Related Prunus Species Research Articles

A trnH-psbA barcode genotyping assay for the detection of common apricot (Prunus armeniaca L.) adulteration in almond (Prunus dulcis Mill.)

ABSTRACT Almond kernels can be mixed with apricot (Prunus armeniaca L.) seeds while preparing certain almond-based products such as marzipan, without an identifiable alteration in the final product’s sensory attributes. Consequently, apricot kernels are the most commonly utilized almond substitutes for the economic adulteration of almonds. In the present work, the trnH-psbA intergenic spacer was evaluated as a potential PCR-based authenticity marker of almond. The barcode enabled the straightforward discrimination of almond and apricot in their blends, based on diagnostic length polymorphisms between the two closely related Prunus species. The proposed assay simultaneously amplifies and distinguishes almond- and apricot-specific trnH-psbA barcode fragments from mixed samples. The barcode size of almond segregates significantly from peach as well, which is a secondary source for the economic adulteration of almond kernels. The proposed trnH-psbA genotyping assay is easy and selective for the discrimination of almond from its potential adulterants when present as admixtures.

Combination of Simple Sequence Repeat, S-Locus Polymorphism and Phenotypic Data for Identification of Tunisian Plum Species (Prunus spp.).

Plums (Prunus spp.) are among the first fruit tree species that attracted human interest. Artificial crosses between wild and domesticated species of plums are still paving the way for creation of new phenotypic variability. In Tunisia, despite a considerable varietal richness of plum as well as a high economic value, the plum sector is experiencing a significant regression. The main reason of this regression is the absence of a national program of plum conservation. Hence, this work was aimed to phenotypically and genetically characterize 23 Tunisian plum accessions to preserve this patrimony. Closely related Prunus species from the same subgenus may be differing at two characteristics: ploidy level and phenotypic traits. In this study, single sequence repeat (SSR) markers allowed distinguishing between eighteen diploid accessions and five polyploid accessions, but SSR data alone precluded unambiguous ploidy estimation due to homozygosity. In contrast, S-allele markers were useful to identify the ploidy level between polyploid species, but they did not distinguish species with the same ploidy level. Seven out of 12 phenotypic traits were shown to be discriminant traits for plum species identification. Molecular and phenotypic traits were significantly correlated and revealed a powerful tool to draw taxonomic and genotypic keys. The results obtained in this work are of great importance for local Tunisian plum germplasm management.

Development of a LC-MS method for the discrimination between trace level Prunus contaminants of spices

The need for an analytical procedure for the identification of allergens present at trace levels in foods was highlighted by conflicting results in a case of contamination of the spice cumin. The application of a bottom-up proteomics experiment was investigated to identify marker peptides for potential contaminant nuts which could then be monitored with high specificity and sensitivity by selective reaction monitoring experiments. The method developed allowed for the distinction between two closely related Prunus species, almond and mahaleb, in two different spices, cumin and paprika. The paprika sample was found to be contaminated with almond and the cumin sample, contaminated at a much lower level, was found to be contaminated with mahaleb. The method could be applied to any protein-dense food matrix allergen so long as suitable control and reference samples can be acquired.

The Peach v2.0 release: high-resolution linkage mapping and deep resequencing improve chromosome-scale assembly and contiguity

BackgroundThe availability of the peach genome sequence has fostered relevant research in peach and related Prunus species enabling the identification of genes underlying important horticultural traits as well as the development of advanced tools for genetic and genomic analyses. The first release of the peach genome (Peach v1.0) represented a high-quality WGS (Whole Genome Shotgun) chromosome-scale assembly with high contiguity (contig L50 214.2 kb), large portions of mapped sequences (96%) and high base accuracy (99.96%). The aim of this work was to improve the quality of the first assembly by increasing the portion of mapped and oriented sequences, correcting misassemblies and improving the contiguity and base accuracy using high-throughput linkage mapping and deep resequencing approaches.ResultsFour linkage maps with 3,576 molecular markers were used to improve the portion of mapped and oriented sequences (from 96.0% and 85.6% of Peach v1.0 to 99.2% and 98.2% of v2.0, respectively) and enabled a more detailed identification of discernible misassemblies (10.4 Mb in total). The deep resequencing approach fixed 859 homozygous SNPs (Single Nucleotide Polymorphisms) and 1347 homozygous indels. Moreover, the assembled NGS contigs enabled the closing of 212 gaps with an improvement in the contig L50 of 19.2%.ConclusionsThe improved high quality peach genome assembly (Peach v2.0) represents a valuable tool for the analysis of the genetic diversity, domestication, and as a vehicle for genetic improvement of peach and related Prunus species. Moreover, the important phylogenetic position of peach and the absence of recent whole genome duplication (WGD) events make peach a pivotal species for comparative genomics studies aiming at elucidating plant speciation and diversification processes.

Novel Approach to the Rapid Differentiation of Common <i>Prunus</i> Allergen Species by PCR Product Melt Analysis

Food allergies represent a clear threat to the general health and wellbeing of those affected which place increasing pressure on food producers and regulatory authorities. Current analytical techniques typically find difficulties distinguishing between closely related Prunus species which include almond (Prunus dulcis), an EU listed allergenic species. This study describes a proof of principle real-time PCR approach utilising DNA melt analyses that targets the internal transcribed spacer (ITS) sequence to differentiate between a panel of Prunus test species. The method was successfully applied to the characterisation of a commercial paprika sample suspected of having being adulterated with almond, referred to the UK Government Chemist in 2015 in its advisory capacity. Subject to further validation work, the method appears to specifically amplify Prunus species and is capable of discrimination based on the resultant melt profiles. The developed method provides analysts with a simple and broad molecular tool to identify common Prunus species for food authenticity and allergen testing purposes. Initial development work demonstrates a promising approach with the potential to improve discrimination between Prunus species not easily resolved by routine analytical methods.

Parallel consideration of SSRs and differentially expressed genes under abiotic stress for targeted development of functional markers in almond and related Prunus species

RNA-Seq approach is widely used to study plant transcriptome responses to different environmental stresses. RNA-Seq datasets have also become valuable resources to develop SSR markers and other types of markers in plant species. However, there are challenges such as the validation of SSR polymorphisms, and translation of these information into a functional approach for plant breeding programs. In our recent work, the first de novo transcriptome assembly of almond have been reported in response to freezing stress, and thousands of differential expression (DE) genes have been identified. Here, for the first time, we have suggested a parallel consideration of genes with DE under frost stress and SSR markers to find functional markers in almond (Prunus dulcis Mill.) and other related Prunus species. The term “RNA-Seq SSR” was used in the current study, replacing the previous term “EST-SSR” (expressed sequence tagged), for the distinction between traditional EST sequencing and the new RNA-Seq methods. Eleven RNA-Seq SSR markers were identified as polymorphic markers. Some of SSR loci were found on genes which are responsive in cold and other abiotic stresses, including calmodulin, trihelix transcription factor GT-1-like and delta-(8)-fatty-acid desaturase. Furthermore, these markers revealed high polymorphism in population of Prunus arabica, Prunus scoparia and Prunus haussknechtii. Our overall results suggest potential application of DE genes carrying SSR sequences as functional markers. The developed workflow and the new findings presented here are likely to open new opportunity for future genetic diversity, association studies and breeding projects of almond and other plants grown under environmental stresses. This workflow can also be applied to targeted validation and development of SNP and/or indel markers.

Whole-Genome Analysis of Diversity and SNP-Major Gene Association in Peach Germplasm.

Peach was domesticated in China more than four millennia ago and from there it spread world-wide. Since the middle of the last century, peach breeding programs have been very dynamic generating hundreds of new commercial varieties, however, in most cases such varieties derive from a limited collection of parental lines (founders). This is one reason for the observed low levels of variability of the commercial gene pool, implying that knowledge of the extent and distribution of genetic variability in peach is critical to allow the choice of adequate parents to confer enhanced productivity, adaptation and quality to improved varieties. With this aim we genotyped 1,580 peach accessions (including a few closely related Prunus species) maintained and phenotyped in five germplasm collections (four European and one Chinese) with the International Peach SNP Consortium 9K SNP peach array. The study of population structure revealed the subdivision of the panel in three main populations, one mainly made up of Occidental varieties from breeding programs (POP1OCB), one of Occidental landraces (POP2OCT) and the third of Oriental accessions (POP3OR). Analysis of linkage disequilibrium (LD) identified differential patterns of genome-wide LD blocks in each of the populations. Phenotypic data for seven monogenic traits were integrated in a genome-wide association study (GWAS). The significantly associated SNPs were always in the regions predicted by linkage analysis, forming haplotypes of markers. These diagnostic haplotypes could be used for marker-assisted selection (MAS) in modern breeding programs.

QTL mapping of pomological traits in peach and related species breeding germplasm

Peach is an economically important fruit tree crop that exhibits high phenotypic variability yet suffers from diversity-limited gene pool. Genetic introgression of novel alleles from related species is being pursued to expand genetic diversity. This process is, however, challenging and requires the incorporation of innovative genomic and statistical tools to facilitate efficient transfer of these exotic alleles across the multiple generations required for introgression. In this study, pedigree-based analysis (PBA) in a Bayesian QTL mapping framework was applied to a diverse peach pedigree introgressed with almond and other related Prunus species. The aim was to investigate the genetic control of eight commercially important fruit productivity and fruit quality traits over two subsequent years. Fifty-two QTLs with at least positive evidence explaining up to 98 % of the phenotypic variance across all trait/year combinations were mapped separately per trait and year. Several QTLs exhibited variable association with traits between years. By using the peach genome sequence as a reference, the intrachromosomal positions for several QTLs were shown to differ from those previously reported in peach. The inclusion of introgressed germplasm and the explicit declaration of the genetic structure of the pedigree as covariate in PBA enhanced the mapping and interpretation of QTLs. This study serves as a model study for PBA in a diverse peach breeding program, and the results highlight the ability of this strategy to identify genomic resources for direct utilization in marker-assisted breeding.

Genetic diversity and distribution patterns of Ecuadorian capuli (Prunus serotina)

Prunus serotina subsp. capuli (Cav.) is an arboreal species with promising economic prospects in the timber, health-food and neutraceutical markets. Despite its cultural and commercial significance, limited information exists with regards to the degree of genetic variation and ecological history of P. serotina in Ecuador. The main objective of this study was to evaluate the degree of genetic diversity and population structure of Ecuadorian P. serotina, as a preliminary step towards understanding the distribution history of this species in Ecuador and establishing germplasm conservation programs. P. serotina samples (217, representing 8 provinces from the Ecuadorian highlands) were characterized using 8 heterologous SSR markers derived from related Prunus species. Expected heterozygosity across samples (He = 0.71) reveals a moderate level of genetic diversity for Ecuadorian P. serotina. Nevertheless, simple allele-count analysis indicates that Ecuadorian capuli has a narrower degree of allelic richness relative to collections from the species’ center of origin in North America. Furthermore, pairwise F statistics (0.0069 ≤ Fst ≤ 0.0319) and Nei genetic distance estimates (0.02 ≤ Ds ≤ 0.09) indicate minimal population differentiation within Ecuadorian capuli. However, Bayesian population structure analysis suggests a subtle genetic contrast between germplasm from the Northern and Southern highlands. Certainly, it is of interest to analyze whether this underlying genetic differentiation between the Northern- and Southern-Highland groups is also manifested in morphological, agronomic or other phenotypic characters that could indicate adaptive differences to divergent agro-ecosystems.

Genetic diversity in peach [Prunus persica (L.) Batsch] at the University of Florida: past, present and future

The University of Florida (UF) stone fruit breeding and genetics program was created in 1952 to develop early ripening stone fruit cultivars with high quality, adaptation to summer rainfall, low chilling requirements, and the ability to withstand high disease pressure. Diverse germplasm sources were used to introduce desirable traits in UF breeding pool. The main objective of this research was to determine the genetic diversity and population structure of the breeding germplasm, and to search for loci under selection. A total of 195 peach genotypes were used: UF cultivars and advanced selections (n = 168), cultivars and selections from the UF-UGA-USDA joint breeding effort (n = 13), landrace cultivars (n = 4), high-chilling cultivars released by NCSU (n = 5), and related Prunus (n = 5) species. A total of 36 SSR markers distributed across the peach genome amplified 423 alleles. An average of 18 genotypes were detected per marker: A (number of observed alleles) of 11.43, Ae (effective number of alleles) of 2.58, Ho (observed heterozygosity) of 0.4, He (expected heterozygosity) of 0.52, F (Wright’s fixation index) of 0.25, and PIC (polymorphism information content) of 0.48. UPGMA cluster analysis based on Nei’s genetic distance represented best the known pedigree information for the germplasm pools. Two major groups were observed across the germplasm corresponding to melting and non-melting flesh cultivars/selections. Population structure results supported these two major groups. Several loci closely located to genome regions where different phenotypic traits have been previously mapped were detected to be under selection.

A PCR BASED RFLP SURVEY OF S-ALLELES IN WILD AND CULTIVATED ALMOND AND RELATED PRUNUS SPECIES

Almond is one of the most important nut crops in the world. The origin of almond is central and west Asia. About 30 wild species are identified in this region which represent a rich source of desirable characteristics useful in almond breeding programs. However, almonds express gametophytic self- and cross-incompatibility which affects breeding parent selection. Incompatibility in almond is controlled by a multi-allelic, single gene (S-locus). Recently chemical and molecular methods have been utilized to survey the S-alleles in almond cultivars and closely related species. The restriction fragment length polymorphism method using CAPs protocol was used to determin S-alleles in multiple accessions of wild almond and related Prunus species. Polymerase chain reactions were carried out using one set of degenerate primer pairs (EM-PC2consFD/EM-PC3consRD), and PCR products were digested by five restriction enzymes (TaqI, HinfI, DpnI, ApoI and AluI). The genotypes revealed different cutting paterns. ApoI and DpnI produced the most and the least bandings respectively. AluI, ApoI and HinfI digested the S7. ApoI was the only enzyme which could digest the S9. TaqI, ApoI and HinfI digested the S8 and S12.

Development and Evaluation of a 9K SNP Array for Peach by Internationally Coordinated SNP Detection and Validation in Breeding Germplasm

Although a large number of single nucleotide polymorphism (SNP) markers covering the entire genome are needed to enable molecular breeding efforts such as genome wide association studies, fine mapping, genomic selection and marker-assisted selection in peach [Prunus persica (L.) Batsch] and related Prunus species, only a limited number of genetic markers, including simple sequence repeats (SSRs), have been available to date. To address this need, an international consortium (The International Peach SNP Consortium; IPSC) has pursued a coordinated effort to perform genome-scale SNP discovery in peach using next generation sequencing platforms to develop and characterize a high-throughput Illumina Infinium® SNP genotyping array platform. We performed whole genome re-sequencing of 56 peach breeding accessions using the Illumina and Roche/454 sequencing technologies. Polymorphism detection algorithms identified a total of 1,022,354 SNPs. Validation with the Illumina GoldenGate® assay was performed on a subset of the predicted SNPs, verifying ∼75% of genic (exonic and intronic) SNPs, whereas only about a third of intergenic SNPs were verified. Conservative filtering was applied to arrive at a set of 8,144 SNPs that were included on the IPSC peach SNP array v1, distributed over all eight peach chromosomes with an average spacing of 26.7 kb between SNPs. Use of this platform to screen a total of 709 accessions of peach in two separate evaluation panels identified a total of 6,869 (84.3%) polymorphic SNPs.The almost 7,000 SNPs verified as polymorphic through extensive empirical evaluation represent an excellent source of markers for future studies in genetic relatedness, genetic mapping, and dissecting the genetic architecture of complex agricultural traits. The IPSC peach SNP array v1 is commercially available and we expect that it will be used worldwide for genetic studies in peach and related stone fruit and nut species.

Cultivar Identification, Pedigree Verification, and Diversity Analysis among Peach Cultivars Based on Simple Sequence Repeat Markers

Information on genetic relationships and pedigree structure in germplasm collections is vital to breeders in crop improvement programs. In this study, we assessed genetic identity, kinship distance, and parentage–sibship relationships among 37 peach (Prunus persica) accessions and breeding lines using simple sequence repeat (SSR) markers. Pairwise comparisons based on multilocus SSR profiles led to the identification of two synonymous groups including five accessions. Two pairs of parent–offspring and one full sibling relationships were identified using the likelihood method, and Bayesian cluster analysis partitioned the accessions into groups that were partially compatible with the known pedigree, origin, and flesh color. The 37 accessions were grouped into four clusters, which were largely supported by the known pedigree and origin of these accessions. Although the observed mean heterozygosity was 0.219, mean inbreeding coefficient was 0.635, indicating a high degree of inbreeding among the accessions. Eleven of the 15 SSR markers (73.3%) tested were transferable to nine related Prunus species. Results of the study demonstrate that these SSRs could facilitate the assessment of genetic identity and pedigree structure.

Genetic diversity of some wild almonds and related Prunus species revealed by SSR and EST-SSR molecular markers

Almond and its related wild species, which are widely distributed in Central and West Asia, have high genetic variation. This is an important source of genetic diversity for crop improvement. In this study a set of 32 SSR and 12 EST-SSR primer pairs were used to determine genetic diversity in 89 accessions of almond and other Prunus species. Most of the accessions (68) were collected from natural habitats of Iran. SSR primers amplified higher numbers of alleles than EST-SSR markers and discriminated genotypes more effectively. Results indicated high diversity among accessions. Observed heterozygosity (Ho) was 0.581. Nei’s index of diversity (He) and average number of alleles per locus (na) were 0.885 and 34, respectively. The mean value of polymorphism information content (PIC) was 0.874. The average Fst (F-statistics index) was 0.271 and the fixation index (Fis) was 0.151. Estimated variance among putative populations (AP) and individuals (AI) and within individuals (WI) were 5, 35, and 60%, respectively, which revealed that most of the variation was distributed among individuals rather than groups. Cultivated almonds were highly similar to P. fenzeliana, which is native to West Asia, supporting the importance of these regions in almond domestication. In the dendrogram of groups, minimum genetic distance was observed between Amygdalus and Orientalis groups from the Euamygdalus section. The Leptopus and Chameamygdalus sections were more distant from almonds than plums. The results also showed Dodecandra (Lycioides) series should be taxonomically classified closer to the section Euamygdalus.

Microsatellite variability between apricot and related Prunusspecies

Background Apricots, family Rosaceae, are economically important representatives of the genus Prunus. The number of apricot species ranges from three to ten, depending on the classification system adopted. The major obstacles to expansion of apricot production are irregular yields and low resistance to diseases. Compared with the high genetic variability in related species, apricots are not so variable and thus interspecific hybrids were proposed as a means to overcome deficiencies inherent in the common apricot. To estimate the extent of variation in apricot germplasm, between ecogeographical groups and related species, is very useful for planning breeding programmes, through diversity analysis, cultivar identification or marker-assisted selection.

Morphological and molecular variability in some Iranian almond genotypes and related Prunus species and their potentials for rootstock breeding

In this study, in order to know the variability for a rootstock breeding program genetic diversity and relationships among 55 Iranian almond genotypes and seven related Prunus species were investigated. Morphological and molecular analyses were used. Principal component analysis showed that three components explained 67.6% of the total morphological variation for the first year and 68.06% for the second year of the study. Leaf traits were predominant in the first component and contributed most of the total variation. Leaf length and width, as well as, leaf area were highly correlated with each other and correlated to vigor. Also a negative correlation was found between leaf length/width ratio and vigor. Ward's method was used to construct cluster from morphological data which allocated individuals into their respective species. Out of 100 pre-screened RAPD primers, 16 with reproducible bands and maximum polymorphism were selected. Two-hundred and sixty bands were scored of which 250 of them were polymorphic. Average value of polymorphism per primer was 95.81% and maximum value for polymorphism (100%) was obtained from TIBMBA-14, TIBMBA-17, TIBMBB-05, TIBMBB-08, TIBMBD-09, and TIBMBD-10. On the other hand, the minimum value was obtained from TIBMBB-16 (86%). Primer TIBMBB-5 gave the maximum number of bands (25 fragments) and the minimum obtained from TIBMBE-18 (11 fragments). Genetic similarity based on Jaccard's coefficient ranged from 0.28 to 0.79 with an average of 0.53. Molecular analysis revealed a high degree of separation among samples regarding their geographical origin. Correlation between two approaches was low ( R = −0.38). High molecular and morphological variability indicated that this collection includes rich and valuable plant materials for almond rootstock breeding.

Relationships among Peach, Almond, and Related Species as Detected by Simple Sequence Repeat Markers

The genetic relationships among peach [Prunus persica (L.) Batsch], almond [P. dulcis (Mill.) D.A. Webb or P. amygdalus (L.) Batsch] and 10 related Prunus species within the subgenus Amygdalus were investigated using simple sequence repeat (SSR) markers. P. glandulosa Pall. was included as an outgroup. Polymorphic alleles were scored as present or absent for each accession. The number of alleles revealed by the SSR analysis in peach and almond cultivars ranged from one to three whereas related Prunus species showed a range of one to 10 alleles. Results demonstrated an extensive genetic variability within this readily intercrossed germplasm as well as the value of SSR markers developed in one species of Prunus for the characterization of related species. Mean character difference distances were calculated for all pairwise comparisons and were used to construct an unrooted dendogram depicting the phenetic relationships among species. Four main groups were distinguished. Peach cultivars clustered with accessions of P. davidiana (Carr.) Franch. and P. mira Koehne. The second group contained almond cultivars. A third group included accessions of P. argentea (Lam) Rehd., P. bucharica Korschinsky, P. kuramica Korschinsky, P. pedunculata Pall, P. petunikowii Lits., P. tangutica (Spach) Batal., and P. webbii (Spach) Vieh.. P. glandulosa and P. scoparia Batal. were included in a fourth group.

Characterization of the ribosomal DNA units in two related Prunus species (P. Cerasifera and P. Spinosa)

The genetic relationships between two Prunus species, involved in rootstock breeding, were examined at the level of the ribosomal RNA genes. Twenty clones of P. cerasifera, a diploid species, and 12 clones of P. spinosa, a tetraploid wild species, were studied. The use of three heterologous ribosomal DNA probes covering different regions of the ribosomal tandem repeats enabled us to construct restriction maps for EcoRI and BamHI. We identified two unit types (unit I and unit II) in P. cerasifera. In P. spinosa, P. cerasifera units were present in addition to a third ribosomal unit type (unit III). These results appeared to confirm previous cytological studies (Salesses 1973) indicating that one of the genomes in P.spinosa has homology with the one from P. cerasifera.