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Mol Breeding (2010) 26:693–709 DOI 10.1007/s11032-010-9405-5

Analysis of S-RNase gene diversity based on a comparative study of old and modern and European wild apple

Rozemarijn S. G. Dreesen • Bartel T. M. Vanholme • Katrien Luyten • Lobke Van Wynsberghe • Gennaro Fazio • Isabel Rolda´n-Ruiz • Johan Keulemans

Received: 11 March 2009 / Accepted: 29 January 2010 / Published online: 16 February 2010 Ó Springer Science+Business Media B.V. 2010

Abstract Malus S-RNase genetic diversity was genes under negative frequency-dependent selection. analyzed in Malus 9 domestica cultivars and com- The majority of the M. 9 domestica S-alleles has pared to European wild apple (). been found in M. sylvestris as well, which points to Using PCR-based approaches, the S-RNase genotype strong conservation of the S-locus gene structure. of 140 M. 9 domestica cultivars, 196 M. sylvestris Based on the sequence of all different SCAR- and 27 M. sylvestris—M. 9 domestica hybrids fragments, which comprise both the hypervariable was determined. S-RNase allelic richness in M. PS1 region and the single intron, S-RNase genetic sylvestris was much higher than in M. 9 domestica, diversity was further explored. It provided some clues indicating the negative influence of domestication on to the occurrence of new S-alleles among the S-RNase diversity. Heterogeneity of the S-RNase multitude of novel S-RNase sequences that have allelic distribution is much higher in cultivated apple been identified, which were mostly unique for the than in wild apple, which shows that breeding leads group of M. sylvestris individuals. The determination to strong departure from the expected homogeneity of of the S-RNase genotypes of old cultivars and M. sylvestris will enable their introduction into new breeding strategies. As M. sylvestris has become an R. S. G. Dreesen (&) K. Luyten endangered in Belgium, the knowledge L. Van Wynsberghe J. Keulemans gathered in this study will be an important tool for Laboratory for Breeding and Biotechnology, KULeuven, Division of Crop Biotechnics, Willem de selecting useful genotypes for a core collection. Croylaan 42, box 2427, 3001 Heverlee, Belgium e-mail: [email protected] Keywords Malus 9 domestica Malus sylvestris Gametophytic self-incompatibility B. T. M. Vanholme Department of Systems Biology, Flemish Institute Hypervariable region SCAR S-allele-specific PCR of Biotechnology, Technologiepark 927, 9052 Ghent, Belgium Introduction G. Fazio Plant Genetic Resources Unit, USDA-ARS, North Street, Geneva, NY 14456, USA Woody fruit crops, such as apple, are grown and cultivated to produce fruit for human consumption I. Rolda´n-Ruiz purposes. But from a biological point of view, fruit Institute for Agricultural and Fisheries Research, Unit PLANT, Growth and Development, Caritasstraat 21, formation is also an important aspect of the repro- 9090 Melle, Belgium ductive cycle, since these plant organs contain the 123 694 Mol Breeding (2010) 26:693–709 seeds. Fruit set is a complex process which is initiated wild apple (Malus sylvestris (L.) Mill.). Both are by pollination and subsequent fertilization of the assumed to be closely related and the occurrence of ovules. For this, a pollen tube has to grow through hybridization between M. sylvestris and local apple stigmatic tissue and style, after which it enters the cultivars has been reported (Coart et al. 2006). The ovule. In apple, completion of this process is production of apple fruit is economically very dependent on a robust crossing barrier which is important for Belgian agriculture. However, com- active in the style and impedes outgrowth of those mercial apple cultivation relies on a small group of pollen grains which are genetically identical to the cultivars mostly grown in monocultures. The need for style tissue (de Nettancourt 1977). As such, out- the development and introduction of new cultivars breeding is guaranteed. This process is commonly has increased recently. The backbone of a successful referred to as gametophytic self-incompatibility breeding program is the availability of diverse (GSI) and is active in many members of the germplasm with useful attributes. Old, obsolete Solanaceae and Scrophulariaceae families, in addi- M. 9 domestica cultivars and wild relatives such as tion to the (Sims 2005). The underlying M. sylvestris are potential sources since these consti- mechanism is based on the interaction between two tute a reservoir of genetic variability, which is narrow gene products encoded by the S-locus, both expressed in current cultivars (Noiton and Alspach 1996). in a haplotype-specific fashion (de Nettancourt 1977): However, in line with the current drop in biodiversity a pistil-expressed T2-type S-RNase protein with due to loss of habitat, M. sylvestris has become an ribonuclease activity (Certal et al. 1999; Sassa et al. endangered species in many European regions, 1996) and a pollen-specific F-box type protein SLF including Flanders (North Belgium), revealing the (Cheng et al. 2006). urgent need for conservation measures. A direct consequence of the GSI mechanism is that Neutral DNA markers such as single sequence S-locus genes are multi-allelic. Indeed, population repeats (SSRs) have been used to describe genetic studies have shown that S-alleles are subject to diversity in both wild and cultivated apple (Garkava- negative frequency-dependent selection because rare Gustavsson et al. 2008; Koopman et al. 2007; alleles have access to more mates while common Vanwynsberghe 2006). However, the use of func- alleles are more frequently rejected (Castric and tional, gene-related markers is very important since Vekemans 2004). S-RNases have been explored these are directly related to traits. This study extensively in cultivated fruit-bearing species, includ- describes the genetic diversity of the S-RNase gene, ing apple (Malus 9 domestica). As such, many apple which is related to fertility, in a large collection S-RNase alleles have been cloned (Broothaerts 2003; composed of M. 9 domestica cultivars, M. sylvestris Broothaerts et al. 2004). These molecular data, genotypes and interspecific hybrids between these together with those from Pyrus and Prunus, have two species. For cultivated apple, the GSI mechanism made it possible to analyze the structure of S-RNase represents an obstacle to regular cropping, meaning genes in Rosaceae (Sassa et al. 1996; Ishimizu et al. that a balanced S-RNase diversity is crucial in a 1998a; Ushijima et al. 1998; Ortega et al. 2006). commercial orchard. It may also constitute a barrier Basically, the S-RNase protein consists of five for certain crosses between apple cultivars. As such, conserved regions (C1–C5) and one hypervariable knowledge of the S-RNase genotype is useful for region (RHV), located between C2 and C3. Sliding breeding strategies. S-RNases have never been stud- window analysis showed that four regions are subject ied before in M. sylvestris. At the onset of the to positive amino acid selection (defined as PS1 to research it was not known whether the GSI system is PS4—RHV is located within PS1; Ishimizu et al. functional in this species and thus to what extent this 1998b). These PS regions are believed to be key constitutes a genetic barrier for the introgression of factors in discrimination between self and non-self useful genetic variation into the cultivated gene pool. pollen, as all four are located on the sides of the Furthermore, knowledge of S-RNase diversity is active site cleft, making them possible interactors essential for the development of conservation guide- with substrates (Matsuura et al. 2001). lines for M. sylvestris. It would enable us to establish The present study is part of a large survey of a core collection of individuals which contains a large biodiversity in the cultivated apple and the European diversity of possible mating partners to ensure 123 Mol Breeding (2010) 26:693–709 695 persistence once reintroduced in nature. Suitable on the sequence diversity of the single Malus intron habitats for M. sylvestris are getting too scarce in region which is located in the RHV, data on which Flanders (Vander Mijnsbrugge et al. 2008). However, were non-existent at the onset of this research. Their the possibility remains that the disappearance of this analysis could be very useful in developing specific species in Flemish woods is also caused by the lack genotyping assays since such regions are known to be of suitable breeding partners. The comparison of hot spots for intra-allelic variation such as tandem S-RNase genetic diversity in wild and cultivated repeats. apple will provide us with data on the impact of domestication on this locus. Based on results of SSR diversity analyses in other crops and their wild Materials and methods relatives (Brown-Guedira et al. 2000; Sonnante et al. 1993; Reif et al. 2005), we expect it to lead to a Plant materials narrowing of genetic diversity. The rich diversity of the S-RNase allelic sequences A total of 37 modern cultivars and 103 old has led to the development of PCR assays using M. 9 domestica cultivars were included in this study. allele-specific primers, in some cases combined with The modern cultivars had been S-genotyped previ- restriction digestion (Broothaerts 2003; Kitahara and ously (Broothaerts et al. 2004) and were selected as a Matsumoto 2002a, b). Twenty different apple reference based on their S-allele diversity. Old S-alleles can be detected by this approach, making cultivars are defined here as cultivars and landraces it an important tool for S-genotype apple cultivars. which originated before 1940. Modern cultivars An alternative PCR-based approach is based on the resulted from controlled breeding practices after this use of primer sequences annealing to conserved period. Old cultivars were sampled from ‘‘Nationale regions flanking PS1 (Ishimizu et al. 1999; Matsum- Boomgaarden Stichting’’, ‘‘le Centre Wallon de oto and Kitahara 2000). In Malus and Pyrus the Recherches Agronomiques’’ and from the breeding single intron present interrupts the RHV region of the collection of the ‘‘Centre for Fruit Culture’’ S-RNase gene, which is a source of length polymor- (KULeuven). phisms (Igic and Kohn 2001). Using this SCAR- Most of the 223 M. sylvestris individuals included marker strategy, nine different apple S-alleles were in this study were sampled at ten sites in Belgian visualized in a selection of cultivars by Matsumoto woods and hedges (Fig. 1). Ninety-eight trees were and Kitahara (2000). In recent years, S-RNases have sampled in seven Walloon locations and are almost been isolated from other, Asiatic wild apple species all located in the geographical region ‘‘Les Ard- such as S34 and S35 from (P. Zhang ennes’’ which is characterized by large woods, a low 0 et al., unpublished data) and Sg from Malus transi- human population and almost no industry. Ninety- toria (Matsumoto et al. 2001). Preliminary analysis of one trees were sampled from three Flemish sites. these gene sequences indicated that the SCAR- Flanders is an industrialized region with a very high genotyping approach should be successful in deter- population density. These are the only places in this mining the S-RNase genotype of M. sylvestris.In region where small groups of wild apple trees are addition to the description of S-allelic frequencies present. Fourteen German and 20 French (Metz) and allelic richness, the detection and sequencing of genotypes were also included. If unknown, the ploidy SCAR-marker alleles would provide additional data. level was determined by flow cytometry. To identify First, it would become possible to estimate to which M. sylvestris—M. 9 domestica hybrids among the extent similar or identical S-RNase allelic sequences putative M. sylvestris trees, SSRs in combination with are circulating in M. sylvestris and M. 9 domestica STRUCTURE software (Pritchard et al. 2000) were germplasm. Second, our knowledge of the structure used as described in Coart et al. (2006). As such, and diversity of the RHV would improve significantly twenty-seven putative hybrids were identified in all and provide us with some clues about presumptive subgroups, except for ‘‘Heverleebos’’ and ‘‘Rance’’. novel functional S-RNase proteins that will be These trees were handled as a separate group for data identified in this study. Third, data could be gathered analysis.

123 696 Mol Breeding (2010) 26:693–709

agarose electrophoresis. In a second round, those samples which displayed long SCAR fragments ([600 bp) were reloaded onto 0.8% agarose gel whereas those displaying shorter fragments were re- analyzed by capillary electrophoresis on an ABI-3130 capillary sequencer (6-FAM-labeled FTQQYQ; Applied Biosystems, Foster City, CA USA).

S-allele-specific PCR- and PCR-RFLP-assays

When possible, results of the SCAR-genotyping approach were confirmed by available S-allele-specific PCR-genotyping assays. S-allele-specific PCR reac-

tions for S1, S2, S4, S5, S6, S7, S9, S11, S16, S17, S19, S20, Fig. 1 Sampling of Belgian M. sylvestris individuals. Leaf S , S , S and S were performed as described by samples were collected and processed as described in Materials 21 23 24 26 and Methods. Belgian sampling locations are indicated on the Broothaerts (2003). S3 was genotyped using primers map. The border between Flanders and Wallonia is indicated OWB149-OWB145 (Janssens et al. 1995) and the by a dotted line. Three sites are situated in Flanders cycling conditions of Broothaerts (2003). Specific PCR (Meerdaalwoud, Heverleebos and Voeren) and seven sites are 0 0 screenings for the S1 - and S9 -alleles were developed located in Wallonia (Spa, Verviers, Vielsalm, La Roche, 0 Houyet, Treignes, Rance). Numbers of trees sampled at each in this study (Fig. 2a, b). S1 /S1 genotyping was done location are indicated between brackets (bold: M. sylvestris, using S1-specific primers (Broothaerts 2003) followed italics: M. sylvestris—M. 9 domestica hybrids) by restriction digestion with FokI (600 at 37°C; Roche, 0 Basel, Switzerland). S9 /S9 genotyping occurred by 0 For each of these genotypes, DNA was extracted combining S9-specific PCR with BamHI-digestion (60 from lyophilized leaf material as described in Dum- at 37°C; Roche). In both cases the S0-allele fragment is 0 0 olin et al. (1995). cut in two fragments (S1 : 209 ? 290 bp and S9 : 313 ? 30 bp) whereas the S1 and S9 products remain S-RNase genotyping with a SCAR marker uncut. S17 can be distinguished from S6 by using an earlier described S-specific PCR assay (Broothaerts 0 The S-RNase genotype was determined using a 2003). The presence of S23 was confirmed using S23- SCAR-marker strategy that had been applied previ- specific primers of Broothaerts (2003), while S40 gives ously to characterize Malus and Pyrus S-RNases a positive result using an S4-specific assay (Broothaerts (Ishimizu et al. 1999; Matsumoto and Kitahara 2000) 2003). using primers against the conserved S-RNase regions In addition, 165 S-alleles (21.6%) among the FTQYQQ and IIPNV. As such, the resulting amplicon samples could not be amplified using the SCAR-based spans the intron region. Based on additional apple S- approach. Because of the large size of the predicted RNase allelic sequences and some preliminary results, SCAR-fragments of the alleles S8, S10, S16, S22 and S25, we introduced a subtle change to the reverse-oriented there were amplification difficulties under the PCR primer (Anti-IIPNV-FTC (50-ACRTTYGGCCAAA conditions applied. To check for their presence, all TMATT-30)). Additionally, we increased the anneal- samples for which the genotype was incomplete were ing temperature from 48 to 50°C to improve repro- screened using pre-existing S-allele-specific assays for ducibility. All reactions were done using 25 ng of S22 and S16 (Broothaerts 2003) and S10 and S25 DNA, 1 unit Taq (Genecraft, Lu¨dinghausen, Ger- (Kitahara and Matsumoto 2002a, 2002b). We devel- many) with its buffer, 0.8 lM of each primer, 2 mM oped an S8-specific assay based on partial mRNA MgCl2 and 0.4 mM dNTP mixture. Cycling condi- sequence information (AY744080; Fig. 2c) using tions were [20 94°C; 10x (1500 94°C/3000 50°C/20 72°C); oligonucleotides S8-F (50-CGATTATTTTCAATTTA 20 9 (1500 94°C/3000 50°C/203000 72°C); 70 72°C]. In a CGCTTCA-30) and S8-R (50-AGGTTGTTTCTTTG- first round, PCR products were separated by 0.8% CAATACTCTG-30) in an identical reaction mixture as 123 Mol Breeding (2010) 26:693–709 697

smallest sample was used as a rarefaction factor (26; representing the group of interspecific hybrids). To test the null hypothesis of equal allele frequencies among the Malus subgroups, modified chi-square (v2) statistics (P = 0.05) were computed as described in Campbell and Lawrence (1981) instead of the conventional v2 goodness-of-fit proce- dure. The method of Campbell and Lawrence has been especially developed to test the distribution of S-alleles among samples as it deals with the fact that in the sampled , these alleles occur in pairs and the occurrence of the second allele cannot be independent of the first since all individuals must be heterozygous for this gene. The modified formula for v2 goodness of fit is "#, Xn 2 2 2 v ¼ ðÞn 1 Cj 4r =n ðÞ2r 4r=n 0 0 j¼1 Fig. 2 S-allele-specific PCR-assays for S1 , S9 and S8. Within this study, S-allele-specific genotyping methods were devel- where (n-1) is the degrees of freedom, n the total oped. This figure shows PCR band patterns after amplification 0 number of different alleles found in the sample and r with specific primer sets. a S1 -genotyping. Agarose electro- phoresis of FokI-cut S1-specific PCR-products. Uncut PCR the number of plants which have been sampled. bands of 499 bp correspond to S1, while restriction leads to 2 bands of 209 and 290 bp in the case of S 0. 1. Hernaut 1 Sequencing of SCAR fragments (S1S3S17), 2. Joseph Musch (S1S10S46), 3. Boelpaep (S1S7S10), 0 0 4. Speeckaert NBS (S1 S17), 5. M. sylvestris no.171 (S1 S46), 6. 0 (S1S9), 7. (negative control, S9S24). b S9 - To link specific length polymorphisms to S-RNase genotyping. Agarose electrophoresis of BamHI-cut S9-specific alleles, more than half of the visualized SCAR PCR-products. Uncut PCR bands of 343 bp correspond to S9, while restriction leads to 2 bands of 313 and 30 bp in the case fragments were sequenced. For this, SCAR fragments 0 of S9 .1.M. sylvestris no.167 (S9Sx), 2. M. sylvestris no.454 were excised from agarose gel, eluted with the TM (S3S9), 3. Roter Eiserapfel (S6S9S24), 4. M. sylvestris no.185 Invisorb Spin DNA Extraction kit (Invitek, Berlin, 0 (S9 S25), 5. Fuji (S1S9), 6. (negative control, S3S5). c S8- Germany) and cloned into the pGEM-TÒ plasmid genotyping. Agarose electrophoresis of S -specific PCR 8 vector (Promega Corporation, Madison, Wisconsin products (155 bp). 1. Ontario (S1S8), 2. Zijden Hemdje (S6S8), 3. Pladei (S8S23Sx), 4. M. sylvestris no.180 (S8S41), 5. USA). Sequencing was done on 300 ng of purified Braeburn (negative control, S9S24) plasmid DNA and for three clones per fragment. Consensus sequences were obtained by assembling data from independent replicate sequences. Homol- above at [20 94°C; 35 9 (3000 94°C/3000 60°C/3000 ogy searches were performed using different BLAST 72°C); 70 72°C] yielding a product of 155 bp. algorithms (Altschul et al. 1990). Multiple sequence alignments were done by ClustalW (Thompson et al. S-allelic richness and S-allelic distribution 1994) using default parameter settings. Results were manually corrected with the BioEdit sequence align- Allelic richness is highly dependent on sample size ment editor. Boundaries between intron and exon (Nei et al. 1975). Therefore, the calculation of S- region of the SCAR fragments were determined by allelic richness was standardized to cope with uneven aligning mRNA against genomic sequences. When sample sizes. This was done by determinating the mRNA sequence information was not available, allelic richness after rarefaction (Pb) to a sample size splice sites were predicted on genomic sequences chosen by the investigator (El Mousadik and Petit using FSPLICE software implemented at the Soft- 1996). Calculations were done using the CONTRIB Berry server (www.softberry.com). Graphical pre- software (Petit et al. 1998). Half the size of the sentation of intron–exon boundaries was done using 123 698 Mol Breeding (2010) 26:693–709

WebLogo (Crooks et al. 2004). The percentage of Genetic diversity of the Malus S-RNase gene pairwise amino acid identity was calculated by Mat- GAT v2.02 (Campanella et al. 2003) using default Calculations of S-RNase allelic frequencies and parameters. allelic richness are displayed in Table 2. In order to cope with the large differences in population size which influence allelic diversity, we calculated allelic Results richness after rarefaction (Pb; Nei et al. 1975;El Mousadik and Petit 1996). From the results in Determination of the S-locus genotype Table 2 it is clear that M. sylvestris samples represent a high allelic richness (37 different S-RNase alleles The S-RNase genotype of 196 M. sylvestris individu- scored; Pb: 23.6), which is much higher than that als, 103 old and 37 modern M. 9 domestica cultivars observed for M. 9 domestica (28 different S-RNase and 27 interspecific M. sylvestris—M. 9 domestica alleles scored; Pb: 17.4). When comparing old with hybrids was determined by means of a modified modern cultivars we see that the Pb value for the first SCAR-marker strategy based on Ishimizu et al. (1999) group is higher (Pb: 18.0, 27 different S-RNase and Matsumoto and Kitahara (2000) in combination alleles) than for the second (Pb: 13.6; 17 different S- with S-allele-specific PCR-genotyping assays. Using RNase alleles). The Pb value of the hybrid group this approach, 714 out of the 770 S-Rnase alleles (21.0) is situated in between both Malus species and which were expected to be present in these 363 Malus represents 22 different S-RNase alleles. individuals were identified. Among the old cultivars, Using a modified chi-square goodness-of-fit test multiple triploids (41) were present, which is in developed to study the distribution of S-alleles in contrast to the modern cultivars (2), M. sylvestris (1) plants (Campbell and Lawrence 1981), we observed and hybrids (0). Fifty-six S-RNase alleles were coded that there is a large heterogeneity in S-allele as null-allele as they could not be identified by the frequencies for all groups, except the hybrids. Indeed, methods used. the v2 values calculated to test for goodness of fit of In total we detected 39 different Malus S-RNase each allele distribution against the hypothesis of genomic sequences in our dataset (Table 1; hereafter equal allele frequencies showed that S-allelic distri- named S-RNase alleles). Furthermore, the sequenc- bution is significantly distorted for both M. sylvestris ing of SCAR fragments revealed that S-RNase and M. 9 domestica. The significantly high v2 value allelic diversity is more complex than indicated by (142.7; df: 36) observed for M. sylvestris is caused by the visualized length polymorphisms. First, SCAR the S-RNase allele distribution in the subgroup of markers of identical length can correspond to individuals isolated from Meerdaalwoud (65 out of different S-RNase alleles, which appeared to be the 196 individuals). On comparison with the much 0 0 2 the case for S1/S1 (538 bp), S9/S9 (344 bp), S17/S6 higher v -value for M. 9 domestica (374.8; df: 27), 0 (367 bp) and S23 /S40 (345 bp). In order to be able to we predict the heterogeneity of S-RNase allele discriminate between these alleles, additional distribution to be much higher in the latter group, S-allele-specific genotyping methods were devel- even though the individuals were basically selected to oped. Second, the S5-specific genotyping method represent a large S-RNase allelic variety. (Broothaerts 2003) gave a positive result for two The distorted S-allelic distribution in the Malus SCAR fragments with respective lengths of 1,359 subgroups is caused by the strong over-representation and 1,284 bp. The first fragment corresponds to the of a subset of S-alleles. S2 is over-represented in both functional S5-allele from ‘‘’’. The second frag- M. sylvestris and M. 9 domestica, whereas this is not ment corresponds to an S-RNase allele, which we the case in the hybrid group. In addition to this allele, 0 named S5 , from which the DNA sequence of the S4, S9, S10 and S37 have been frequently detected ([5%) cloned partial exon region is identical to that of S5, in M. sylvestris. Additional, frequent alleles in the which points to the possibility that these are collection of old and modern apple cultivars are S1, S2, identical alleles, since all sequence differences are S3, S5, S7,S9,S10 and S19. In the hybrid individuals, the located within the intron. alleles S1, S7, S19 and S45 are the most frequent.

123 o reig(00 26:693–709 (2010) Breeding Mol Table 1 S-alleles detected by SCAR-marker analysis in this study, based on the S-allele numbering of Broothaerts (2003) Allele SCAR-fragment S-specific Accession number Accession number Reference Occurrence length (bp) assaya genomic sequence mRNA sequence

S1 538** a EU427454 D50837 Fuji (S1S9) M. 9 domestica, M. sylvestris, hybrids 0 0 S1 *** 538 This work EU419866 – Speeckaert (S1 S17) M. 9 domestica old, M. sylvestris

S2/S33 347** a DQ219464 U12199 (S2S3) M. 9 domestica, M. sylvestris, hybrids

S3 1,491 a EU427455 U12200 Golden Delicious (S2S3) M. 9 domestica, M. sylvestris, hybrids

S4 337 a EU427456 AF327223 Gloster (S4S19) M. 9 domestica, M. sylvestris

S5 1,359 a EU427460 U19791 Gala (S2S5) M. 9 domestica, M. sylvestris, hybrids 0 S5 *** 1,284 cf. S5 EU419870 – – M. sylvestris

S6/S12 (S6a*) 367 a AB094495, – Wintercitroenen (S3S5S6) M. 9 domestica old, M. sylvestris EU427461

S7 318** a EU427457 AB032246 (S2S7) M. 9 domestica, M. sylvestris, hybrids

S8 ± 2,300 This work – AY744080 Ontario (S1S8) M. 9 domestica old, M. sylvestris, hybrids

S9 344** a AY187627 U19793 Fuji (S1S9) M. 9 domestica, M. sylvestris, hybrids 0 S9 *** 344 This work EU419868 – – M. sylvestris

S10 ± 2,300 b – AF327221 McIntosh (S10S25) M. 9 domestica, M. sylvestris, hybrids

S11/S14 372 a – AB105060 (S4S11S31) M. 9 domestica old, M. sylvestris

S16 2,415 a AB126322 AF016919 Baskatong (S16S26) M. 9 domestica modern, M. sylvestris, hybrids

S17(S6b*) 367 a AB094493 – Bohnapfel (S9S16S17) M. 9 domestica old, M. sylvestris, hybrids

S19(S28) 369 a AF201748/AB050636 – Delicious (S9S19) M. 9 domestica, M. sylvestris, hybrids S20 511** a EU427458 AB019184 (S2S3S20) M. 9 domestica, M. sylvestris, hybrids

S21 374 a AB094494 – (S1S9S21) M. 9 domestica old, M. sylvestris, hybrids

S22 [2,500 a AF327222 – (S5S22) M. 9 domestica, hybrids

S23 347 a AF239809 – (S3S23) M. 9 domestica, M. sylvestris, hybrids 0 S23 *** 345 cf. S23 EU419867 – – M. sylvestris

S24 533 a AB050635 AB032247 Braeburn (S9S24) M. 9 domestica, M. sylvestris, hybrids

S25 [2,500 c – AB062100 McIntosh (S10S25) M. 9 domestica, M. sylvestris, hybrids

S26 357 a EU427459 AF016918 Baskatong (S16S26) M. 9 domestica old, M. sylvestris, hybrids

S29 426 – AY039702 – – M. 9 domestica modern, M. sylvestris

S31 470 – DQ135990 – Gravenstein (S4S11S31) M. 9 domestica old, M. sylvestris, hybrids

S34 938 – DQ649477 (M. sieversii) – Radoux (S19S34) M. 9 domestica old, M. sylvestris 123 S36*** 377 – EU419865 – – M. sylvestris, hybrids

S37*** 364 – EU419864 – – M. sylvestris, hybrids 699 S38*** 361 – EU419863 – – M. sylvestris 700 Mol Breeding (2010) 26:693–709

The high number of null-alleles in the M. sylvestris (12.5%) and hybrids group (11.1%) is intriguing. In

, hybrids contrast, we were able to provide complete S- genotypes for all M. 9 domestica individuals except the old triploid cultivar ‘‘Pladei’’ (Tables 3, 4). Our analysis revealed a large set of S-RNase alleles M. sylvestris M. sylvestris among the M. sylvestris trees which appear to be old, old, 0 0 0 unique for this group: S5 , S9 , S23 , S29, S36, S37, S38,

, hybrids , hybrids S39, S40, S41, S44, S45 and S47. S29, however, has been isolated previously as well from M. 9 domestica cv. domestica domestica domestica ‘‘’’ by Matityahu et al. (2005). In the collection 9 9 9 of old cultivars, the allele S34, previously identified in M. M. M. M. sylvestris Occurrence M. sylvestris M. sylvestris M. sylvestris M. sylvestris M. sylvestris M. sieversii (unpublished; DQ649477),was present in

three individuals. S22 and S42 are exclusively present ) 46

S in M. 9 domestica (except for one hybrid). S16, ) 10 S 43 which is frequently present in M. sylvestris (3.3%) 1 S S 5 )

S was only detected in ‘‘Bohnapfel’’, ‘‘Maypole’’ 42 S

10 (‘‘Wijcik’’ 9 ‘‘Baskatong’’) and crabapple varieties S ‘‘Baskatong’’, ‘‘Golden Hillieri’’ and ‘‘Malus flori- bunda’’ (data not shown). Reference cultivar Sequence analysis of presumptive new Malus S-alleles

Our study revealed 16 novel S-RNase allelic ) 2002b sequences. In several cases these novel S-alleles are quite distinct from those which have been identified in 2000

Accession number mRNA sequence previous studies and thus might have led to the differentiation to functionally different S-alleles. By cloning and sequencing of corresponding SCAR fragments, partial genomic sequences were obtained. Based on results of BLAST and ClustalW analysis, 0 0 0 0

Kitahara and Matsumoto they were named S1 , S5 , S9 , S23 , S36, S37, S38, S39, S40, c

, S41, S42, S43, S44, S45, S46 and S47. ) EU419859 – – Accession number genomic sequence An alignment of the amino acid region between 2002a 2006

, the conserved motifs FTQQYQ and IIWPNV (which both correspond to the SCAR-marker primers that a 2003a 3 were used for S-allelic genotyping) was made S -specific

S assay between these and already known S-RNase alleles (Fig. 3). Consequently, this analysis included a total of 45 known Malus S-allelic sequences. This align- ment clearly illustrates the large sequence diversity of

Kitahara and Matsumoto the RHV, in which only one residue (cysteine) is fully b , conserved for the whole set of aligned Malus 2,100 – EU419872 – – 2003 S-RNases. Furthermore, the RHV is not only charac- ± length (bp) -alleles that have been cloned in this work S terized by the considerable variation in amino acid continued composition but also by length polymorphisms. From -nomenclature by Matsumoto et al. ( ********* 1,636*** 345****** 2,003*** 1,018 –*** 912 –*** 1,342 – 670 1,285 EU419871 – EU419869 – – EU427453 – – EU427452 cf. this – EU419862 alignment EU419861 – EU419860 – –and – – the – – calculation Murray ( ( – of – the Joseph Musch ( pairwise Broothaerts S 39 40 41 42 43 44 45 46 47 ** SCAR marker that has been reported before by Matsumoto and Kitahara ( * *** Novel S S S S S S S S S Table 1 Allele SCAR-fragment a identity matrix over this region, it is clear that 123 Mol Breeding (2010) 26:693–709 701

Table 2 Allelic frequency, absolute amounts observed and allelic richness of S-RNase alleles detected in this study S-RNase allele Allelic frequency in % (absolute amount) M. sylvestris M. 9 domestica M. sylvestris— M. 9 domestica Old Modern Old ? modern Hybrids

Sx 12.47 (49)* 0.41(1) 0 (0) 0.32 (1) 11.11 (6)*

S1 2.04 (8) 14.94 (36)* 2.63 (2) 11.99 (38)* 7.41 (4)* 0 S1 0.09 (9) 0.41(1) 0 (0) 0.32 (1) 0 (0)

S2 5.09 (20)* 5.39 (13)* 14.47 (11)* 7.57 (24)* 3.7 (2)

S3 2.8 (11) 14.94 (36)* 15.79 (12)* 15.14 (48)* 5.56 (3)*

S4 5.09 (20)* 2.9 (7) 1.32 (1) 2.52 (8) 5.56 (3)*

S5 0.76 (3) 7.47 (18)* 18.42 (14)* 10.09 (32)* 1.85 (1) 0 S5 2.04 (8) 0 (0) 0 (0) 0 (0) 0 (0)

S6 4.33 (17) 1.66 (4) 0 (0) 1.26 (4) 0 (0)

S7 1.78 (7) 6.22 (15)* 5.26 (4)* 5.99 (19)* 9.26 (5)*

S8 3.05 (12) 3.32 (8) 0 (0) 2.52 (8) 3.7 (2)

S9 4.83 (19) 4.15 (10) 9.21 (7)* 5.36 (17)* 1.85 (1) 0 S9 0.76 (3) 0 (0) 0 (0) 0 (0) 0 (0)

S10 4.58 (18) 7.05 (17)* 13.16 (10)* 8.52 (27)* 3.7 (2)

S11 3.05 (12) 4.15 (10) 0 (0) 3.15 (10) 0 (0)

S16 3.05 (12) 0.41(1) 1.32 (1) 0.63 (2) 1.85 (1)

S17 2.04 (8) 4.56 (11) 0 (0) 3.47 (11) 0 (0)

S19 3.31 (13) 5.81 (14)* 1.32 (1) 4.73 (15) 7.41 (4)*

S20 3.31 (13) 2.07 (5) 1.32 (1) 1.89 (6) 5.56 (3)*

S21 0.25 (1) 0.83 (2) 0 (0) 0.63 (2) 1.85 (1)

S22 0 (0) 2.9 (7) 3.95 (3) 3.15 (10) 1.85 (1)

S23 4.07 (16) 0.83 (2) 3.95 (3) 1.58 (5) 3.7 (2) 0 S23 0.76 (3) 0 (0) 0 (0) 0 (0) 0 (0)

S24 1.53 (6) 2.9 (7) 3.95 (3) 3.15 (10) 3.7 (2)

S25 2.54 (10) 1.24 (3) 1.32 (1) 1.26 (4) 0 (0)

S26 1.53 (6) 0 (0) 1.32 (1) 0.32 (1) 3.7 (2)

S29 0.51 (2) 0 (0) 0 (0) 0 (0) 0 (0)

S31 2.04 (8) 0.83 (2) 0 (0) 0.63 (2) 1.85 (1)

S34 0 (0) 1.24 (3) 0 (0) 0.95 (3) 0 (0)

S36 3.05 (12) 0 (0) 0 (0) 0 (0) 0 (0)

S37 5.6 (22)* 0.41(1) 0 (0) 0.32 (1) 1.85 (1)

S38 1.53 (6) 0 (0) 0 (0) 0 (0) 0 (0)

S39 1.02 (4) 0 (0) 0 (0) 0 (0) 3.7 (2)

S40 0.76 (3) 0 (0) 0 (0) 0 (0) 0 (0)

S41 0.76 (3) 0 (0) 0 (0) 0 (0) 0 (0)

S42 0 (0) 0.41(1) 1.32 (1) 0.63 (2) 0 (0)

S43 0.51 (2) 1.24 (3) 0 (0) 0.95 (3) 0 (0)

S44 1.27 (5) 0 (0) 0 (0) 0 (0) 0 (0)

S45 0.76 (3) 0 (0) 0 (0) 0 (0) 5.56 (3)*

S46 3.82 (15) 1.24 (3) 0 (0) 0.95 (3) 3.7 (2)

S47 1.02 (4) 0 (0) 0 (0) 0 (0) 0 (0)

123 702 Mol Breeding (2010) 26:693–709

Table 2 continued S-RNase allele Allelic frequency in % (absolute amount) M. sylvestris M. 9 domestica M. sylvestris— M. 9 domestica Old Modern Old ? modern Hybrids

# S-RNase alleles detected 393 241 76 317 54 Allelic richness (Pb) 23.6 18.0 13.6 17.4 21.0 Chi-square value (v2)** 142.7 (36; 55.1) 258.7 (26; 38.9) 77.4 (16; 26.3) 374.8 (27; 40.1) 13.1 (21; 38.9) * Frequent alleles ([5%) ** v2 statistics have been performed with P = 0.05. Values indicated in italics are significantly different from the null hypothesis, meaning that allelic frequencies are significantly different. Degrees of freedom (number of different S-alleles detected minus 1) and the threshold value for v2 to accept the hypothesis of equal S-allele frequencies are indicated in parentheses

0 0 S-RNase allele pairs S1 and the novel S1 , S5 and S5 , by a sequence logo in Fig. 4. Both the first (donor S26 and S35 (M. sieversii), St/S30 (M. transitoria; site) and last 10–12 nucleotides (acceptor site) are Matsumoto et al. 2000) and the novel S36-RNase strongly conserved (respectively G|GTAATATTATT allele share 100% identity for this part of the protein. and TTATNTTGTCAG|AT). The region near the

S37, S38, S39, S40, S41, S42, S43, S44, S45, S46 and S47,on acceptor site tends to be very T-rich. the other hand, are significantly different from Novel genomic sequences obtained in this study previously identified S-RNase alleles. The amino have been deposited in the NCBI-database. Corre- acid similarity between these and the other S-RNase sponding accession numbers are listed in Table 1. alleles ranged from 56.9% (S29 vs. S38) to 98.2% (S42 vs. S44). Discussion Characterization of the Malus S-RNase intron region Description of S-allele genotypes in wild and cultivated apple Using SCAR-fragment sequencing, we obtained complete sequence information of the single intron This study presents a large-scale S-allele genotyping region for the majority of the S-RNase alleles for old and modern M. 9 domestica cultivars, identified in this collection of samples. Previously M. sylvestris, and M. sylvestris—M. 9 domestica characterized intron sequences of known alleles S16, hybrids. The (partial) S-genotype of the modern St/S30, S32 and S35, were included in this analysis as cultivars was already known at the onset of the well. The size of the intron is extremely variable and experiments and was determined by S-allele-specific ranges between 118 (S7) and 2,218 bp (S16). As a genotyping (Broothaerts et al. 2004). Our results are consequence, alignment of this region was impossi- mainly based on the use of an SCAR marker that ble. The intron region is very AT-rich: an average of generates co-dominant S-allelic length polymor- 77.66%, compared to 55.36% for the surrounding phisms. It proved to be essential to combine these mRNA region of the derived SCAR fragments. This results with S-allele-specific assays for accurate percentage tends to be much conserved. Tandem genotyping, as S-RNase genetic diversity is more repeats ([6 repeats) do occur, but only in a minority complex than visualized by length polymorphisms of the introns studied (S3, S4, S24, S39 and S42). All the alone. This led to the development of some new intron–exon junctions studied followed the canonical S-allele specific assays. Interestingly, for many GT/AG-rule for cis-splicing (Breatnach and Cham- individuals in the M. sylvestris subgroup incomplete bon 1981). Despite the large size variation of the SCAR S-genotypes were scored (i.e., presence of intron, the nucleotide sequence of the extremities null-alleles). The majority of these were caused by tends to be conserved. This is graphically displayed difficulties in the amplification of the alleles S8, S10, 123 o reig(00 26:693–709 (2010) Breeding Mol

Table 3 S-RNase genotypes of old apple cultivars surveyed in this study Cultivar S- Cultivar S- Cultivar S- Cultivar S- genotype genotype genotype genotype

Blenheim Orange S1S3S17 Graafappel S1S46 Pomme Jeanne S1S8 Rode Superman S3S7

Adam’s S1S3S10 Gravenstein type S4S11S20 Paradijs S1S10 Rode Wintercalville S2S10 Dendermonde

Appel Cronenbergs S3S10S25 Gravenstein type Martens S4S11S31 Pater van der Elzen S3S10S25 Roem van Vlaanderen S5S19

Appel Hoecke S4S5S9 Groninger Kroon S3S5 Pladei S8S23Sx Roter Cardinal S3S7S19

Appel Janssen S1S19 Grosse de St Cle´ment S2S3 Pomme Obus Vivier S7S11 Roter Eiserapfel S6S9S24

Belle de Pontoise S4S43 Halfoogstappel S7S24 Pre´sident Damseaux S1S22 Rouville S8S25S42

Belle-Fleur de France S5S19 Hondekop S1S11 Purpurroter Cousinot S1S10 Saint-Bernard S17S20

Belle-fleur Large Mouche (Rambeau) S1S7S34 Ingrid Marie S5S43 Radoux S19 S34 Schaapsneus S5S19

Berglander CRA1 S3S5S19 Jacques Lebel S1S3S17 Rambour de Flandre S1S19S31 Siau S10S22

Berglander CRA2 S5S10S19 S5S8 Rambour d’Hiver S5S23 Spe´che S1S3 0 Reinette Descardre (Boelpaep/Zoete S1S7S10 S7S9 Rammelaar S4S11S20 Speeckaert NBS S1 S17 Rabau)

Bohnapfel S9S16S17 Joseph Eersels S1S22 Reinette Coulon S1S2S3 Speeckaert CRA S1S10

Bon-Pommier Nouveau S1S11 Joseph Musch S1S10S46 Reinette de Cheˆne´e S1S9S21 Spokane Beauty S2S7

Boutersem S2S3S5 Keiing S1S4 Reinette de France S3S19S24 Stafner Rozenapfel S3S9S17

Bru¨nnerling S5S7S10 Keuleman NBS1 S5S6 Reinette de Waleffe S3S9S19 Sterwirtz S1S3

Calville Blanc d’Ete´ S1S7 Keuleman NBS2 S17S19S24 Reinette de Grez- S3S9 Van Dooveren’s Renette S1S5S10 Doiceau

Cellini S3S11 Keuleman Jaune S5S19 Reinette de Tournay S2S7 Vondeling Lovendegem S3S7 Clemens S3S20 Kortessems Grijske S10S24 Reinette d’Ohain S1S2S3 Wagner S3S8

Court-Pendu Doux S10S19 Leuenapfel S22S24 Reinette Duquesne S3S43 Wellington bloomless S8S9

Danziger Kant S2S7 Limburgse Bellefleur S1S17 Reinette Hernaut S1S3S17 Wintercitroenen (Citron S3S5S6 d’Hiver)

De Hert I S1S3S7 Marie Joseph d’Othee S1S17 Reinette Jaeghers S1S2S22 Yvette Detier S3S11

Dore´e de Tournai S3S4S22 Mme Macors S5S24S34 Reinette Rouge Etoile´e S1S2 Zaailing V. S2S22

Fameuse S1S3 Mutsu S2S3S10 Reinette van Zorgvliet S2S3S37 Zijden Hemdje S6S8

Fenoucelet Rouge S11S17 S1S3 Reinette Vuurveld S1S3 123 Frieslandse Reinette S3S46 Ontario S1S8 Rode Keiing S3S5 703 704 Mol Breeding (2010) 26:693–709

Table 4 S-RNase Cultivar S-genotype Cultivar S-genotype genotypes of modern apple cultivars which have been Alkmene S S ** S S S surveyed in this study. 5 22 3 5 9 Results are compared to Arlet S2S7 Maypole S10S16 those of Broothaerts et al. Belgica S3S5 Merlijn S3S22 (2004) Boskoop S2S3S5 Moiria S7S10

Braeburn S9S24 Murray S10S42

Charlotte S5S10 Nabella S5S9

Chusenrainer S2S3 Nicogreen (Greenstar) S3S23

Clivia S5S9 Nicoter () S5S24

David S1S26 Nova Easy Grow S5S10

Delbard Jubile´ S2S22 Pink Lady S2S23

Delbarestivale (Delcorf)* S2S10 S2S9

Discovery S10S24 Rubinola S2S3

* S-genotype formerly Ecolette S3S10 Selena S2S5 reported as S3S10 and Elstar/Elshof S3S5 Summerred S2S9 corrected in this study Fie¨sta S3S5 Telamon S3S10 ** S-genotype formerly Fuji S1S9 Vanda S5S7 reported as S3S5S10 and corrected in this study. Gloster S4S19 Varka S2S5 ‘‘Liberty’’ is a diploid Granny S3S23 Wijcik S10S25 cultivar with a triploid S7S20 S-genotype

S16, S22 and S25 as a result of their large size. We identical at the DNA level for both intron and exon solved this by screening for these alleles by S-allele- region, which points to the extreme conservation of specific assays. The remaining individuals with an this gene between these two Malus species. In incomplete genotype are not likely to have a homo- addition we detected an S-RNase allele which is zygous S-RNase genotype: multilocus SSR-data identical to the S34-allele, previously identified in (E. Coart et al., unpublished results) for these trees M. sieversii (P. Zhang et al., unpublished data), show that M. sylvestris, like M. 9 domestica, is a in three old apple cultivars. The results on S-allelic strongly heterozygous species (Fis: 0.027). This is as diversity of the hybrid trees confirm their hybrid expected for obligate cross-pollinators. Therefore, we character which has been demonstrated before by argue that these null-alleles correspond to unknown multi-locus SSR analysis (Coart et al. 2006). Our data S-RNase alleles with either a very large intron or an show that these hybrids share 91% of their S-alleles aberrant sequence in the primer region (which is with M. 9 domestica, compared to 81% with assumed to be highly conserved; Ishimizu et al. M. sylvestris. 1998a). Among all triploids, only one was found to The cloned partial gDNA sequence of some novel contain a null-allele. For the remaining triploids, the S-RNase alleles is very similar to previously known 0 S-RNase genotype consisted of three different alleles, M. 9 domestica S-alleles, which is the case for S1 0 therefore being the result of fertilization with a (similar to S1), S9 (similar to S9), and S23 (similar to 0 heterozygous diploid gamete. In conclusion, the S23). S5 and S5 are represented by different SCAR strong heterozygosity of the S-genotypes in polymorphisms but their sequences only differ in the M. sylvestris points to the existence of a functional intron region. All these similar S-RNase alleles 0 GSI system in wild apple. coexist within M. sylvestris. The S1 -RNase allele is Twenty-six of the 40 different S-RNase alleles the only one of these, which is also present in detected in this study are common to M. sylvestris M. 9 domestica (‘‘Speeckaert’’). Unfortunately, data and M. 9 domestica. Sequence analysis of cloned on a possible hybrid nature of this genotype are SCAR fragments showed that these are in fact 100% lacking.

123 Mol Breeding (2010) 26:693–709 705

Fig. 3 Alignment of deduced partial amino acid sequences of primers used for SCAR-marker analysis (arrows). The RHV the Malus S-RNase defined by the SCAR marker. Alignment of and PS1 regions described by Sassa et al. (1996) and Ishimizu the deduced partial amino acid sequences for 45 Malus et al. (1998a) are indicated by a white and black bar, S-RNase alleles. Identical residues are shown in black and respectively. The single conserved cysteine residue of the similar residues in grey. Dashes correspond to gaps. The RHV region is marked by an asterisk. The position of the conserved motifs FTQQYQ and IIWPN correspond to the single intron is indicated by an inverted black triangle

123 706 Mol Breeding (2010) 26:693–709

Fig. 4 Sequence logo plot of the 50- and 30-end of the Malus of consensus sequences (logos). The donor and acceptor region S-RNase intron. The first and last 30 nucleotides (donor and of the intron region are conserved with respective consensus acceptor) of the intron are shown. The relative occurrence of sequences G|GTAATATTATT and TTATNTTGTCAG|AT each nucleotide is displayed, enabling graphical representation

The richness of Malus S-RNase genetic diversity present in old cultivars but not in modern ones. The most plausible explanation is that they differ in their By calculating allelic richness, it was shown that origin. Modern cultivars are the result of breeding S-RNase genetic diversity is much higher for with a very limited set of parents, such as ‘‘Golden

M. sylvestris and the hybrid trees than for M. 9 Delicious’’ (S2S3), ‘‘Jonathan’’ (S7S9) and ‘‘Fuji’’ domestica. The percentage of null alleles (12.6%) (S1S9), while old cultivars are local cultivars or indicates that in reality the allelic richness in wild landraces of which the parentage is largely unknown. apple is even higher than predicted. Based on the This explains the over-representation of these alleles frequencies of the known alleles (0.3–5.8%), one can and the absence of others (such as S6 and S8)in assume that at least two other S-RNase alleles could modern cultivars. One can argue that the number of be present. Altogether, our results point to the fact modern apple cultivars in our survey is too small to that domestication of apple leads to higher levels of make correct assumptions about S-RNase allelic genetic uniformity. This confirms similar observa- diversity. However, this proves not to be the case; tions in other crops such as soybean, common bean if, based on literature data, results are extrapolated to and wheat (Brown-Guedira et al. 2000; Reif et al. a total of 198 cultivars (Broothaerts et al. 2004; 2005; Sonnante et al. 1993) using neutral genetic Matityahu et al. 2005; Matsumoto et al. 2003b, 2006 markers. Furthermore, the large heterogeneity of and 2007), only four more alleles (S29,S32, Skb, and S-RNase allelic frequencies in M. 9 domestica in S1-20-24-like) are found, while S2, S3, S5 and S9 remain comparison to M. sylvestris illustrates the influence of the most frequent ones. Moreover, the results of our controlled breeding practices. Indeed, it has been study are in line with those of Garkava-Gustavsson demonstrated that in natural populations of species et al. (2008)onS-RNase allelic diversity of indige- with a GSI system the S-locus is subject to a strong nous Swedish apple cultivars. frequency-dependent natural selection (Castric and Vekemans 2004; Wright 1939). In our analysis, The Malus S-RNase PS1 and intron regions however, the allelic frequency was not equal for the are highly diverse M. sylvestris group of individuals since this does not represent a true population. When analyzing the This study revealed SCAR fragments that have not subgroups from different locations (data not shown), been identified before. Except for S42 from ‘‘Mur- we found all allelic distributions to be equal, except ray’’, all these were cloned from M. sylvestris and old for ‘‘Meerdaalwoud’’ where these trees have been apple cultivars. Although the sequence information planted as food for residing game. was derived from a relatively small part of the In addition, we demonstrated that the S-RNase S-RNase gene, our data are relevant as they comprise allelic richness of modern cultivars is poor compared the hypervariable region PS1/RHV. Indeed, Ishimizu to old cultivars. This is also demonstrated by the fact and coworkers (1998a) demonstrated that the high that one finds all (except S26) S-RNase alleles of variability points to the fact that this is an important modern cultivars in old cultivars while 11 alleles are determinant of S-allele specificity. This statement 123 Mol Breeding (2010) 26:693–709 707 was made based on the alignment of deduced amino M. transitoria. It is very probable that these three acid sequences of different Rosaceae S-alleles includ- novel S-RNase alleles in fact represent intra-allelic ing five M. 9 domestica alleles. Our alignment of 45 variations of previously identified S-alleles. Malus S-allelic sequences confirms the extreme In order to prove functional distinctiveness of all variability of this region and showed that this is not the identified novel S-RNase allelic sequences, full- only at the sequence level since we observe length length sequence information is a prerequisite and variations as well. The inclusion of a much larger pollination tests need to be performed in the field. group of alleles in the alignment shows that in fact Recent literature shows that, despite the importance only one amino acid (aa) residue (cysteine) is fully of PS1 for recognition, sequence differences which conserved in PS1. This was already noted as being are located in this region are not always sufficient to conserved within the PS1 region of Rosaceae S- decide on functional distinctiveness of S-alleles. In RNases by Ishimizu et al. (1998a) and has been European pear, a pollination assay showed that the observed to be conserved in almond by Ortega et al. Sn- and Si-RNases are functionally distinct, although (2006) which included 23 S-RNase sequences. In the the deduced amino acid sequences of their hyper- analysis of Decuyper et al. (2005) of the genetic variable region PS1 are identical (Zisovich et al. diversity of wild cherry S-alleles (Prunus avium), this 2004). In this case, the differences in the regions PS2 cysteine residue was conserved in 21 out of the 22 and PS3 proved to be critical for specification. Ortega alleles. Altogether, these data point to a crucial role et al. (2006) reached a similar conclusion for the S11 for this amino acid in this hypervariable region, and S24 S-RNases of almond. On the other hand, for 0 which is predicted to form a loop structure in the Sg from M. transitoria it was shown that, compared tertiary structure of the S-RNase (Matsuura et al. to S20 (Sg), a difference of one single amino acid 2001). The importance of cysteine residues for the located in the RHV was not sufficient to generate a structure and functionality of ribonucleases has been new specificity (Matsumoto et al. 2001). pointed out previously by other studies too (Horiuchi The Malus S-RNase intron is highly variable in et al. 1988; Kawata et al. 1988). length, similarly to what has been reported for intron regions of both almond (Ortega et al. 2006) and Identification of presumptive functionally novel cherry S-RNases (Sonneveld et al. 2006). For a Malus S-alleles minority of the Malus S-alleles studied, tandem repeats were detected in the intron region. This has The identification of a multitude of novel S-RNase also been reported by Sonneveld et al. (2006) for the sequences in this study makes it probable that novel cherry S13-allele. Tandem repeats are a source for functional S-allelic classes have been discovered as intra-allelic variation. Its occurrence in S3, which is a well. ClustalX analysis showed that the derived very frequent allele in modern apple cultivars, could protein sequences of the S39, S40, S46 and S47 alleles be useful to develop specific assays for paternity from M. sylvestris are clearly distinct from other S- analysis and breeder’s rights protection. alleles. For other S-RNase sequences derived from novel SCAR polymorphisms, the situation is more complex. Comparison of the derived amino acid Concluding remarks and future perspectives sequences of novel S-RNase sequences S42 to S44 and S37 to S38 shows that these are distinct from The large-scale study presented here has provided previously identified S-alleles, but mutual differences valuable insights about the S-allele structure and its in the PS1 regions are limited to 1 and 3 aa residues, rich genetic diversity in Malus. The richness of respectively, which might not be enough for func- S-allelic diversity in this is far larger then has 0 tional diversification from each other. S45 and S23 are been assumed up till now. The description of the also unlikely to represent new S-alleles, since differ- S-allelic genotype of M. sylvestris and old cultivars ences with the respectively similar S-RNase alleles enables the introduction of specific individuals into

S19 and S23 are limited to a few aa residues, mainly modern breeding programs in order to extend 0 located outside the PS1 region. The S1 protein is the narrow genetic variation of modern apple culti- 0 identical to S1, S5 to S5 and S36 to St/S30 cloned from vars and introduce interesting new traits. The 123 708 Mol Breeding (2010) 26:693–709 heterozygosity of the S-allelic genotype indicates the Campanella JJ, Bitincka L, Smalley J (2003) MatGAT: an functionality of the GSI system in wild apple and application that generates similarity/identity matrices using protein or DNA sequences. BMC Bioinform 4:29 implies that the results of this work are important to Campbell JM, Lawrence MJ (1981) The population genetics of include in the establishment of a core collection of the self-compatibility polymorphism in Papaver rhoeas. M. sylvestris which will be established to protect this II. The number and frequency of S-alleles in a natural endangered species. It will be very important to population (R106). Heredity 46:81–90 Castric V, Vekemans X (2004) Plant self-incompatibility in include individuals of which the S-RNase genotype is natural populations: a critical assessment of recent very diverse, in order to guarantee the presence of theoretical and empirical advances. Mol Ecol 13:2873– enough suitable breeding partners. Furthermore, it 2889 was shown that the disappearance of wild apple in Certal AC, Sanchez AM, Kokko H, Broothaerts W, Oliveira MM, Feijo´ JA (1999) S-RNases in apple are expressed in Flanders cannot be the consequence of a narrow the pistil along the pollen tube growth path. Sex Plant S-RNase diversity. The fact that these trees are Reprod 12:94–98 residing in unsuitable habitats as a result of fruit Cheng J, Han Z, Xu X, Li T (2006) Isolation and identification cultivation and forestry management is the sole of the pollen expressed polymorphic F-box genes linked to the S-locus in apple (Malus 9 domestica). Sex Plant important reason for their disappearance in nature Reprod 19:175–183 (Vander Mijnsbrugge et al. 2008). Coart E, Van Glabeke S, De Loose M, Larsen AS, Rolda´n-Ruiz The marker approach which has been developed I (2006) Chloroplast diversity in the genus Malus: new and applied in this study could be a useful tool for insights into the relationships between the European Wild apple (Malus sylvestris (L.) Mill.) and the domesticated similar S-RNase diversity studies in other related apple (Malus domestica Borkh.). Mol Ecol 15:2171–2182 Malus species such as M. sieversii. The description of Crooks GE, Hon G, Chandonia JM, Brenner SE (2004) Weblogo: genetic diversity of the S-locus could contribute a sequence logo generator. Genome Res 14:1188–1190 significantly to unraveling the evolutionary relation- de Nettancourt D (1977) Incompatibility in angiosperms. 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