Abstract
A traditional cultivar, Besztercei Bt.2, and a clone of an autochthonous landrace (Nemtudom P3) of the hexaploid European plum (Prunus domestica) were studied to highlight their breeding perspectives. Five self-incompatibility ribonuclease (S-RNase) alleles were detected in both cultivars, with one allele shared. DNA sequence analysis confirmed it as a new, previously unidentified allele in P. domestica, which we labeled as S18. This allele was found to share ∼99% identity with the Prunus spinosa SB-RNase allele. Because Prunus species are readily hybridizing, sequence variations in 10 chloroplast DNA regions and nuclear internal transcribed spacers were studied to check if ‘Nemtudom P3’ and ‘Besztercei Bt.2’ are indeed P. domestica. The majority-rule consensus tree of maximum likelihood and Bayesian inferences confirmed it, and also indicated genetic differentiation with ‘Nemtudom P3’ and ‘Besztercei Bt.2’ forming a statistically supported subclade within the P. domestica germplasm. Our results pointed to some regions of the P. domestica chloroplast genome (trnS-trnG-trnG, trnC-ycf6, and trnD-trnT) that can be used to detect intraspecific variations. The proportion of parsimony informative characters compared with the total length of amplified regions was the highest in the case of nrITS with 12.1%. The S-genotyping of 68 wild-growing Nemtudom trees showed the genetic consequences of long-term vegetative propagation and occasional crossing between Besztercei and Nemtudom accessions. Controlled pollinations confirmed the self-compatibility of ‘Nemtudom P3’. By clarifying their phylogenetic position, and characterizing the S-locus, our results will help breeding P. domestica cultivars and pave the way to understanding how the S-locus works in a hexaploid Prunus species.
The attractive and high-economic-value fruit of different species belonging to the genus Prunus have been extensively studied in recent years (Abanoz and Okcu 2022; Delialioglu et al. 2022; Rampáčková et al. 2021). European plum (P. domestica) is a highly valuable fruit-bearing plant belonging to the Rosaceae family, and grown predominantly in Europe (Sottile et al. 2022). It takes a significant proportion of production quantity in many European regions, like all Balkan countries, or central (Hungary, Poland, Czech Republic, etc.) and western Europe (France, Germany, etc.) (Food and Agriculture Organization of the United Nations 2020). In Hungary, European plum is the second Prunus species in production quantity with ∼27,000 t/year. Plums are consumed in varied ways as fresh fruit or processed into jam, marmalade, juice, prunes (dried fruit), or spirits. Plums are also used for making popular pálinkas. Pálinka is a special distilled product, “Hungaricum” (this collective term indicates an officially registered product that represents the high performance of Hungarian people thanks to its typically Hungarian attribute, uniqueness, specialty, and quality) with deep traditions and is protected as a geographical indication of the European Union.
P. domestica is a hexaploid (2n = 6× = 48) species about the origin of which to this day we have only hypotheses. The widely accepted proposition of the species’ emergence included a relatively recent hybridization between some types of Prunus cerasifera and P. spinosa, which itself is also a hybrid of P. cerasifera and a yet unidentified species (Rybin 1936; Zhebentyayeva et al. 2019). The hexaploid Damson plum (P. domestica ssp. insititia) is also grown in Hungary and is currently considered a subspecies within P. domestica.
Self-incompatibility is determined by the S-locus, harboring a pistil-expressed S-ribonuclease (S-RNase) and a pollen-expressed S-haplotype-specific F-box gene (Sutherland et al. 2008). Both self-incompatible (SI) and self-compatible (SC) cultivars were described in P. domestica (Nikolić and Milatović 2010; Nyéki and Szabó 1996). Its genetic background is complex because six S-alleles are expected to be present in a hexaploid plum genome. In plums, several self-incompatibility ribonuclease (S-RNase) alleles were identified (Abdallah et al. 2019; Fernandez i Marti et al. 2021; Halász et al. 2014; Sutherland et al. 2004b, 2008) and from two to six alleles were assigned to the studied cultivars. However, the molecular background of self-compatibility in this species is still to be clarified.
Plum species are readily hybridizing and hence the taxonomic classification of accessions might be problematic. Sequence variations in the noncoding regions of chloroplast DNA (cpDNA) and the nuclear ribosomal internal transcribed spacers (nrITS) are the most often used for phylogenetic studies. Variations in cpDNA were first studied by restriction site polymorphism, while currently the chloroplast genome sequence is available for many plant species (Geng et al. 2020; Horvath et al. 2011). Rate heterogeneity became evident among cpDNA regions and the information content of specific regions also varies greatly according to lineages (Shaw et al. 2005). Based on such findings, several primers that might be used efficiently were selected and exploited for many purposes in Prunus genetic analyses (Batnini et al. 2019; Bortiri et al. 2001; Horvath et al. 2011; Li et al. 2021; Reales et al. 2010; Shaw and Small 2004). ITSs are noncoding regions between ribosomal RNA genes, which sequences were efficiently used for phylogenetic analyses in a range of plant taxa including Prunus (Bortiri et al. 2001; Gilani et al. 2010), for the discrimination among species (Quan and Zhou 2011) and cultivars (Yoon et al. 2009).
Traditional plum cultivars in Hungary are of outstanding taste properties with some undesirable characteristics, including small fruit size and plum pox virus (PPV) sensitivity. Besztercei is of unknown origin and it was the major cultivar in Hungarian plum production with a massive presence in other central European countries where it was named as Pozegaca, German Prune, Küstendili, and Hauszwetschen (Faust and Surányi 1998). The original genotype and its variants are grown in Hungary as landraces (‘Besztercei szilva’ and ‘Sárga besztercei szilva’), whereas only selected clones (‘Besztercei Bt. 2’ and ‘Korai Besztercei’) are planted in commercial orchards (National Food Chain Safety Office 2022). The popularity of ‘Besztercei’ declined due to its severe susceptibility to PPV. Nemtudom (its synonymous name is Penyigei) is native to the northeastern part of Hungary where wild growing forests can be found in the flood plain of river Tisza. It shows a high level of tolerance to PPV with only mild symptoms on fruit surface (Pethő et al. 2010). The sweet (13% to 20% soluble solid content) and moderately acidic fruits are rich in aroma and are mainly used to make premium quality traditional spirits (pálinka) and jam. Based on phenotypic characteristics, Nemtudom is thought to be P. domestica ssp. insititia. From the autochthonous populations, ‘Nemtudom P3’ has received cultivar recognition (National Food Chain Safety Office 2022).
The aim of this study was the S-genotyping of two traditional plum cultivars, Besztercei Bt. 2 and Nemtudom P3, to assist their future improvement, as both present a combination of excellent and unfavorable characteristics. We also wanted to characterize if the allelic profiles at the S-locus and other nuclear and cpDNA regions can help estimate the genetic variability of breeding material and the origin of the tested germplasm.
Materials and Methods
Plant materials.
Young leaves of the P. domestica cultivars Besztercei Bt. 2 and Nemtudom P3 were frozen in liquid nitrogen, transported to the laboratory, and stored at −80 °C until used for DNA extractions. A total of 68 trees of a wild-growing Nemtudom landrace accession were sampled in the northeastern Hungarian region. The sample was named based on the location where it was collected as shown in the map (Supplemental Fig. 1). These samples were used for DNA extraction.
DNA extraction.
Total genomic DNA was extracted from 200-mg leaf samples using the DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The concentrations of the DNA were determined using a spectrophotometer (NanoDrop ND-1000; Thermo Fisher Scientific, Waltham, MA, USA). The extracted DNA was stored at −20 °C until used.
Polymerase chain reactions.
For S-allele polymerase chain reaction (PCR) analysis, the consensus primers PaConsII-F with PaConsII-R (Sonneveld et al. 2003) and EM-PC2consFD with EM-PC5consRD (Sutherland et al. 2004a) were used to amplify the conserved regions C2 and C5 of the S-RNase gene including the second intron. The region between C2 and C3 was amplified using EM-PC2consFD and EM-PC3consRD primer pair by Sutherland et al. (2004a). For the analysis of S-RNase gene first intron region, the fluorescently labeled PaConsI-F (FAM) and EM-PC1consRD (Ortega et al. 2005; Sonneveld et al. 2003) primers were applied. For the marker analysis of chloroplast genome, the following 10 primer pairs were used: matk5/matk6, psbA5′R/matk8F, psbA/trnHGUG, psbB/psbH, rpL16F71/rpL16R1516, 5′rpS12/rpL20, rps16F/rps16R, trnDGUCF/trnTGGU, trnCGCAF/ycf6R, trnSGCU/3′trnGUUC (Shaw et al. 2005). ITS regions were amplified using ITS-Leu and ITS4 primers (Baum et al. 1998). All PCRs were performed in a 12.5-μL reaction volume containing ∼40–60 ng of genomic DNA, 10 × DreamTaq™ Green buffer (Thermo Fisher Scientific) with final concentrations of 1.5 mM MgCl2, 0.2 mM of dNTPs, 0.4 μM of the adequate primers, and 0.625 U of DreamTaq™ DNA polymerase (Thermo Fisher Scientific). The PCR amplification was carried out in a Swift MaxPro thermocycler (ESCO Healthcare, Singapore, Republic of Singapore). Cycling parameters in the case of the previously published primer pairs were the same as described by Baum et al. (1998), Shaw et al. (2005), Sonneveld et al. (2003), and Sutherland et al. (2004a). Before direct sequencing, PCR products of ITS and chloroplast regions were purified using ExoSAP- IT Express reagent (Thermo Fisher Scientific) with 5-µL post-PCR reaction product.
Agarose gel electrophoresis and evaluation.
The PCR products were separated by electrophoresis on 1% TBE agarose gel after ethidium bromide staining at 80 V for 40 min and then the DNA bands were visualized by ultraviolet illumination and documented by Gel Documentation System (Bio-Rad, Hercules, CA, USA). The approximate fragment sizes were determined by comparison with the GeneRuler™ 1-kb DNA Ladder (Thermo Fisher Scientific). The evaluation of the first intron region of S-RNase was made using an automated capillary sequencer (ABI PRISM 3100 Genetic Analyzer; Thermo Fisher Scientific) for fragment length analysis, ABI Peak Scanner 1.0 software and GS500 LIZ (Thermo Fisher Scientific) internal size standard were used for data analysis.
Cloning, transformation, and sequencing.
Cloning of PCR products was carried out using pTZ57R/T vector (Thermo Fisher Scientific). The ligated plasmid vectors were transformed into JM109 Escherichia coli competent cells (Zymo Research, Irvine, CA, USA). The nucleotide sequences were determined for each fragment in both directions by using M13 sequencing primers. The plasmid DNA fragments were purified using the EZ-10 Spin Column Plasmid DNA kit (Bio Basic, Markham, ON, Canada) and then sequenced in an automated sequencer ABI PRISM 3100 Genetic Analyzer (Thermo Fisher Scientific). The sequences have been deposited in GenBank under accession numbers OP647098, OP647099, OP709316-OP709323, OP709349-OP709360, OR161115, and OR161116 (Supplemental Tables 1–3).
Sequence analyses.
The Basic Local Alignment Search Tool (BLAST) of the National Center for Biotechnology Information (NCBI, Bethesda, MD, USA) was used for homology searches (Altschul et al. 1990). Alignments were created using MEGA7 (Kumar et al. 2016), and were presented with the BioEdit program v.7.2.5 (Hall 1999). The P. domestica chloroplast genome (NC_050959) was visualized by Chloroplot (Zheng et al. 2020).
Phylogenetic inference.
Phylogenetic inference among the studied taxa was carried out using maximum-likelihood (ML) and Bayesian (BA) approaches. The ML and BA methods were chosen for phylogenetic analysis because of their robustness, efficiency, and demonstrated success in former studies (Xu et al. 2022; Ye et al. 2020). Before the analysis, the cpDNA and nuclear (nrITS) sequences were concatenated into separate matrices, then indel polymorphisms were coded following the simple gap coding algorithm developed by Simmons et al. (2001) using FastGap 1.2 (Borchsenius 2009). Matrices were visually checked and formatted using the SeaView 4.6.4 program (Gouy et al. 2010). ML analysis was performed using the RAxML-NG web server (Kozlov et al. 2019) with 10 random starting trees and an autoMRE bootstrap search with 0.01 cutoff value (Pattengale et al. 2010). Statistical selection of the best-fit model of sequence evolution was carried out using jModelTest version 2.1.10 (Darriba et al. 2012). Likelihood scores were corrected using Akaike Information Criterion (AIC) as implemented in jModelTest. The best-fitting models of substitution for the cpDNA [AIC = 37685.54, delta-AIC (ΔAIC) = 0.00] and the nrITS (AIC = 4004.05, ΔAIC = 0.00) data sets were the transversion model with a gamma distribution (TVM + G) and the general time reversible model with a gamma distribution (GTR + G), respectively. The BA analysis was run in MrBayes v3.2.7 (Ronquist et al. 2012) and consisted of four Markov Chain Monte Carlo (MCMC) chains, one heated (temperature = 0.05) and three unheated, and were run for 2 million generations, with sampling at every 1000 generations. In MrBayes, we used the GTR + G substitution model and we generated the posterior consensus tree when the runs had reached convergence (i.e., the standard deviation of split frequencies had stabilized and was much below the recommended threshold of 0.01) (Mackiewicz et al. 2022; Ronquist et al. 2012). In both analyses, Malus domestica and Pyrus communis were used as outgroups. Majority-rule consensus trees were created in Dendroscope 3 (Huson and Scornavacca 2012), and were visualized using the iTOL v6 web server (Letunic and Bork 2021).
Field pollination tests.
The pollen used for self-pollination was collected from balloon-stage flowers of a 10-year-old ‘Nemtudom P3’ tree growing in a germplasm collection at Kemenessömjén, Hungary (lat. 47°19′42.20″N, long. 17°6′50.90″E). Anthers were desiccated for 24 h at ambient temperature, and the released pollen was used for pollination. A total of 173 flowers of the same tree were emasculated, self-pollinated, and bagged to exclude bees. Another 183 flowers were counted on a marked branch to determine the fruit set ratio from open pollination by bees. The percentage of fruit set was recorded before and after June drop (23 May and 4 Jul 2021).
Results
Characterization of the self-incompatibility locus.
Sizing the fluorescently labeled amplicons of the S-RNase gene first intron region, we detected five fragment lengths in each cultivar: 208, 232, 351, 360, and 376 base pairs (bp) in ‘Nemtudom P3’ and 351, 373, 388, 390, and 412 bp in ‘Besztercei Bt.2’ (Fig. 1A). One of the five fragment lengths (351 bp) was shared by both cultivars. The second intron region is larger, and three primer combinations were used to amplify as many alleles as possible. In ‘Besztercei Bt.2’, the EM-PC2/PC3 primer pair amplified four alleles, whereas the primers amplifying a bigger fragment between C2 and C5 conserved regions could only detect the three smaller-sized alleles (Fig. 1B). However, in ‘Nemtudom P3’ all three combinations amplified five alleles, although the amplification intensity was somewhat lower in case of the higher-sized alleles. PaConsII-F/R produced more intense bands around 1500 bp than EM-PC2/PC5. A seemingly common fragment was also noted in the patterns of cultivars obtained by each of the primer combinations.
Determination of the self-incompatibility ribonuclease (S-RNase) alleles in Hungarian plum (Prunus domestica) cultivars. (A) Genotyper traces of the first intron amplicons and (B) electrophoretic patterns of the second intron region of S-RNase alleles (in negative) using three sets of consensus primers. A common allele shared by the two cultivars is highlighted in green, the asterisks indicate faint but visible bands; M = 1-kb DNA ladder; B = ‘Besztercei Bt.2’; N = ‘Nemtudom P3’.
Citation: J. Amer. Soc. Hort. Sci. 148, 5; 10.21273/JASHS05330-23
The common band amplified by the EM-PC2/PC3 primers was cloned and sequenced from both cultivars to check their identity. BlastN analysis indicated significant homology to other Prunus S-RNase alleles, and they were the most similar to the P. spinosa SB-RNase (E values of 4e−141 and 4e−136). Of the seven differing positions, two were found in the intron regions, four represented synonymous base substitutions in exons 2 and 3, and only one resulted in a conserved amino acid replacement (Lys→Arg) in the P. domestica ‘Besztercei Bt.2’ S18-RNase (Fig. 2).
Alignment of Prunus domestica ‘Besztercei Bt.2’ (B) and ‘Nemtudom P3’ (N) S18-ribonuclease allele with Prunus spinosa SB-RNase. Deduced amino acids are indicated under the center of the corresponding codons, base substitutions, and conserved amino acid replacements are shown against black background color. The S-RNase second intron is found between the vertical lines, exons 2 and 3 are indicated above the corresponding regions.
Citation: J. Amer. Soc. Hort. Sci. 148, 5; 10.21273/JASHS05330-23
Because of the lack of careful pollination studies to verify the self-(in)compatibility status of ‘Nemtudom P3’, controlled self- and open pollination of ‘Nemtudom P3’ flowers were performed. Before June drop, open pollination resulted in higher fruit set ratio than self-pollination. However, after June drop, both pollination types showed a similar fruit set ratio of 8% to 9% (Table 1), confirming the self-compatible phenotype of ‘Nemtudom P3’ plum.
The fruit set ratios of self- and open-pollinated flowers of Prunus domestica ‘Nemtudom P3’ plum after different lengths of time.
Nemtudom is native to and grows wild in northeastern Hungary along the rivers Tisza and Szamos (Supplemental Fig. 1), where 68 trees were sampled to determine if there were any genetic variations in the germplasm. The PCR analysis of the S-RNase second intron region of 35 Nemtudom accessions and ‘Besztercei Bt.2’ revealed different S-genotypes only in four individuals from the villages of Gacsály, Barabás, Tarpa, and Tiszaszalka (each shown in Fig. 3). All those trees carried a fragment under 750 bp, which was also detected in ‘Besztercei Bt.2’. Another allele of ‘Besztercei Bt.2’ was found in the trees sampled in Gacsály and Tarpa (with an approximate size of 900 bp). This latter genotype also amplified a fragment of 1500 bp, which was not seen in any other samples. The analysis of the polymorphic S-locus confirmed that most trees had the same genotype and only a small number of them carried one to three different S-alleles.
Polymerase chain reaction analysis of the autochthonous Hungarian Nemtudom landrace accessions and ‘Besztercei Bt.2’ plum (Prunus domestica). The second intron of the Prunus S-RNase gene in 35 presumed accessions of Nemtudom and ‘Besztercei Bt.2’ plum was amplified using the PaConsII-F/R consensus primers and separated on 1% TBE agarose gel after ethidium bromide staining (M = 1-kb DNA ladder). Genotypes are labeled by the village names of collection sites.
Citation: J. Amer. Soc. Hort. Sci. 148, 5; 10.21273/JASHS05330-23
Phylogenetic position of cultivar Nemtudom P3.
The concatenated cpDNA sequences of the ‘Nemtudom P3’ and ‘Besztercei Bt. 2’ had a total length of 8.4 kb, which covers 9.8% of the large single copy (LSC) region of the P. domestica chloroplast genome (Fig. 4). The size of the amplified regions ranged between 344 bp (trnH-psbA) and 1100 bp (trnS-trnG-trnG). The concatenated sequence involved 4143 (49.3%) and 3088 (36.8%) nucleotides of noncoding intergenic and intron regions, respectively, and the coding regions covered 1169 nucleotides (13.9%). Sequence alterations were not detected between the ‘Nemtudom P3’ and ‘Besztercei Bt.2’ in any region; however, 25 indels of 1–11 bp in size and 26 single-base substitutions (11 transversions and 15 transitions) were identified when compared with the database P. domestica sequences (Table 2). The cpDNA majority-rule consensus tree of ML and BA inferences is shown in Fig. 5A and the bootstrap values from the ML analyses and posterior probability values from the BA are given as percentages (ML/BA) next to the branches.
The name and position of the analyzed sequences on the Prunus domestica chloroplast genome. The genes are color coded according to their functional groups; genes shown outside the circle are transcribed clockwise, and those inside counterclockwise. The most and least informative loci are labeled by asterisks and blue color, respectively.
Citation: J. Amer. Soc. Hort. Sci. 148, 5; 10.21273/JASHS05330-23
Total number (T), conserved (C), variable (V), parsimony informative (PIC), and singleton (S) sites in the genic (CDS), intergenic (non-CDS), and intron regions of the analyzed Prunus chloroplast DNA sequence alignment, the number of indels and single nucleotide polymorphisms (SNPs) as well as the sequence alterations (SA) between the Hungarian and other plum cultivars.
Phylogenetic analysis of Prunus domestica using majority-rule consensus trees based on the combined maximum-likelihood (ML) and Bayesian inference (BA). (A) The phylogenetic tree based on the chloroplast DNA dataset. Bootstrap values from the ML analyses (ML ≥60) and posterior probability values from the BA analyses (BA ≥0.60) are given as percentages (ML/BA) on or next to the branches. The “−” symbol indicates support values less than 60%. (B) The phylogenetic tree based on the internal transcribed spacers 1 and 2 of P. domestica nuclear ribosomal DNA dataset. Bootstrap values from the ML analyses (ML ≥50) and posterior probability values from the BA analyses (BA ≥ 0.50) are given as percentages (ML/BA) on or next to the branches. The “−” symbol indicates support values less than 50%. The GenBank accession number for the sequence of each species is given in brackets. The ‘Nemtudom P3’ and ‘Besztercei Bt.2’ sequences were determined in this study; others were downloaded from the National Center for Biotechnology Information nucleotide database.
Citation: J. Amer. Soc. Hort. Sci. 148, 5; 10.21273/JASHS05330-23
The reconstructed tree confirmed the expected phylogenetic positions of ‘Nemtudom P3’ and ‘Besztercei Bt.2’, although they formed a statistically supported subclade that was sister to other P. domestica cultivars (Fig. 5A). P. cerasifera was the closest positioned accession to P. domestica, followed by other strongly supported monophyletic groups containing 1) Prunus salicina and Prunus simonii; 2) Prunus persica, Prunus dulcis, Prunus tennella, and Prunus ussuriensis; as well as 3) Prunus humilis, Prunus japonica, and Prunus glandulosa. P. spinosa f. macrocarpa was positioned more distantly.
The nrITS amplicons of ‘Nemtudom P3’ and ‘Besztercei Bt.2’ were of 639 bp in length, containing 15- and 13-bp fragments of 18S and 26S ribosomal RNA genes, respectively, as well as the complete sequence of the 5.8S ribosomal RNA gene (160 bp). The lengths of ITS1 and ITS2 were 244 bp and 207 bp, respectively. There was a single-base substitution (A/G transition) between the ITS1 regions of the two studied cultivars and an indel and a base substitution compared with database plum accessions. The alignment with homologous NCBI database sequences of related species required 23 independent insertions/deletions, 14 and 9 of which were introduced into ITS1 and ITS2, respectively. The phylogenetic tree based on the nrITS sequences also confirmed the two Hungarian plum cultivars forming a separate and statistically supported monophyletic group within a bigger, less reliable paraphyletic group of other P. domestica, P. domestica ssp. insititia, P. spinosa, and P. tenella (Fig. 5B).
The highest ratio of parsimony informative characters (PICs) to the total length of sequence was identified in the nrITS region with 12.1%. In cpDNA, the proportion of PICs was the highest in trnS-trnG-trnG, trnC-ycf6, and trnH-psbA (Table 2). Although trnH-psbA had a considerable proportion of PICs, its short length resulted in a relatively small number of PICs. The ratio of PICs was the smallest in the psbB-psbH sequence, which contained the smallest noncoding portion (43%). However, for the additional sequences, the noncoding region occupied between 80% and 100% of the sequence, and their PIC ratios varied considerably between 3.4% and 9.5%.
Discussion
The analysis of the self-incompatibility locus.
Because P. domestica is hexaploid, a maximum of six alleles were expected to be carried by the cultivars. The amplification of S-RNase alleles using consensus PCR primers might be challenging (Halász et al. 2021a) and hence we applied several pairs of robust primers to compare the amplification efficiencies of the second intron regions. The PaConsII (Sonneveld et al. 2003) and EM-PC2/PC5 (Sutherland et al. 2004a) primer pair produced ∼220-bp bigger amplicons than those amplified by the EM-PC2/PC3 primers (Sutherland et al. 2004a) for its reverse primer anneals to the C3 region instead of C5 of the S-RNase gene. However, none of the primer pairs could amplify six S-RNase alleles, only five different alleles were detected in both ‘Besztercei Bt.2’ and ‘Nemtudom P3’.
Most studies published so far have assigned fewer than six alleles to a range of P. domestica cultivars (Fernandez i Marti et al. 2021; Halász et al. 2014; Sutherland et al. 2004b), the complete S-genotype could have been only proposed for a single cultivar (Abdallah et al. 2019). The reasons for the fewer S-RNase alleles detected may involve the preferential amplification of a subset of those by consensus primers (Brace et al. 1994), more copies of the same allele might be carried by the polyploid genome (Esselink et al. 2004) or a partial deletion in a chromosome 6 homeolog, similar to what has been reported for Snull in Prunus cerasus (Yamane et al. 2001).
One of the amplicons seemed to be shared by both cultivars and their DNA sequence in the C2-C3 region confirmed their functional identity. Allele-specificity of S-RNases mainly resides in the rosaceous hypervariable region (RHV) (Ushijima et al. 1998), which was identical in the tested sequences. The Prunus S-RNase gene is characterized by an allele-specific intron length, which provided the basis of an efficiently used PCR discrimination system (Tamura et al. 2000; Tao et al. 1999). The sequences determined in ‘Besztercei Bt.2’ and ‘Nemtudom P3’ had matching intron lengths [141 bp (Supplemental Table 3)], further confirming their identity. Only three base substitutions occurred between the pair of sequences, which resulted in a single amino acid replacement, just downstream of the RHV region. Although this conserved amino acid replacement is not likely to change allele specificity, pollination studies will be required for no-doubt clarification; however, it is only possible if the allele has not lost its function. We labeled this allele S18, following the order of P. domestica S-RNase labels introduced by Sutherland et al. (2008) and Fernandez i Marti et al. (2021).
The S-genotyping of 68 Nemtudom landrace accessions detected an overwhelming number of trees sharing a common S-genotype, identical to that of ‘Nemtudom P3’. This is consistent with the fact that plums have been vegetatively propagated by suckers in this region for a long time (Pethő 2011). However, four of the trees sampled around Gacsály, Barabás, Tarpa, and Tiszaszalka villages carried one, two, or three S-RNase alleles not present in most Nemtudom samples. Most of those alleles were also detected in ‘Besztercei Bt.2’, indicating they might be offspring of a cross between Besztercei and Nemtudom accessions. In this region, protected designation of origin was granted to plum spirit (Szatmári szilvapálinka), which stipulates the product can be made only from the fruits of two landraces, Nemtudom and Besztercei (Stéger-Máté 2006). In consequence, people grow exclusively these landraces in this geographic region, and our study provides evidence that hybridization occurs between the two predominant landraces.
The tree sampled in Tarpa amplified only three S-alleles, none of which was carried by Nemtudom. Two of them could be detected in ‘Besztercei Bt.2’; however, the most reliably amplified allele of ‘Besztercei Bt.2’ with the smallest size was missing from the Tarpa accession. Its S-genotype is consistent with the assumption that this tree might be derived from the self-pollination of a Besztercei accession. All clones of Besztercei were long known to be SC (Nyéki and Szabó 1996; Tóth 1975). In such a case, the band around 1500 bp was not detected in ‘Besztercei Bt.2’, presumably because of its great size but having two copies in the genome may explain the increased amplification efficiency. If this genotype indeed originated from the self-pollination of a Besztercei accession and carries two copies of each allele detected, its genotype automatically reveals the three nonfunctional S-haplotypes of Besztercei. In the tetraploid P. cerasus, only the diploid pollen grains carrying two inactivated S-haplotypes are capable of self-fertilization (Hauck et al. 2006). If this model also works in plums, at least three nonfunctional S-haplotypes must be accumulated in the triploid pollen of P. domestica to be SC. Therefore, Tarpa genotype might be of crucial importance in the identification of mutations inducing self-compatibility in P. domestica by putatively carrying the inactivated S-haplotypes.
Interestingly, we did not find an S-genotype that might be characteristic of an offspring from the self-fertilization of Nemtudom. Although Nemtudom is generally believed to be SC, based on its reliable yields in large orchards, no specific data from carefully conducted pollination studies have been found in the literature. Therefore, fruit set ratios were determined after self- and open pollination and found to be higher than 8% both before and after the June drop, hence, self-compatibility could have been confirmed based on the critical values set by Nyéki and Szabó (1996). Probably a larger sample set would detect offspring from Nemtudom self-fertilization or other factors like inbreeding depression (DeBuse et al. 2005) may explain why such individuals were not found in this area.
Phylogenetic position of ‘Nemtudom P3’ and ‘Besztercei Bt.2’.
Regions of the cpDNA and nrITS were frequently used for phylogenetic studies in Prunus (Batnini et al. 2019; Gilani et al. 2010; Quan and Zhou 2011; Reales et al. 2010; Shaw and Small 2004; Uncu 2020). Although recently, many studies have been published to show that some highly variable cpDNA regions can be used to detect intraspecific variability in Prunus and other rosaceous species (Li et al. 2020, 2021; Sevindik et al. 2023; Wei et al. 2021), our study is the first to prove that there is enough information in certain regions of cpDNA to detect differences between landraces or cultivars of P. domestica. From the cpDNA regions, trnS-trnG-trnG and trnD-trnT provided the most parsimony informative sites and were found best-fitting to former low-level systematic studies of angiosperm species (Quan and Zhou 2011; Shaw et al. 2005). Although trnC-ycf6 was identified by Shaw et al. (2005) as one of the regions with the fewest PICs, in our study it was characterized by considerable information content among species while it showed low intraspecific variability. The trnH-psbA was also reported by Shaw et al. (2005) to be among the less polymorphic regions and provided the third smallest number of PICs in our study, but it was mainly due to its small length whereas the relative number of PICs was among the highest values. This region was also variable in P. persica (Quan and Zhou 2011).
The ratios of the noncoding region were the smallest in case of psbB-psbH and psbA-3′trnK-matK. Both were characterized by few PICs, which points to the higher level of conservation of coding regions (Gielly and Taberlet 1994; Wang et al. 2022). However, for the additional sequences, the noncoding region occupied between 80% and 100% of the sequence, whereas their PICs varied greatly between 27 and 107. It gives support for other factors (e.g., sequence structure, mutational hot spots) influencing the mutability of different loci in the chloroplast genome (Kelchner 2000). Because proportions and distributions of variable sites in the chloroplast genome can be strikingly different for plant species (Fan et al. 2018), our results help the identification of regions in the Prunus chloroplast genome that carry the most information for evolutionary inferences.
The phylogenetic analyses based on the nuclear and cpDNA sequences were congruent in grouping the ‘Nemtudom P3’ and ‘Besztercei Bt.2’ in a well-supported subclade that was sister to another highly supported subgroup containing other P. domestica sequences. This topology confirms ‘Nemtudom P3’ belonging to P. domestica. However, resolving the relationship between P. domestica and P. domestica ssp. insititia was not possible, which agrees with previous studies (Reales et al. 2010). It was interesting to see that ‘Nemtudom P3’ and ‘Besztercei Bt.2’ formed a separate clade within all P. domestica sequences, pointing to some degree of genetic differentiation within the P. domestica germplasm. Similar data were obtained from an SSR analysis in which Besztercei clones and ‘Nemtudom P3’ were characteristically differentiated from commercial cultivars like Stanley, President, Hanita, and others (Makovics-Zsohár et al. 2017).
The origin of Besztercei landrace is unknown but most probably it was transported to the Carpathian Basin from the Balkans ∼600 years ago (Faust and Surányi 1998). The cultivar Nemtudom P3 was selected from a landrace population that is growing wild and has been also cultivated for centuries in the Upper Tisza region, Hungary (Pethő 2011). The long and isolated development of such landraces excludes the possibility that they would have crossed with cultivars from other parts of the world, which was confirmed by our present and former (Makovics-Zsohár et al. 2017) results. It draws attention to the breeding value of such unique genotypes that have developed in specific regions and have different genetic backgrounds, similar to the allelic singularity and the rich variability of Tunisian germplasm (Abdallah et al. 2019). The discovery of such genotypes is of crucial importance for the future of P. domestica cultivation. While facing a significant number of challenges, the current plum industry is dominated by a small number of popular cultivars in the main producing countries (Sottile et al. 2022). New sources of resistance to economically devastating diseases like PPV should be found (Neumüller 2011); hence, the naturally existing genetic diversity must be exploited to create the best possible cultivars for future use. However, several factors erode the available genetic variability, including the reproductive barriers between hexaploids and species with other ploidy levels, the consequences of a limited number of founder clones described in western European countries (Horvath et al. 2011; Urrestarazu et al. 2018), and the recent emergence of this putatively allopolyploid species under the influence of human selection in ancient Eurasian societies (Zhebentyayeva et al. 2019). Therefore, the identification and deep characterization of landraces and local germplasm are of high priority.
The S18-RNase allele identified in this study showed 97.9% to 98.9% to P. spinosa SB-RNase. It might be explained by transspecific evolution (Halász et al. 2021a) but this high degree of similarity is also consistent with the supposed origin of P. domestica, that it is a descendant of P. spinosa. Our results are further supported by Fernandez i Marti et al. (2021) who identified two additional P. domestica S-RNase alleles sharing similarity of 95% to 98% with P. spinosa alleles. There were three base substitutions between the DNA sequences of the ‘Besztercei Bt.2’ and ‘Nemtudom P3’ S18-RNase and in each of the three positions ‘Nemtudom P3’ was identical to P. spinosa SB-RNase, which was shown to occur frequently in blackthorn accessions native to the same geographic region as Nemtudom plum (Halász et al. 2021b). It is tempting to consider if Nemtudom as a small-fruited landrace represents a genotype close to the interspecific offspring resulting from the original introgressive hybridization between P. cerasifera and P. spinosa. It requires further analysis, and Nemtudom plum might be an interesting landrace for studies focusing on the origin of P. domestica.
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