The draft chromosome-level genome assembly of tetraploid ground cherry (Prunus fruticosa Pall.) from long reads.

Cherries are stone fruits and belong to the economically important plant family of Rosaceae with worldwide cultivation of different species. The ground cherry, Prunus fruticosa Pall., is an ancestor of cultivated sour cherry, an important tetraploid cherry species. Here, we present a long read chromosome-level draft genome assembly and related plastid sequences using the Oxford Nanopore Technology PromethION platform and R10.3 pore type. We generated a final consensus genome sequence of 366 Mb comprising eight chromosomes. The N50 scaffold was ~44 Mb with the longest chromosome being 66.5 Mb. The chloroplast and mitochondrial genomes were 158,217 bp and 383,281 bp long, which is in accordance with previously published plastid sequences. This is the first report of the genome of ground cherry (P. fruticosa) sequenced by long read technology only. The datasets obtained from this study provide a foundation for future breeding, molecular and evolutionary analysis in Prunus studies.

Identification and Molecular Analysis of Putative Self-Incompatibility Ribonuclease Alleles in an Extreme Polyploid Species, Prunus laurocerasus L.

Cherry laurel (Prunus laurocerasus L.) is an extreme polyploid (2n = 22x) species of the Rosaceae family where gametophytic self-incompatibility (GSI) prevents inbreeding. This study was carried out to identify the S-ribonuclease alleles (S-RNases) of P. laurocerasus using PCR amplification of the first and second intron region of the S-RNase gene, cloning and sequencing. A total of 23 putative S-RNase alleles (S 1-S 20, S 5 m, S 13 m, and S 18 m) were sequenced from the second (C2) to the fifth conserved region (C5), and they shared significant homology to other Prunus S-RNases. The length of the sequenced amplicons ranged from 505 to 1,544 bp, and similar sizes prevented the proper discrimination of some alleles based on PCR analysis. We have found three putatively non-functional alleles (S 5 m, S 18 m, and S 9) coding for truncated proteins. Although firm conclusions cannot be drawn, our data seem to support that heteroallelic pollen cannot induce self-compatibility in this polyploid Prunus species. The identities in the deduced amino acid sequences between the P. laurocerasus and other Prunus S-RNases ranged between 44 and 100%, without a discontinuity gap separating the identity percentages of trans-specific and more distantly related alleles. The phylogenetic position, the identities in nucleotide sequences of the second intron and in deduced amino acid sequences found one or more trans-specific alleles for all but S 10, S 14, S 18, and S 20 cherry laurel RNases. The analysis of mutational frequencies in trans-specific allele pairs indicated the region RC4-C5 accepts the most amino acid replacements and hence it may contribute to allele-specificity. Our results form the basis of future studies to confirm the existence and function of the GSI system in this extreme polyploid species and the alleles identified will be also useful for phylogenetic studies of Prunus S-RNases as the number of S-RNase sequences was limited in the Racemose group of Prunus (where P. laurocerasus belongs to).

Simple Sequence Repeat and S-Locus Genotyping to Assist the Genetic Characterization and Breeding of Polyploid Prunus Species, P. spinosa and P. domestica subsp. insititia.

Polyploid Prunus spinosa (2n = 4 ×) and P. domestica subsp. insititia (2n = 6 ×) represent enormous genetic potential in Central Europe, which can be exploited in breeding programs. In Hungary, 16 cultivar candidates and a recognized cultivar 'Zempléni' were selected from wild-growing populations including ten P. spinosa, four P. domestica subsp. insititia and three P. spinosa × P. domestica hybrids (2n = 5 ×) were also created. Genotyping in eleven simple sequence repeat (SSR) loci and the multiallelic S-locus was used to characterize genetic variability and achieve a reliable identification of tested accessions. Nine SSR loci proved to be polymorphic and eight of those were highly informative (PIC values ˃ 0.7). A total of 129 SSR alleles were identified, which means 14.3 average allele number per locus and all accessions but two clones could be discriminated based on unique SSR fingerprints. A total of 23 S-RNase alleles were identified and the complete and partial S-genotype was determined for 10 and 7 accessions, respectively. The DNA sequence was determined for a total of 17 fragments representing 11 S-RNase alleles. 'Zempléni' was confirmed to be self-compatible carrying at least one non-functional S-RNase allele (SJ). Our results indicate that the S-allele pools of wild-growing P. spinosa and P. domestica subsp. insititia are overlapping in Hungary. Phylogenetic and principal component analyses confirmed the high level of diversity and genetic differentiation present within the analysed accessions and indicated putative ancestor-descendant relationships. Our data confirm that S-locus genotyping is suitable for diversity studies in polyploid Prunus species but non-related accessions sharing common S-alleles may distort phylogenetic inferences.

LC-MS based metabolic fingerprinting of apricot pistils after self-compatible and self-incompatible pollinations.

KEY MESSAGE: LC-MS based metabolomics approach revealed that putative metabolites other than flavonoids may significantly contribute to the sexual compatibility reactions in Prunus armeniaca. Possible mechanisms on related microtubule-stabilizing effects are provided. Identification of metabolites playing crucial roles in sexual incompatibility reactions in apricot (Prunus armeniaca L.) was the aim of the study. Metabolic fingerprints of self-compatible and self-incompatible apricot pistils were created using liquid chromatography coupled to time-of-flight mass spectrometry followed by untargeted compound search. Multivariate statistical analysis revealed 15 significant differential compounds among the total of 4006 and 1005 aligned metabolites in positive and negative ion modes, respectively. Total explained variance of 89.55% in principal component analysis (PCA) indicated high quality of differential expression analysis. The statistical analysis showed significant differences between genotypes and pollination time as well, which demonstrated high performance of the metabolic fingerprinting and revealed the presence of metabolites with significant influence on the self-incompatibility reactions. Finally, polyketide-based macrolides similar to peloruside A and a hydroxy sphingosine derivative are suggested to be significant differential metabolites in the experiment. These results indicate a strategy of pollen tubes to protect microtubules and avoid growth arrest involved in sexual incompatibility reactions of apricot.

Retraction Note: Comparison of traditional and new generation DNA markers declares high genetic diversity and differentiated population structure of wild almond species.

Editor's Note: this Article has been retracted; the Retraction Note is available at https://www.nature.com/articles/s41598-020-72522-x.

Identification, Structural and Functional Characterization of Dormancy Regulator Genes in Apricot (Prunus armeniaca L.).

In the present study, we identified and characterized the apricot (Prunus armeniaca L.) homologs of three dormancy-related genes, namely the ParCBF1 (C-repeat binding factor), ParDAM5 (dormancy-associated MADS-BOX) and ParDAM6 genes. All highly conserved structural motifs and the 3D model of the DNA-binding domain indicate an unimpaired DNA-binding ability of ParCBF1. A phylogenetic analysis showed that ParCBF1 was most likely homologous to Prunus mume and Prunus dulcis CBF1. ParDAM5 also contained all characteristic domains of the type II (MIKCC) subfamily of MADS-box transcription factors. The homology modeling of protein domains and a phylogenetic analysis of ParDAM5 suggest its functional integrity. The amino acid positions or small motifs that are diagnostic characteristics of DAM5 and DAM6 were determined. For ParDAM6, only a small part of the cDNA was sequenced, which was sufficient for the quantification of gene expression. The expression of ParCBF1 showed close association with decreasing ambient temperatures in autumn and winter. The expression levels of ParDAM5 and ParDAM6 changed according to CBF1 expression rates and the fulfillment of cultivar chilling requirements (CR). The concomitant decrease of gene expression with endodormancy release is consistent with a role of ParDAM5 and ParDAM6 genes in dormancy induction and maintenance. Cultivars with higher CR and delayed flowering time showed higher expression levels of ParDAM5 and ParDAM6 toward the end of endodormancy. Differences in the timing of anther developmental stages between early- and late-flowering cultivars and two dormant seasons confirmed the genetically and environmentally controlled mechanisms of dormancy release in apricot generative buds. These results support that the newly identified apricot gene homologs have a crucial role in dormancy-associated physiological mechanisms.

Comparison of traditional and new generation DNA markers declares high genetic diversity and differentiated population structure of wild almond species.

Wild almond species as sources of genetic variation may have crucial importance in breeding. A total of 389 accessions of 18 species have been analysed using inter-retrotransposon amplified polymorphism (IRAP), retrotransposon-microsatellite amplified polymorphism (REMAP), sequence-specific amplification polymorphism (S-SAP), amplified fragment length polymorphism (AFLP), inter simple sequence repeat (ISSR) and simple sequence repeats (SSR). Retrotransposon markers indicated the presence and movement of some Ty3-gypsy and Ty1-copia-elements in almond genome. Since transposable elements are associated with large-scale genome alterations, REMAP produced more reliable phylogenetic inferences than AFLP where homoplasy may affect clustering. In addition, high resolution melting (HRM) analysis was developed to detect SNPs. HRM analysis revealed 1:189 bp frequency of SNPs in exon positions, and the transition-to-transversion proportion was 1.84:1. The low transition bias suggests low methylation levels in almond genome. The polymorphic information content (PIC) was the highest for SSR markers, while SNPs had an average PIC of 0.59, which is close to the values of the rest of the markers. Huge genetic diversity, fragmented population structure and footprints of human selection was confirmed by merging information from all marker strategies. Considering time, cost and performance HRM can be a marker of choice in future studies of Prunus diversity.

Simple Sequence Repeat and S-locus Genotyping to Explore Genetic Variability in Polyploid Prunus spinosa and P. insititia.

Polyploid Prunus spinosa (2n = 4×) and P. insititia (2n = 6×) represent enormous genetic potential in Central Europe, which can be exploited in breeding programmes. In Hungary, 17 cultivar candidates were selected from wild-growing populations including 10 P. spinosa, 4 P. insititia and three P. spinosa × P. domestica hybrids (2n = 5×). Their taxonomic classification was based on their phenotypic characteristics. Six simple sequence repeats (SSRs) and the multiallelic S-locus genotyping were used to characterize genetic variability and reliable identification of the tested accessions. A total of 98 SSR alleles were identified, which presents 19.5 average allele number per locus, and each of the 17 genotypes could be discriminated based on unique SSR fingerprints. A total of 23 S-RNase alleles were identified. The complete and partial S-genotype was determined for 8 and 9 accessions, respectively. The identification of a cross-incompatible pair of cultivar candidates and several semi-compatible combinations help maximize fruit set in commercial orchards. Our results indicate that the S-allele pools of wild-growing P. spinosa and P. insititia are overlapping in Hungary. A phylogenetic and principal component analysis confirmed the high level of diversity and genetic differentiation present within the analysed genotypes and helped clarify doubtful taxonomic identities. Our data confirm that S-locus genotyping is suitable for diversity studies in polyploid Prunus species. The analysed accessions represent huge genetic potential that can be exploited in commercial cultivation.

Identification of a recently active Prunus-specific non-autonomous Mutator element with considerable genome shaping force.

Miniature inverted-repeat transposable elements (MITEs) are known to contribute to the evolution of plants, but only limited information is available for MITEs in the Prunus genome. We identified a MITE that has been named Falling Stones, FaSt. All structural features (349-bp size, 82-bp terminal inverted repeats and 9-bp target site duplications) are consistent with this MITE being a putative member of the Mutator transposase superfamily. FaSt showed a preferential accumulation in the short AT-rich segments of the euchromatin region of the peach genome. DNA sequencing and pollination experiments have been performed to confirm that the nested insertion of FaSt into the S-haplotype-specific F-box gene of apricot resulted in the breakdown of self-incompatibility (SI). A bioinformatics-based survey of the known Rosaceae and other genomes and a newly designed polymerase chain reaction (PCR) assay verified the Prunoideae-specific occurrence of FaSt elements. Phylogenetic analysis suggested a recent activity of FaSt in the Prunus genome. The occurrence of a nested insertion in the apricot genome further supports the recent activity of FaSt in response to abiotic stress conditions. This study reports on a presumably active non-autonomous Mutator element in Prunus that exhibits a major indirect genome shaping force through inducing loss-of-function mutation in the SI locus.

Self-(in)compatibility genotypes of Moroccan apricots indicate differences and similarities in the crop history of European and North African apricot germplasm.

BACKGROUND: Allelic diversity of the S-locus is attributed to the genetic relationships among genotypes and sexual reproduction strategy. In otherwise self-incompatible Prunus species, the emergence of loss-of-function in S-haplotypes has resulted in self-compatibility. This information may allow following major stages of crop history. The genetic diversity in the S-locus of local apricots (Prunus armeniaca L.) from different oasis ecosystems in Morocco and the comparison of the occurrence and frequency of S-alleles with other regions may allow testing the validity of previous theories on the origin and dissemination of North African apricots. RESULTS: The S-genotypes of 55 Moroccan apricot accessions were determined, resulting in 37 self-compatible genotypes, from which 33 were homozygotes for self-compatibility. SC was the most frequent S-allele in this germplasm, followed by S13, S7, S11, S2, S20, S8, and S6. New approaches (CAPS or allele-specific PCR) were designed for a reliable verification of the rare or unexpected alleles. The frequency and distribution of the S-alleles differed among the oases. Some of these alleles, S8, S11, S13 and S20, were formerly detected only in the Irano Caucasian germplasm and are not present in Europe. CONCLUSIONS: Our data supports the Irano-Caucasian origin of the Moroccan apricots and their original introduction by Phoenicians and Arabs through the North African shore. North Africa seems to have preserved much higher variability of apricot as compared with Europe. The loss of genetic diversity in apricot might be explained by the occurrence of self-compatibility and the length of time that apricot has spent with this breeding system in an environment without its wild relatives, such as the Moroccan oases or Central Europe.

Antioxidant and antiradical capacities in apricot (Prunus armeniaca L.) fruits: variations from genotypes, years, and analytical methods.

The fruit quality parameters and antioxidant capacity (ferric reducing antioxidant power, FRAP) and total phenolic content (TPC) were determined in 27 apricot cultivars and hybrids of diverse origins. Twenty one- to 35-fold variations were measured among FRAP and TPC values. Besides genotype, harvest year also contributed significantly (P≤0.05) to the variations of TPC presumably due to the climatic differences between years. A subset of genotypes (15) was also analyzed for their antiradical activities (2,2'-diphenyl-1-picrylhydrazyl, DPPH; total radical-scavenging activity, TRSA; water-soluble antioxidant capacity, ACW; and lipid-soluble antioxidant capacity, ACL), and vitamin C contents as well as color indices (CIE H°, L*, and chroma). The hybrid "Preventa" had outstanding FRAP (>10.4 mmol ascorbic acid/L), DPPH (74.45%), TRSA (0.002%), ACW (33587.5 nmol AA/L) and ACL (78.65 nmol Trolox/L), TPC (>2890.0 mg gallic acid/L), and vitamin C (16.17 mg/100 g FW) levels and an average carotenoid content estimated from the hue angle (66.99°). Most antioxidant and antiradical activities correlated significantly except for TRSA; the closest correlation was observed between FRAP and ACW (r=0.952). Only TRSA showed significant correlations with color indices, H° and chroma, suggesting TRSA measures at least a fraction of the antioxidant capacity attributable to apricot carotenoids.

Origin and dissemination of the pollen-part mutated SC haplotype which confers self-compatibility in apricot (Prunus armeniaca).

In China, its centre of origin, apricot (Prunus armeniaca) is self-incompatible. However, most European cultivars are self-compatible. In most cases, self-compatibility is a result of a loss-of-function mutation within the pollen gene (SFB) in the SC haplotype. Controlled pollinations performed in this work revealed that the cross 'Ceglédi óriás' (S8S9)x'Ceglédi arany' (SCS9) set well, as expected, but the reciprocal cross did not. Apricot S8, S9 and SC haplotypes were analysed using a multilevel approach including fruit set evaluation, pollen tube growth analysis, RNase activity assays, polymerase chain reaction (PCR) analysis and DNA sequencing of the S-RNase and SFB alleles. SFB8 was revealed to be the first known progenitor allele of a naturally occurring self-compatibility allele in Prunus, and consequently SC=The first intron of SC-RNase is a phase one intron, indicating its more recent evolutionary origin compared with the second intron. Sequence analysis of different cultivars revealed that more single nucleotide polymorphisms accumulated in SC-RNase than in SFBC. New methods were designed to allow high-throughput analysis of S genotypes of apricot cultivars and selections. S-RNase sequence data from various sources helped to elucidate the putative origin and dissemination of self-compatibility in apricot conferred by the SC haplotype.