture Journal 84 (3): 233–242. 2015.doi: 10.2503/hortj.MI-060

JSHS

�e Japanese Society for

Horticultural Sciencehttp://www.jshs.jp/

© 2015 The Japanese Society for Horticultural Science (JSHS), All rights reserved.

these plant families is the root of about 75% of all di-cots; therefore, S-RNase-based GSI is considered to bean ancestral state of the majority of dicots, which issupposed to have been present approximately 120 mil-lion years ago (Igic and Kohn, 2001; Steinbachs andHolsinger, 2002; Vieira et al., 2008). This would alsoimply that loss of the S-RNase-based GSI system andgain of another SI system could have occurred frequent-ly during lineage speciation (Sherman-Broyles andNasrallah, 2008).

Despite the single origin of S-RNase-based GSI andthe commonality of specificity determinant genes, sev-eral lines of evidence indicate that the SI recognitionmechanism in Prunus in the Rosaceae might differ fromthose in the other species (Hauck et al., 2006; Tao andIezzoni, 2010; Ushijima et al., 2004). It has been pro-posed that the function of multiple (or a single) pollen SF-box genes at the S locus are responsible for detoxifi-cation of all but the self S-RNase in the Solanaceae, thePlantaginaceae, and the subtribe Malinae in theRosaceae (Kakui et al., 2011; Kubo et al., 2010;Minamikawa et al., 2010; Sassa et al., 2007; Zhou et al.,2003), while a single pollen S determinant (SFB) isresponsible for the cytotoxicity of the self S-RNase inPrunus (Tao and Iezzoni, 2010). This hypothesis issupported by the different outcome of hetero-diallelicpollen production and pollen S mutation in Prunuscompared with the outcome in the Solanaceae, thePlantaginaceae, and the subtribe Malinae (Hauck et al.,2006; Ushijima et al., 2004). The presence of a hypo-thetical general inhibitor that detoxifies all S-RNase isproposed in Prunus to explain the Prunus-specific SIrecognition (Tao and Iezzoni, 2010).

Gene duplication has been reported to have played animportant role in the evolution of plants (Flagel andWendel, 2009). A prevailing theory predicts gene loss(pseudogenization) or functional diversification (sub- orneofunctionalization) as the main fates for one of thetwo copies of duplicated genes (Blanc and Wolfe, 2004;Cusack and Wolfe, 2007; Moore and Purugganan,2005). Of the two functional diversification mecha-nisms, subfunctionalization, in which each duplicategene develops a distinct expression pattern as models ofcis-evolution (Carroll, 2008), seems to be the mostcommon phenomenon and takes place soon after geneduplication (Papp et al., 2003). In contrast, neofunction-alization is the result of diversification over the longterm (Rastogi and Liberles, 2005).

Previous studies have demonstrated that S-RNase-based GSI is monophyletic and evolved in eudicotsbefore the divergence of asterids and rosids (Igic andKohn, 2001; Steinbachs and Holsinger, 2002; Vieiraet al., 2008). Several S-RNase-like genes have beenidentified from certain plant species with S-RNase-based GSI. The non-S-RNases, showing homology to S-RNase, have been reported in Petunia inflata (Lee et al.,1992), Nicotiana alata (Kuroda et al., 1994), and

Prunus avium (Yamane et al., 2003). The non-S-RNasesfound in the Solanaceae are assumed to have arisenfrom the duplication of the S locus, but are not associ-ated with self-incompatibility (Golz et al., 1998; Igicand Kohn, 2001; Lee et al., 1992). Although non-S-RNase in Prunus was reported to be a possible candi-date for an ancestral form of S-RNase in Prunus(Yamane et al., 2003), its physiological functions areyet to be clarified. Detailed evolutionary analysis of S-RNase and pollen S F-box genes would be useful to un-derstand the origin and evolutionary paths of S-RNase-based GSI in the Rosaceae, and provide an insight intothe molecular basis of S-RNase-based GSI in rosaceousspecies.

Recent advances in genome sequencing have enabledanalysis of angiosperm genome evolution usinggenome-wide comparative analysis. According to thePhytozome database v9.1 (http://www.phytozome.net/),41 sequenced and annotated plant genomes, which havebeen clustered into 20 evolutionarily significant nodes,are available. In this study, we investigated the evolu-tionary paths of S-RNase and its homologs, mainly bytracking their duplication patterns in genomes. Ourresults indicates that S-RNase and its homologs wereduplicated in the ancestral genome of Rosaceae, andthe S loci in the current Rosaceae species have evolvedindependently from the duplicated S loci. The resultsof this study on rosaceous S locus evolution could ex-plain the distinct recognition mechanisms present inthe Rosaceae and will be valuable for the future devel-opment of artificial SI control and SC breeding inrosaceous species.

Materials and Methods

Gene collectionPutative full-length sequences of 38 S-RNase-like

genes were identified from the genomes of seven angio-sperms and outgroup species, Selaginella andPhyscomitrella, using BLASTp in Phytozome (version9.1, http://www.phytozome.net/). We selected genesthat showed significant homology (e−19 cut-off for allangiosperms and e−10 cut-off for others) to S3-RNase(accession no. AB010306) from Prunus avium. Theamino acid sequences with significant BLASTp hitswere subjected to gene ontology (GO) analysis byscanning the Pfam database (Finn et al., 2014) for thepresence of a ribonuclease T2 motif. Informationconcerning the physical locations and structure of geneswas based on the genome sequence obtained from thePhytozome database.

Putative full-length sequences of pollen S F-box-likegenes were searched for around the regions of the S-RNase-like genes in the Rosaceae genome usingPhytozome Gbrowse. We identified F-box genes show-ing significant homology (e−19 cut-off) to pollen SF‑box genes in asterids and the subtribe Malinae. Thepollen S of Prunus, SFB, was excluded from analyses in

234 T. Morimoto, T. Akagi and R. Tao

this study because it is still unclear whether PrunusSFB or asterid SLF is more closely related to PrunusSLFLs.

Construction of evolutionary topologyAlignment analyses on amino acid sequences were

conducted using MAFFT ver.7 with the L-INS-I model(Katoh and Standley, 2013). The raw alignments weresubjected to manual revision using Sea View ver.4(Gouy et al., 2010). Unnecessarily long gap sequences,which disturb the proper alignment of orthologous se-quences, and genes showing apparently different struc-tures from other S-RNase-like genes were removed. Theneighbor joining (NJ) approach was applied to defineevolutionary topology using Mega v5.05 (Tamura et al.,2011) with 1,000 bootstrap replications.

Estimation of the proxy age of gene divergenceWe aligned the amino acid sequences of each pair of

homologs using MAFFT with the L-INS-I model, andconverted the amino acid alignments to nucleotidealignments using PAL2NAL (Suyama et al., 2006). Wecalculated the transversion rate at fourfold-degeneratesites (4DTv value) between the gene pairs usingMicrosoft Excel 2007 (Microsoft). Here, the fourfold-degenerate sites were the codons of amino acid residuesG, A, T, P, V, R, S, and L. The 4DTv values were calcu-lated for gene pairs retaining at least 23 fourfold-degenerate sites. For calculation of the 4DTv betweenspecific gene groups, we averaged the 4DTv valuesfrom all combinations of the genes between the twogroups. Aligned nucleotide sequences were also ana-lyzed to calculate the values of synonymous substitu-tions per synonymous site (Ks), non-synonymoussubstitutions per non-synonymous site (Ka), and theratio of Ka/Ks, using DnaSP 5.1 (Librado and Rozas,2009).

Gene expression analysisTo perform organ-specific PCR analysis for S-

RNase-like genes, total RNA from leaves, calyxes, pet-als, filaments, pollen, styles, and ovaries was isolatedfrom P. avium (‘Satonishiki’) and P. mume (‘Nanko’)using the cold-phenol extraction method, as describedby Tao et al. (1999). Total RNA of pistil tissue was alsoisolated from P. persica (‘Akatsuki’) and P. salicina(‘Sordum’). cDNA was synthesized from 100 ng oftotal RNA using the ReverTra Ace qPCR RT MasterMix with gDNA Remover (Toyobo, Osaka, Japan),according to the manufacturer’s protocol. Gene-specificprimers for each S-RNase-like gene were designedusing online software Primer3 (http://frodo.wi.mit.edu/primer3/): 5'-GTTGCCCAAGGAAAAGACAA-3' and5'-GTCGCGTTTGTTGGAAAGAT-3' for ppa011133m(non-S-RNase1); and 5'-AGTGCTCCGACGACAAGTTT-3' and 5'-ATTGCTCGCAAAGGAGAAGA-3' forppa024151m (non-S-RNase2). Primers 5'-ACCATAA

CGTTGGAGGTGGA-3' and 5'-GGAGACGAAGGACAAGGTGA-3' for ubiquitin (ppa005507m) were usedas a control. Reverse transcription PCR (RT-PCR) wasperformed in a 20-μL reaction volume including 1×ExTaq buffer (TaKaRa Bio, Shiga, Japan), 0.2 mMdNTPs, 100 nM each of the forward and reverse pri-mers, 0.5 U ExTaq, and cDNA equivalent to the amountsynthesized from 10 ng of total RNA. The PCR cyclewas: initial denaturation at 94°C for 3 min; then 35 cy-cles of 94°C for 30 s, 55°C for 30 s, and 72°C for 1 min;followed by final elongation at 72°C for 5 min. ThePCR products were electrophoresed through a 1.5%agarose gel, stained with ethidium bromide, and visual-ized under UV radiation.

Identification of selective pressure on the S-RNase-likegenes

Full-length allele sequences of S-RNase in theSolanaceae and the subtribe Malinae, and S-RNase,non-S-RNase1, and non-S-RNase2 in Prunus, were ob-tained from the NCBI and Phytozome databases. Genesequence orthologous to ppa024151m in P. avium andP. salicina were obtained by direct sequencing of PCRproducts. Primers were designed outside of the ORF re-gion in ppa024151m, using sequences from the peachgenome database (Genome Database for Rosaceae;GDR, Prunus_persica_v.1.0). Amino acid sequencealignments were constructed using MAFFT with theL‑INS-I model, and amino acid alignments were con-verted to nucleotide alignments using PAL2NAL.Informative single-nucleotide polymorphisms (SNPs)in the alleles were analyzed by DnaSP 5.1 and used tocalculate the evolutionary speed (Ka/Ks). Window-average Ka/Ks values were calculated from the startcodon (ATG) in 90-bp windows with a 20-bp walkingstep, until the walking window reached the stop codon.To calculate amino acid variability level, normed vari-ability indices (NVIs) for each site of the aligned alleleswere calculated as described by Kheyr-Pour et al.(1990) using Microsoft Excel 2007. Window-averageNVI values were calculated from the first amino acid(M) in the 11-AA window.

Results

Evolutionary patterns of S-RNase and its homologs inangiosperm genome

Comprehensive detection of the genes that showedsignificant homology to Prunus S-RNase with the ribo-nuclease T2 motif was performed by BLAST and geneontology (GO) analyses. To obtain a wide range ofphylogenetic data for S-RNase-like genes across angio-sperms, we selected representative genomes accordingto Angiosperm Phylogeny Group III (APGIII) (TheAngiosperm Phylogeny Group, 2009) as follows: Oryzasativa (monocot) as an outgroup of eudicots, Mimulusguttatus from asterids, Arabidopsis thaliana frommalvids, and Populus trichocarpa and three genomes

Hort. J. 84 (3): 233–242. 2015. 235

of the Rosaceae (Prunus persica, Fragaria vesca, andMalus × domestica) for fabits. Thirty-eight genes (onegene per locus) were identified as S-RNase-like genes.

Phylogenetic analysis of S-RNase genes in theSolanaceae, the Plantaginaceae, the subtribe Malinae,and Prunus, and the 38 S-RNase-like genes found inangiosperm genomes, indicated that the representativeS-RNase-like genes in angiosperms could be dividedinto three major classes [Angiosperm (AG) I–III], as re-ported previously (Igic and Kohn, 2001) (Fig. 1). Wecould define at least three S-RNase-like genes in themost recent common ancestor (MRCA) of the eudicots(triangles in Fig. 1), which corresponded to the diver-gence of the three major classes. For the AGI and AGIIclasses, the divergence patterns of the genes corre-sponded well to the lineage speciation in APGIII.For the AGIII class, which includes S-RNases of theSolanaceae, the Plantaginaceae, and the Rosaceae,

along with non-S-RNase1 (Yamane et al., 2003) fromPrunus, we could find only one gene in the MRCA ofeudicots, supporting the single origin of the S-RNase-based GSI system. The gene divergence patterns were,however, inconsistent with the lineage speciation, espe-cially for the Rosaceae species, in which the S-RNasesof the subtribe Malinae and Prunus diverged earlierthan the root of the Rosaceae species (circle in Fig. 1).

The detailed phylogenetic tree constructed to focuson the AGIII class resulted in almost the same topology,with significant statistical support (Fig. 2), comparedwith that obtained using all S-RNase-like gene se-quences (Fig. 1). Importantly, we could find only asingle gene in the MRCA of Rosaceae for the non-S-RNase2 clade, which included genes from threeRosaceae species (Fragaria, Prunus, and Malus)(circled in Fig. 2). Here, we reconfirmed that the diver-gence of the S-RNases of the subtribe Malinae and

Fig. 1. Phylogenetic tree of S-RNase-like genes and S-RNases in the angiosperm genome. Phylogenetic tree for S-RNase-like genes from sevenangiosperm genomes, along with S-RNases from asterids and Rosaceae, and outgroup species, Physcomitrella patens and Selaginellamoellendorffii, constructed using amino acid sequences and the neighbor joining method. The orthologous genes in monocots are shown asstars using rice (Oryza sativa) genes. The putative genes in the MRCA of eudicots and Rosaceae are indicated by triangles and circles,respectively. The prefixes “LOC”, “mgv”, “AT”, “Potri”, “ppa”, “MDP”, and “mrna” correspond to Oryza sativa, Mimulus guttatus,Arabidopsis thaliana, Populus trichocarpa, Prunus persica, Malus × domestica, and Fragaria vesca, respectively.

236 T. Morimoto, T. Akagi and R. Tao

Prunus predated the divergence of the Rosaceae spe-cies.

Proxy age of gene divergenceWe calculated the genetic distance of each gene clade

in the AGIII class (Fig. 2). The mean 4DTv value be-tween the S-RNases from Prunus and the subtribeMalinae (0.39 ± 0.03) was significantly higher than thatbetween the non-S-RNase2 genes from Prunus and thesubtribe Malinae (0.28 ± 0.03). Furthermore, the mean4DTv value between mrna00224.1-v1.0-hybrid andother genes in the non-S-RNase2 clade (0.42 ± 0.01)was higher than that between the non-S-RNase2 genesfrom Prunus and the subtribe Malinae, while no signifi-cant difference was observed between the mean 4DTvvalue between mrna00224.1-v1.0-hybrid and othergenes in the non-S-RNase2 clade and that between theS-RNases from Prunus and the subtribe Malinae. Themean Ks value between the S-RNase genes fromPrunus and the subtribe Malinae (0.78 ± 0.004) wassignificantly higher than that between the non-S-RNase2 genes from Prunus and the subtribe Malinae(0.61 ± 0.015) and that between mrna00224.1-v1.0-

hybrid and other genes in the non-S-RNase2 clade(0.70 ± 0.013) (Table 1). Collectively, these results sup-ported the topology of the phylogenetic tree (Fig. 2),and indicated that the current S-RNases of the subtribeMalinae and Prunus may have been generated by agene duplication event that occurred before the estab-lishment of the Fragaria, Prunus, and Malinae.

It was reported that S-RNases in asterids and the sub-tribe Malinae have one intron, whereas Prunus S-RNasehas an additional intron located in the upstream regionof the common intron present in all S-RNases (Igic andKohn, 2001). Prunus non-S-RNase1, on the other hand,has only one intron, located at the same position as theintrons of asterids and the subtribe Malinae S-RNases(Yamane et al., 2003). The genes in the non-S-RNase2clade also have only one intron at the same position asthe common introns of all S-RNases, indicating thatnon-S-RNases have maintained the original structure ofS-RNase (Fig. 2). The additional intron found only inPrunus S-RNase may have been produced after the geneduplication that generated non-S-RNase1 and Prunus S-RNase.

Fig. 2. Phylogenetic tree, genetic distance and gene structure of S-RNase-like genes and S-RNases in Angiosperm class III. Phylogenetic tree forS-RNase-like genes in Angiosperm class III (Fig. 1), along with S-RNases from asterids and Rosaceae, and non-S-RNase1 and non-S-RNase2genes from Prunus, constructed using amino acid sequences and the neighbor joining method. The putative genes in the MRCA of Rosaceaeare shown in a circle. Intron presence/absence data for each clade are shown as a schematic diagram.

Hort. J. 84 (3): 233–242. 2015. 237

Evolutionary patterns of pollen S F-box-like genesIn the surrounding regions of the S-RNase-like genes

in the Rosaceae, we found F-box genes showing signifi-cant homology to pollen S F-box genes in asterids andthe subtribe Malinae (Fig. 3). The phylogenetic treesconstructed from pollen S F-box-like genes and theircounterpart S-RNases showed almost the same topolo-gies, with significant statistical support (Figs. 2 and 3),suggesting that the F-box/RNase segments of the Slocus were duplicated. Conversely, no F-box gene wasdetected in the proximity of the non-S-RNase1 gene inthe P. persica genome.

Expression of duplicated S-RNase-like genes in PrunusTo explore the possibility of change in cis-functions

between duplicated S-RNase-like genes, expressionanalysis was conducted with non-S-RNase1 and non-S-RNase2 genes of Prunus species. Although the S-RNase-like genes were expressed mainly in the style(Fig. 4A), the expression patterns differed among spe-cies in Prunus (Fig. 4B). Expression of non-S-RNase1(ppa011133m) was detected in the styles of P. avium,P. mume, and P. persica, but not in those of P. salicina.The non-S-RNase2 (ppa024151m) showed significantexpression in the styles of P. mume and P. persica, butnot in those of P. avium and P. salicina.

Selective pressure on duplicated S-RNase-like genesSelective pressure on non-S-RNase1 and non-S-

RNase2 genes in Prunus, and S-RNases in asterids, inthe subtribe Malinae, and in Prunus, was investigated interms of the Ka/Ks and NVI, which were previouslyused to assess the amino acid variable sites of S-RNases(Ioerger et al., 1991; Kheyr-Pour et al., 1990; Ushijimaet al., 1998). A sliding window analysis indicated thatKa/Ks peaks appeared in the hypervariable (HV) region(Ishimizu et al., 1998a, b; Ushijima et al., 1998) in S-RNases of asterids and Prunus, while no such indica-tion was observed for S-RNases of the subtribe Malinae(Fig. 5A). The window-averaged plot of NVI clearlyindicated that the HV region of all S-RNases showed

significantly high NVI values (NVI > 0), indicatingpositive selection in this region (Fig. 5B). No sign ofpositive selection was detected in the region corre-sponding to the Rosaceae hypervariable (RHV) regionsof duplicated S-RNase-like genes.

Discussion

Previous studies suggested that the S-RNase-basedGSI system evolved once in eudicots (Igic and Kohn,2001; Steinbachs and Holsinger, 2002; Vieira et al.,2008). Consistent with this, our results supported a sin-gle origin of the S-RNase-based GSI system in eudicots(Figs. 1 and 2). However, we identified segmental du-plications of the S locus in the Rosaceae lineage. Figure6 shows a schematic model of the duplications of S-RNase and pollen S F-box genes. The current S-RNasesin asterids and Rosaceae are thought to have originatedfrom a single original S-RNase. After the divergence ofthe asterid and rosid ancestors, the original S-RNasewas duplicated in the ancestral early-stage genome ofthe Rosaceae, producing the S-RNases of the subtribeMalinae (Dupli. S-I in Fig. 6). Subsequently, anotherancestral S-RNase produced by this duplication wasthought to have been duplicated again to produce theancestral S-RNases of Prunus and the non-S-RNase2genes (Dupli. S-II). Note that we could not find genesorthologous to the S-RNases of the subtribe Malinae inPrunus and Fragaria, nor those to S-RNase of Prunusin the subtribe Malinae and Fragaria. Similar patternsof duplication and evolutionary paths have been sug-gested for the S locus F-box gene divergence. As ob-served in the evolutionary paths of S-RNases, twoduplications that generated SFBB of the subtribeMalinae and Prunus SLFLs (Entani et al., 2003;Matsumoto et al., 2008; Ushijima et al., 2003) were de-fined by Dupli. S-I and Dupli. S-II, respectively. Withinthe range of the genes analyzed in this study, the originof SFB, the pollen S F-box gene of Prunus, remains un-known.

Recent studies suggested that duplicated genes ex-perience subfunctionalization, mainly in their cis-

Table 1. Ka/Ks values for S-RNase and S-RNase-like genes in Rosaceae.

Ksz (Jukes-Cantory) Kaz (Jukes-Cantory) Ka/Ksz (Jukes-Cantory)

Within Prunus S-RNase 0.15 ± 0.026 (0.21 ± 0.018) 0.12 ± 0.025 (0.13 ± 0.027) 0.75 ± 0.093 (0.69 ± 0.146)Within Malinae S-RNase 0.23 ± 0.038 (0.27 ± 0.053) 0.21 ± 0.016 (0.25 ± 0.023) 0.97 ± 0.104 (0.96 ± 0.122)Within Prunus non-S-RNase1 0.07 ± 0.016 (0.08 ± 0.017) 0.05 ± 0.007 (0.05 ± 0.008) 0.78 ± 0.120 (0.77 ± 0.126)Prunus S-RNase vs. Malinae S-RNase 0.78 ± 0.004 (n.a.x) 0.46 ± 0.003 (0.71 ± 0.007) 0.59 ± 0.006 (n.a.)Non-S-RNase1 vs. Malinae S-RNase 0.79 ± 0.006 (n.a.) 0.53 ± 0.005 (0.92 ± 0.017) 0.67 ± 0.007 (n.a.)Non-S-RNase1 vs. Prunus S-RNase 0.68 ± 0.008 (1.81 ± 0.104) 0.43 ± 0.002 (0.65 ± 0.004) 0.64 ± 0.008 (0.36 ± 0.018)

Prunus non-S-RNase2 vs. Malinae non-S-RNase2 0.61 ± 0.015 (1.28 ± 0.080) 0.32 ± 0.003 (0.41 ± 0.005) 0.51 ± 0.017 (0.32 ± 0.024)mrna00224.1-v1.0-hybrid vs. other genes in non-S-RNase2 clade (the MRCA of Rosaceae) 0.70 ± 0.013 (2.03 ± 0.214) 0.45 ± 0.003 (0.69 ± 0.007) 0.65 ± 0.014 (0.35 ± 0.035)

z Values between the two genes were calculated as described by Nei and Gojobori (1986).y Values are Jukes-Cantor-corrected.x Not available.

Table 1. Ka/Ks values for S-RNase and S-RNase-like genes in Rosaceae.238 T. Morimoto, T. Akagi and R. Tao

Fig. 3. Phylogenetic tree and gene location of pollen S F-box-like and pollen S F-box genes in Prunus, Fragaria, and the subtribe Malinae in theRosaceae, along with the asterid pollen S F-box gene. For pollen S F-box genes in the Solanaceae and the tribe Malinae, we included allSLFs identified in the S11 haplotype of Petunia × hybrida (Kubo et al., 2015) and all SFBBs from the S3 haplotype of Pyrus pyrifolia (Kakuiet al., 2011). The phylogenetic tree was constructed using amino acid sequences and the neighbor joining method. The pollen S of Prunus,SFB, was not used to construct this tree because it is still unclear whether Prunus SFB or asterid SLF should be used as the outgroup. Theputative genes in MRCA of Rosaceae are shown in a circle. Each symbol in the phylogenetic tree corresponds to those in the schematicdiagram of S-RNase evolution shown in the inset figure (upper left).

Fig. 4. Expression profiling of the S-RNase-like genes in Prunus. (A) Organ-specific expression profiles of non-S-RNase1 and non-S-RNase2 inP. avium (left) and P. mume (right). RT-PCR was performed on samples from leaves (Lf), calyx (C), petal (Pe), filament (Fi), pollen (Po),style (Sty), and ovary (Ov). The expression of ubiquitin (ppa005507m) was used as a reference control. (B) Species-specific expression ofS‑RNase-like genes was determined by RT-PCR using cDNA prepared from the styles of P. persica, P. salicina, P. avium, and P. mume.

Hort. J. 84 (3): 233–242. 2015. 239

functions (Liu and Adams, 2010; Roulin et al., 2013).This study demonstrated that non-S-RNase1 and non-S-RNase2 genes, which are S-RNase-like genes, showedstyle-specific expression in some Prunus species, as didS-RNase in Prunus, while expression was not detectedin styles of other Prunus species tested, indicating thatcis-evolution or pseudogenization had taken place inthe duplicated genes. Regarding the maintenance of thetrans-functions in duplicated S-RNase-like genes, nosign of positive selection (balancing selection) on theHV region was observed for non-S-RNase1 and non-S-RNase2 genes. Given that the HV region is necessaryfor self/nonself recognition (Ishimizu et al., 1998a, b;Ushijima et al., 1998), the duplicated S-RNase-likegenes are thought to have lost the self/nonself recogni-tion function, leading to the presence a single S locusstate in Prunus.

Recently, a diploid Fragaria was reported to havetwo independent self-incompatibility loci (S and T) con-trolling S-RNase-based GSI (Bošković et al., 2010).Comparative mapping analysis showed partial syntenybetween the T (LG6) and S (LG1) loci and Prunus LG6on which the S locus lies (Bošković et al., 2010). In our

phylogenetic analysis, five genes were identified as S-RNase-like genes in the genome of Fragaria vesca, ofwhich mrna00224 and mrna00227 are located aroundthe T locus on chromosome 6 and are clustered closelytogether with the Prunus S-RNase in the phylogenetictree. Mrna13010, located on chromosome 1, appearedto diverge at an early stage of the ancestral genome ofthe Rosaceae. Therefore, it is possible that the S-RNase-based GSI system in Fragaria has evolved dif-ferently from those of the subtribe Malinae and Prunus.It should be noted, however, that Fragaria vesca is anSC species, and was reported to have no RNase activityin its style (Bošković et al., 2010). No mutation produc-ing a premature stop codon could be observed in codingsequences of mrna00224, mrna00227, and mrna13010within the range of our analyses, so it is possible thatthere are other mutations that affect RNase activity or,alternatively, these genes are not related to self-incompatibility of Fragaria.

In conclusion, the S locus and its specificity determi-nant genes have experienced several duplications dur-ing the evolution of the Rosaceae. Furthermore, the S-RNase-based GSI in the Rosaceae species have evolved

Fig. 5. Selective pressure on S-RNase-like genes in Prunus and S-RNases in asterids, the subtribe Malinae, and Prunus. Window-average Ka/Ksand NVI values in the S-RNase gene of asterids, the subtribe Malinae, and Prunus, and non-S-RNase1 and non-S-RNase2 in Prunus. Con-served (C1–C5) and hypervariable (HV) regions are shown by closed and hatched bars, respectively. For non-S-RNase1 and non-S-RNase2,those regions were estimated by alignment with the S-RNase sequence. (A) Window-average Ka/Ks values in 90-bp sliding windows with a20-bp walking step. (B) Window-average NVI values in 11-AA sliding windows.

240 T. Morimoto, T. Akagi and R. Tao

independently from the duplicated S loci, recruiting dif-ferent duplicated genes, which may explain the genus-specific SI recognition systems present in the Rosaceae.The genus-specific SI recognition mechanisms, in turn,should be considered for the future development of arti-ficial SI controls and for SC breeding.

Literature CitedAllen, A. M. and S. J. Hiscock. 2008. Evolution and phylogeny of

self-incompatibility systems in Angiosperms. p. 73–102. In:V. E. Franklin-Tong (ed.). Self-incompatibility in floweringplants. Springer-Verlag, Berlin.

Blanc, G. and K. H. Wolfe. 2004. Widespread paleopolyploidy inmodel plant species inferred from age distributions of dupli-cate genes. Plant Cell 16: 1667–1678.

Bošković, R., D. J. Sargent and K. R. Tobutt. 2010. Genetic evi-dence that two independent S-loci control RNase-based self-incompatibility in diploid strawberry. J. Exp. Bot. 61: 755–763.

Carroll, S. B. 2008. Evo-Devo and an expanding evolutionarysynthesis: A genetic theory of morphological evolution. Cell134: 25–36.

Cusack, B. P. and K. H. Wolfe. 2007. When gene marriages don’twork out: divorce by subfunctionalization. Trends Genet. 23:270–272.

de Nettancourt, D. 2001. Incompatibility and incongruity in wildand cultivated plants. Springer, Berlin.

Entani, T., M. Iwano, H. Shiba, F. S. Che, A. Isogai and S.Takayama. 2003. Comparative analysis of the self-incompatibility (S-) locus region of Prunus mume: identifi-

cation of a pollen-expressed F-box gene with allelicdiversity. Genes Cells 8: 203–213.

Finn, R. D., A. Bateman, J. Clements, P. Coggill, R. Y. Eberhardt,S. R. Eddy, A. Heger, K. Hetherington, L. Holm, J. Mistry,E. L. L. Sonnhammer, J. Tate and M. Punta. 2014. Pfam: theprotein families database. Nucl. Acids Res. 42: D222–D230.

Flagel, L. E. and J. F. Wendel. 2009. Gene duplication and evolu-tionary novelty in plants. New Phytol. 183: 557–564.

Golz, J. F., A. E. Clarke, E. Newbigin and M. Anderson. 1998. Arelic S-RNase is expressed in the styles of self-compatibleNicotiana sylvestris. Plant J. 16: 591–599.

Gouy, M., S. Guindon and O. Gascuel. 2010. SeaView version 4:A multiplatform graphical user interface for sequence align-ment and phylogenetic tree building. Mol. Biol. Evol. 27:221–224.

Hauck, N. R., H. Yamane, R. Tao and A. F. Iezzoni. 2006. Accu-mulation of nonfunctional S-haplotypes results in the break-down of gametophytic self-incompatibility in tetraploidPrunus. Genetics 172: 1191–1198.

Igic, B. and J. R. Kohn. 2001. Evolutionary relationships amongself-incompatibility RNases. Proc. Natl. Acad. Sci. USA 98:13167–13171.

Ioerger, T. R., J. R. Gohlke, B. Xu and T. H. Kao. 1991. Primarystructural features of the self-incompatibility protein insolanaceae. Sex. Plant Reprod. 4: 81–87.

Ishimizu, T., T. Endo, Y. Yamaguchi-Kabata, K. T. Nakamura, F.Sakiyama and S. Norioka. 1998a. Identification of regions inwhich positive selection may operate in S-RNase ofRosaceae: Implication for S-allele-specific recognition sitesin S-RNase. FEBS Letters 440: 337–342.

Ishimizu, T., T. Shinkawa, F. Sakiyama and S. Norioka. 1998b.Primary structural features of rosaceous S-RNases associ-

Fig. 6. Schematic diagram of the establishment of S-RNase and pollen S F-box genes in the Rosaceae. After the divergence of the asterid androsid ancestor, the first S-RNase duplication occurred in the early-stage ancestral genome of Rosaceae, producing the S-RNase of the subtribeMalinae (Dupli. S-I). Subsequently, another S locus was duplicated into the ancestral S locus of Prunus and non-S-RNase2 (Dupli. S-II). Asimilar evolutionary pattern was found for pollen S F-box gene divergence. Two duplication events that generated SFBB of the subtribeMalinae and SLFLs of Prunus correspond to Dupli. S-I and Dupli. S-II, respectively, indicating the segmental duplication of the S locus.Prunus S-RNase lineage-specific gene duplication may have produced non-S-RNase1 of Prunus before Rosaceae species divergence (trian-gle). Dotted line indicates the time of the establishment of Fragaria, Prunus, and the subtribe Malinae. Putative models of the evolution ofthe S-RNase and pollen S F-box genes in Fragaria are shown in thin lines as suspicious branches. Within the range of the genes used in thisstudy, the origin of the pollen S of Prunus, SFB, could not be determined.

Hort. J. 84 (3): 233–242. 2015. 241

ated with gametophytic self-incompatibility. Plant Mol. Biol.37: 931–941.

Kakui, H., M. Kato, K. Ushijima, M. Kitaguchi, S. Kato andH. Sassa. 2011. Sequence divergence and loss-of-functionphenotypes of S locus F-box brothers genes are consistentwith non-self recognition by multiple pollen determinants inself-incompatibility of Japanese pear (Pyrus pyrifolia). PlantJ. 68: 1028–1038.

Katoh, K. and D. M. Standley. 2013. MAFFT Multiple SequenceAlignment Software Version 7: Improvements in Perfor-mance and Usability. Mol. Biol. Evol. 30: 772–780.

Kheyr-Pour, A., S. B. Bintrim, T. R. Ioerger and R. Remy. 1990.Sequence diversity of pistil S-proteins associated with game-tophytic self-incompatibility in Nicotiana alata. Sex. PlantReprod. 3: 88–97.

Kubo, K., T. Entani, A. Takara, N. Wang, A. M. Fields, A. Hua,M. Toyoda, S. Kawashima, T. Ando, A. Isogai, T. H. Kaoand S. Takayama. 2010. Collaborative non-self recognitionsystem in S-RNase-based self-incompatibility. Science 330:796–799.

Kubo, K., T. Paape, M. Hatakeyama, T. Entani, A. Takara, K.Kajihara, M. Tsukahara, R. Shimizu-Inatsugi, K. K. Shimizuand S. Takayama. 2015. Gene duplication and genetic ex-change drive the evolution of S-RNase-based self-incompatibility in Petunia. Nature Plants 1: 14005.

Kuroda, S., S. Norioka, M. Mitta, I. Kato and F. Sakiyama. 1994.Primary structure of a novel stylar RNase unassociated withself-incompatibility in tobacco plant, Nicotiana alata. J.Protein Chem. 13: 438–439.

Lee, H. S., A. Singh and T. H. Kao. 1992. RNase X2, a pistil-specific ribonuclease from Petunia inflata, shares sequencesimilarity with solanaceous S proteins. Plant Mol. Biol. 20:1131–1141.

Librado, P. and J. Rozas. 2009. DnaSP v5: a software for compre-hensive analysis of DNA polymorphism data.Bioinformatics 25: 1451–1452.

Liu, S. L. and K. L. Adams. 2010. Dramatic change in functionand expression pattern of a gene duplicated by polyploidycreated a paternal effect gene in the Brassicaceae. Mol. Biol.Evol. 27: 2817–2828.

Matsumoto, D., H. Yamane and R. Tao. 2008. Characterization ofSLFL1, a pollen-expressed F-box gene located in the PrunusS locus. Sex. Plant Reprod. 21: 113–121.

McClure, B. 2009. Darwin’s foundation for investigating self-incompatibility and the progress toward a physiologicalmodel for S-RNase-based SI. J. Exp. Bot. 60: 1069–1081.

Meng, X., P. Sun and T. H. Kao. 2011. S-RNase-based self-incompatibility in Petunia inflata. Ann. Bot. 108: 637–646.

Minamikawa, M., H. Kakui, S. Wang, N. Kotoda, S. Kikuchi, T.Koba and H. Sassa. 2010. Apple S locus region represents alarge cluster of related, polymorphic and pollen-specific F-box genes. Plant Mol. Biol. 74: 143–154.

Moore, R. C. and M. D. Purugganan. 2005. The evolutionarydynamics of plant duplicate genes. Curr. Opin. Plant Biol. 8:122–128.

Nei, M. and T. Gojobori. 1986. Simple methods for estimating thenumbers of synonymous and nonsynonymous nucleotidesubstitutions. Mol. Biol. Evol. 3: 418–426.

Papp, B., C. Pál and L. D. Hurst. 2003. Evolution of cis-regulatory elements in duplicated genes of yeast. TrendsGenet. 19: 417–422.

Rastogi, S. and D. A. Liberles. 2005. Subfunctionalization of du-plicated genes as a transition state to neofunctionalization.BMC Evol. Biol. 5: 28.

Roulin, A., P. L. Auer, M. Libault, J. Schlueter, A. Farmer, G.

May, G. Stacey, R. W. Doerge and S. A. Jackson. 2013. Thefate of duplicated genes in a polyploid plant genome. Plant J.73: 143–153.

Sassa, H., H. Kakui and M. Minamikawa. 2010. Pollen-expressedF-box gene family and mechanism of S-RNase-based game-tophytic self-incompatibility (GSI) in Rosaceae. Sex. PlantReprod. 23: 39–43.

Sassa, H., H. Kakui, M. Miyamoto, Y. Suzuki, T. Hanada, K.Ushijima, M. Kusaba, H. Hirano and T. Koba. 2007. S locusF-box brothers: Multiple and pollen-specific F-box geneswith S haplotype-specific polymorphisms in apple andJapanese pear. Genetics 175: 1869–1881.

Sherman-Broyles, S. and J. B. Nasrallah. 2008. Self-incompatibility and evolution of mating systems in theBrassicaceae. p. 123–147. In: V. E. Franklin-Tong (ed.).Self-incompatibility in flowering plants. Heidelberg:Springer-Verlag, Berlin.

Steinbachs, J. E. and K. E. Holsinger. 2002. S-RNase-mediatedgametophytic self-incompatibility is ancestral in eudicots.Mol. Biol. Evol. 19: 825–829.

Suyama, M., D. Torrents and P. Bork. 2006. PAL2NAL: robustconversion of protein sequence alignments into the corre-sponding codon alignments. Nucl. Acids Res. 34: W609–W612.

Tamura, K., D. Peterson, N. Peterson, G. Stecher, M. Nei and S.Kumar. 2011. MEGA5: Molecular evolutionary geneticsanalysis using maximum likelihood, evolutionary distance,and maximum parsimony methods. Mol. Biol. Evol. 28:2731–2739.

Tao, R. and A. M. Iezzoni. 2010. The S-RNase-based gameto-phytic self-incompatibility system in Prunus exhibits distinctgenetic and molecular features. Sci. Hortic. 124: 423–433.

Tao, R., H. Yamane, A. Sugiura, H. Murayama, H. Sassa and H.Mori. 1999. Molecular typing of S-alleles through identifica-tion, characterization and cDNA cloning for S-RNases insweet cherry. J. Amer. Soc. Hortic. Sci. 124: 224–233.

The Angiosperm Phylogeny Group. 2009. An update of theAngiosperm Phylogeny Group classification for the ordersand families of flowering plants: APGIII. Bot. J. Linn. Soc.161: 105–121.

Ushijima, K., H. Sassa, A. M. Dandekar, T. M. Gradziel, R. Taoand H. Hirano. 2003. Structural and transcriptional analysisof the self-incompatibility locus of almond: identification ofa pollen-expressed F-box gene with haplotype-specific poly-morphism. Plant Cell 15: 771–781.

Ushijima, K., H. Sassa, R. Tao, H. Yamane, A. M. Dandekar,T. M. Gradziel and H. Hirano. 1998. Cloning and characteri-zation of cDNAs encoding S-RNases from almond (Prunusdulcis): primary structural features and sequence diversity ofthe S-RNases in Rosaceae. Mol. Gen. Genet. 260: 261–268.

Ushijima, K., H. Yamane, A. Watari, E. Kakehi, K. Ikeda, N. R.Hauck, A. F. Iezzoni and R. Tao. 2004. The S haplotype-specific F-box protein gene, SFB, is defective in self-compatible haplotypes of Prunus avium and P. mume. PlantJ. 39: 573–586.

Vieira, J., N. A. Fonseca and C. P. Vieira. 2008. An S-RNase-based gametophytic self-incompatibility system evolvedonly once in eudicots. J. Mol. Evol. 67: 179–190.

Yamane, H., R. Tao, H. Mori and A. Sugiura. 2003. Identificationof a non-S RNase, a possible ancestral form of S-RNases, inPrunus. Mol. Gen. Genomics 269: 90–100.

Zhou, J., F. Wang, W. Ma, Y. Zhang, B. Han and Y. Xue. 2003.Structural and transcriptional analysis of S-locus F-boxgenes in Antirrhinum. Sex. Plant Reprod. 16: 165–177.

242 T. Morimoto, T. Akagi and R. Tao