PCR amplification for S-RNase alleles of 17 sour cherry selections. (A) Genomic DNA was amplified by PCR with Pru-C2 () and PCE-R () primer set. (B) Genomic DNA was amplified by PCR with EM-PC2consFD and EM-PC5consRD () primer set. PCR products were separated on 2% agarose gels and detected with ethidium bromide staining. The colour of black and white is inverted in this image. The asterisks indicate the band of PCR product of S₃₅-RNase. M, 123 bp DNA ladder (Invitrogen, Carlsbad, CA, USA). Lane abbreviations are: C59, ‘Cigány 59’; Cri, ‘Crisana’; ET, ‘Englaise Timpurii’; EB, ‘Erdi Botermo’; EJ, ‘Erdi Jubileum’; EN, ‘Erdi Nagygyumolcsu’; Met, ‘Meteor’; Mon, ‘Montmorency’; P38, ‘Pandy 38’; P114, ‘Pandy 114’; RS, ‘Rheinische Schattenmorelle’; Sur, ‘Surefire’; Tam, ‘Tamaris’; Tar, ‘Tarina’; Tsc, ‘Tschernokorka’; UF, ‘Újfehértói fűrtős’; III 18 (12), ‘MSU III 18 (12)’. (This figure is available in colour at JXB online).

Amino acid sequence alignment of three novel S-RNases obtained from sour cherry and that of other functional S-RNases from sour cherry. The alignment was generated by DNASIS version 3.5 (Hitachi Software Engineering Co. Ltd., Tokyo, Japan). Gaps are marked by dashes. Conserved amino acids are shown on a darkened background. The five conserved regions, C1, C2, C3, RC4, and C5 () are marked with solid boxes, and the hypervariable region, RHV () reported in the rosaceous S-RNases, is marked with a dotted box. The positions and directions of the four consensus primers used in genomic PCR are indicated by arrows.

Amino acid sequence alignment of three novel SFBs obtained from sour cherry and that of other functional SFBs from sour cherry. The alignment was generated by DNASIS version 3.5 (Hitachi Software Engineering Co. Ltd., Tokyo, Japan). Gaps are marked by dashes. Conserved amino acids are shown on a darkened background. The locations of the F-box motif, V1, V2, HVa, and HVb (), and Vn () are indicated by solid boxes.

PCR amplification with S₃₃-, S₃₄-, and S₃₅-allele specific primer pair for S-RNase and SFB in 17 sour cherry selections. PCR products were separated on 2% agarose gel and detected with ethidium bromide staining. M, 123 bp DNA ladder (Invitrogen, Carlsbad, CA, USA). Lane abbreviations are: C59, ‘Cigány 59’; Cri, ‘Crisana’; ET, ‘Englaise Timpurii’; EB, ‘Erdi Botermo’; EJ, ‘Erdi Jubileum’; EN, ‘Erdi Nagygyumolcsu’; Met, ‘Meteor’; Mon, ‘Montmorency’; P38, ‘Pandy 38’; P114, ‘Pandy 114’; RS, ‘Rheinische Schattenmorelle’; Sur, ‘Surefire’; Tam, ‘Tamaris’; Tar, ‘Tarina’; Tsc, ‘Tschernokorka’; UF, ‘Újfehértói fűrtős’; III 18 (12), ‘MSU III 18 (12)’.

The deduced amino acid sequence alignment for the Prunus cerasus S₃₄-RNase (PcS34-RNase), P. domestica S₅-RNase (PdoS5-RNase), P. avium S₁-RNase (PaS1-RNase), P. dulcis S₁₁-RNase (PdS11-RNase), and P. tenella S₈-RNase (PtS8-RNase). The asterisks indicate the five amino acid residues that are different between P. cerasus S₃₄-RNase and P. domestica S₅-RNase. Conserved nucleotides are shown on a darkened background. The five conserved regions, C1, C2, C3, RC4, and C5 () are marked with solid boxes, and the hypervariable region, RHV () reported in the rosaceous S-RNases, is marked with a dotted box.

The deduced amino acid sequence alignment for the Prunus cerasus SFB₃₄ (PcSFB34), P. domestica SFB₅ (PdoSFB5), P. avium SFB₁ obtained from ‘Skeena’ (PaSFB1-Skn), P. avium SFB₁ obtained from ‘Seneca’ (PaSFB1-Snc), P. cerasus SFB₁ obtained from ‘Pandy 114’ (PcSFB1), P. tenella SFB₈ (PtSFB8), and P. dulcis SFB₁₁ (PdSFB11). The asterisks indicate the 15 amino acid residues that are different between P. cerasus SFB₃₄ and P. domestica SFB₅. Conserved nucleotides are shown on a darkened background. The locations of the F-box motif, V1, V2, HVa, and HVb (), and Vn () are indicated by solid boxes.

Phylogenetic trees based on phylogenetic analysis of nucleotide sequences of S-RNase (left) and SFB (right) alleles for species of Prunus. Numbers above, below, or adjacent to branches are bootstrap support values greater than 50%; asterisks indicate branches that collapsed in the strict consensus tree from each analysis. The positions of PcS33, PcS34, and PcS35 are indicated, respectively, by open boxes, lightly shaded boxes, and darkly shaded boxes. Left: Single most parsimonious tree (l=2366, ci excluding autapomorphies=0.3271, ri=0.5263) based on S-RNase alleles. The nucleotide sequences for three novel P. cerasus (PcS-RNase) alleles presented in this study were aligned with 12 S-RNase alleles from P. avium (PaS1-RNase, AB028153; PaS2-RNase, AB010304; PaS3-RNase, AB010306; PaS4-RNase, AB028154; PaS5-RNase, AJ298314; PaS6-RNase, AB010305; PaS7-RNase, EU035974; PaS12-RNase, AY259115; PaS13-RNase, DQ385842; PaS23-RNase, AY259114; PaS24-RNase, AY259112; PaS25-RNase, AY259113); one from P. cerasus (PcS26-RNase, EU035975); three from P. domestica (PdoS5-RNase, AM746946; PdoS6-RNase, AM746947; PdoS9-RNase, AM746948); nine from P. dulcis (PdSa-RNase, AB026836; PdSb-RNase, AB011469; PdSc-RNase, AB011470; PdSd-RNase, AB011471; PdSk-RNase, AB252409; PdSm-RNase, DQ099895; PdSn-RNase, DQ093825; PdS11-RNase, AM231660; PdS12-RNase, AM746949); two from P. mume (PmS1-RNase, AB101438; PmS7-RNase, AB101439); four from P. armeniaca (ParS1-RNase, AY587561; ParS2-RNase, AY587562; ParS4-RNase, AY587564; ParS17-RNase, EU516388); three from P. cerasifera (PcsfS3-RNase, AM746943; PcsfS9-RNase, AM746944; PcsfS10-RNase, AM746945); ten from P. salacina (PsSa-RNase, AB252411; PsSb-RNase, AB252413; PsSc-RNase, AB084102; PsSd-RNase, AB084103; PsSe-RNase, AB280693; PsSf-RNase, DQ512911; PsSg-RNase, AM746950; PsSh-RNase, DQ512914; PsS7-RNase, AY781290; PsS8-RNase, DQ512913); seven from P. spinosa (PspS8-RNase, DQ677587; PspS9-RNase, DQ677588; PspS10-RNase, DQ677589; PspS12-RNase, DQ677590; PspS3-1-RNase, DQ677584; PspS3-2-RNase, DQ677585; PspS7-1-RNase, DQ677586); and nine from P. tenella (PtS1-RNase, DQ983373; PtS2-RNase, DQ983374; PtS3-RNase, DQ983375; PtS4-RNase, DQ983363; PtS5-RNase, DQ983364; PtS6-RNase, DQ983365; PtS7-RNase, DQ983366; PtS8-RNase, DQ983367; PtS9-RNase, DQ983370). Data set contained 63 taxa and 744 characters, of which 220 were constant, 129 were variable but uninformative, and 395 were parsimony-informative. Right: One of 65 most parsimonious trees (l = 2968, ci excluding autapomorphies = 0.3913, ri = 0.4860) from phylogenetic analysis of nucleotide sequences of SFB alleles. The nucleotide sequences for three novel P. cerasus (PcSFB) alleles presented in this study were aligned with coding sequences for 12 SFB alleles from P. avium (PaSFB1, AY805048; PaSFB2, AB111519; PaSFB3, AB096857; PaSFB4, AB111521; PaSFB5, AB111520; PaSFB6, AB096858; PaSFB7, EU035976; PaSFB9, DQ422809; PaSFB10, AY805053; PaSFB12, AY805054; PaSFB13, DQ385844; PaSFB16, AY805056); two from P. cerasus (PcSFB1, DQ827715; PcSFB26, EU035977); three from P. domestica (PdoSFB5, AM746955; PdoSFB6, AM746956; PdoSFB9, AM746957); seven from P. dulcis (PdSFBa, AB092966; PdSFBb, AB092967; PdSFBc, AB079776; PdSFBd, AB081648; PdSFBk, AB252408; PdSFB11, EF061758; PdSFB12, AM746959); three from P. mume (PmSFB1, AB101440; PmSFB7, AB101441; PmSFB9, AB092645); four from P. armeniaca (ParSFB1, AY587563; ParSFB2, AY587562; ParSFB4, AY587565; ParSFB17, EU516388); three from P. cerasifera (PcsfSFB3, AM746952; PcsfSFB9, AM746953; PcsfSFB10, AM746954); nine from P. salicina (PsSFBa, AB252410; PsSFBb, AB252412; PsSFBc, DQ849084; PsSFBd, AM746962; PsSFBe, AB280794; PsSFBf, DQ849089; PsSFBg, AM746963; PsSFBh, DQ849118; PsSFB7, DQ849085); seven from P. spinosa (PspSFB8, DQ677587; PspSFB9, DQ677588; PspSFB10, DQ677589; PspSFB12, DQ677598; PspSFB3-1, DQ677616; PspSFB3-2, DQ677615; PspSFB7-1, DQ677595), and one from P. tenella (PtSFB8, DQ983369). Data set contained 54 taxa and 1161 characters, of which 337 were constant, 230 were variable but uninformative, and 594 were parsimony-informative.