Special Issue "Prunus Dormancy and Breeding"

Publication date: 05-08-2022

Authors: Guest Editor: Dr. Jose A. Egea; Dra. Raquel Sánchez-Pérez ; Dr. David Ruiz.

Journal: Plants

Prunus is a genus of trees and shrubs that includes a wide range of fruiting trees including those bearing plums, cherries, peaches, nectarines, apricots, almonds, and so on. More than 400 different species are classified under Prunus, and they are native to temperate regions. Many members of the genus are widely cultivated for their fruit. Like many other temperate species, Prunus species undergo a dormancy period in the autumn and winter and must be protected under potentially damaging climatic conditions. This dormancy period is overcome when a sufficient winter chill is accumulated, depending on each cultivar. According to the forecast of the Intergovernmental Panel on the Climatic Change (IPPC), in the next 30 years, there will be an increase in the average temperatures on the planet of the order of 2°C. A lack of winter chilling is a limiting factor for the cultivation of temperate fruit trees. This will require us to release and cultivate new lower-chilling-requiring cultivars to break dormancy. This Special Issue on “Prunus Dormancy and Breeding” aims to improve the knowledge on Prunus dormancy and on breeding strategies to face the forecasted lack of winter chill. We encourage the submission of original research papers as well as review papers dealing with new advances in Prunus adaptive genetics, genomics, genome editing techniques, population genetics, and breeding.


Advancing Endodormancy Release in Temperate Fruit Trees Using Agrochemical Treatments.

Publication date: 14-01-2022

Authors: Jesús Guillamón Guillamón, Federico Dicenta and Raquel Sánchez-Pérez*

Journal: Frontiers in Plant Sciences

Endodormancy in temperate fruit trees like Prunus is a protector state that allows the trees to survive in the adverse conditions of autumn and winter. During this process, plants accumulate chill hours. Flower buds require a certain number of chill hours to release from endodormancy, known as chilling requirements. This step is crucial for proper flowering and fruit set, since incomplete fulfillment of the chilling requirements produces asynchronous flowering, resulting in low quality flowers, and fruits. In recent decades, global warming has endangered this chill accumulation. Because of this fact, many agrochemicals have been used to promote endodormancy release. One of the first and most efficient agrochemicals used for this purpose was hydrogen cyanamide. The application of this agrochemical has been found to advance endodormancy release and synchronize flowering time, compressing the flowering period and increasing production in many species, including apple, grapevine, kiwi, and peach. However, some studies have pointed to the toxicity of this agrochemical. Therefore, other non-toxic agrochemicals have been used in recent years. Among them, Erger® + Activ Erger® and Syncron® + NitroActive® have been the most popular alternatives. These two treatments have been shown to efficiently advance endodormancy release in most of the species in which they have been applied. In addition, other less popular agrochemicals have also been applied, but their efficiency is still unclear. In recent years, several studies have focused on the biochemical and genetic variation produced by these treatments, and significant variations have been observed in reactive oxygen species, abscisic acid (ABA), and gibberellin (GA) levels and in the genes responsible for their biosynthesis. Given the importance of this topic, future studies should focus on the discovery and development of new environmentally friendly agrochemicals for improving the modulation of endodormancy release and look more deeply into the effects of these treatments in plants.


Evolution of fruit and seed traits during almond naturalization.

Publication date: 25-12-2021

Authors: Andrés Barea-Márquez,Francisco J. Ocaña-Calahorro,Rodrigo Balaguer-Romano,José María Gómez,Eugene W. Schupp,Raquel Sánchez-Pérez,Jesús Guillamón,Joanna Zhang,Rafael Rubio de Casa

Journal: Journal of Ecology

ABSTRACT: Cultivated plant species often naturalize and enter wild communities in a process known as feralization. To successfully feralize, crops must overcome ecological barriers and may undergo selection on certain traits, diverging phenotypically and genetically from their crop ancestors. In spite of the agronomic and eco-logical relevance of crop feralization, the eco- evolutionary dynamics driving it remain understudied.2. In this paper, we evaluated phenotypic and genotypic differentiation in fruit and seed traits during the naturalization of the almond tree (Prunus dulcis (Mill.) D.A. Webb) in SE Iberia and evaluated the potential role of natural selection in this process. To do so, we investigated the patterns of genetic divergence between cultivated and feral populations using functional (the cyanogenesis Sk gene) and neutral (17 SSR loci) markers and analysed morphological and biochemical traits in kernels of 342 individuals from 15 cultivated and 24 feral populations.3. We detected very little genetic differentiation in neutral markers between cul-tivated and feral populations. The majority of the observed genetic variation was due to differences within each type. Conversely, the recessive allele sk re-sponsible for seed toxicity was significantly more frequent in feral populations. Phenotypic differentiation between cultivated and naturalized almond popula-tions was also significant. Feral almond kernels were smaller and lighter, had denser and more resistant shells (endocarps) and more toxic seeds. Selection analyses indicated that these genetic and phenotypic patterns might be driven by directional selection on fruit and seed traits, potentially linked to defence against predation.


DOWNLOAD: Evolution of fruit and seed traits during almond naturalization.pdf

Almond diversity and homozygosity define structure, kinship, inbreeding, and linkage disequilibrium in cultivated germplasm, and reveal genomic associations with nut and seed …

Publication date: 10-01-2021

Authors: Stefano Pavan, Chiara Delvento, Rosa Mazzeo, Francesca Ricciardi, Pasquale Losciale, Liliana Gaeta, Nunzio D’Agostino, Francesca Taranto, Raquel Sánchez-Pérez, Luigi Ricciardi, Concetta Lotti

Journal: Horticulture research

Almond [Prunus dulcis Miller (DA Webb)] is the main tree nut species worldwide. Here, genotyping-by-sequencing (GBS) was applied to 149 almond cultivars from the ex situ collections of the Italian Council for Agricultural Research (CREA) and the Spanish National Research Council (CSIC), leading to the detection of 93,119 single-nucleotide polymorphisms (SNPs). The study of population structure outlined four distinct genetic groups and highlighted diversification between the Mediterranean and Californian gene pools. Data on SNP diversity and runs of homozygosity (ROHs) allowed the definition of kinship, inbreeding, and linkage disequilibrium (LD) decay in almond cultivated germplasm. Four-year phenotypic observations, gathered on 98 cultivars of the CREA collection, were used to perform a genome-wide association study (GWAS) and, for the first time in a crop species, homozygosity mapping (HM …



Ascorbic acid and prunasin, two candidate biomarkers for endodormancy release in almond flower buds identified by a nontargeted metabolomic study

Publication date: 01-12-2020

Authors: Jesús Guillamón Guillamón, Ángela Sánchez Prudencio, José Enrique Yuste, Federico Dicenta, Raquel Sánchez-Pérez

Journal: Horticulture research

Temperate fruit trees belonging to Prunus species have the ability to suspend (induce dormancy) and resume growth periodically in response to environmental and seasonal conditions. Endodormancy release requires the long-term accumulation of chill. Upon accumulation of cultivar-specific chill requirements, plants enter the state of ecodormancy, which means the ability to grow has been restored, depending on the fulfilment of heat requirements. As many different metabolic pathways are implicated in endodormancy release, we have performed a metabolomic analysis, using the ultra-high-performance liquid chromatography–quadrupole time-of-flying (UPLC–QToF) technique. We assayed flower buds in different stages of endodormancy in four almond cultivars with different flowering times: the extra-early Desmayo Largueta, the late Antoñeta, the extra-late Penta, and the ultra-late Tardona. An orthogonal …



Identification of early and late flowering time candidate genes in endodormant and ecodormant almond flower buds

Publication date: 16-11-2020

Authors: Ángela S Prudencio, Frank A Hoeberichts, Federico Dicenta, Pedro Martínez-Gómez, Raquel Sánchez-Pérez

Journal: Tree Physiology

Flower bud dormancy in temperate fruit tree species, like almond [Prunus dulcis (Mill.) D.A. Webb], is a survival mechanism that ensures flowering will occur under suitable weather conditions for successful flower development, pollination and fruit set. Dormancy is divided into three sequential phases: paradormancy, endodormancy and ecodormancy. During the winter, buds need cultivar-specific chilling requirements to overcome endodormancy and heat requirements to activate the machinery to flower in the ecodormancy phase. One of the main factors that enables the transition from endodormancy to ecodormancy is transcriptome reprogramming. In this work, we therefore monitored three almond cultivars with different chilling requirements and flowering times by RNA sequencing during the endodormancy release of flower buds and validated the data by qRT-PCR in two consecutive seasons. We were thus …



Special Issue "The Molecular Mechanism and Approaches to Break Dormancy in Fruit Trees"

Publication date: 01-08-2020

Authors: Dr. Raquel Sánchez-Pérez (Guest Editor)

Journal: Agronomy

Dear Colleagues, Fruit production directly depends on flowering success, which in turn depends on climate conditions. Perennial plants, such as temperate fruit trees, have the ability to induce and release/break dormancy, which means suspending and resuming growth periodically in response to changing environmental and seasonal conditions. The break of endodormancy (also called winter dormancy) demands the long-term accumulation of low temperatures, recorded as chill requirements (CR), followed by the accumulation of high temperatures (heat requirements) to restore the ability to grow. In other words, only when chill + heat requirements have been fulfilled will flowering take place, by the initiation of an extensive reprogramming of transcriptional and metabolic pathways. Temperate deciduous fruit tree crops are cultivated in different climate conditions over the world. Consequently, breeders are making a big effort to develop new varieties with either lower CR to combat mild winters, or with higher CR with a late flowering time to combat late frosts. In addition, growers aim to develop management practices that can help to overcome shortages in winter chill or to regulate flowering time. Unfortunately, weather conditions are often unpredictable, and undergoing dynamic change, making fruit production a real challenge when taking into consideration global warming as consequence of climate change. Severe effects of climate change on fruit production are projected for warmer regions, in particular around the Mediterranean Sea and Southwestern North America, and more dramatically in South Africa, Southern Australia, and Northern Africa, where most of the required winter chill conditions to release dormancy are projected to be lost. Moreover, global warming due to climate change is also increasing the risk of exposure of forest and fruit trees to delayed spring/late frosts, which often leads to loss of the entire yield. Please share your success stories from research in regions around the world in this Special Issue. We welcome submissions on topics including (but not limited to): (1) the novel application of agrochemicals to advance or delay dormancy and, therefore, flowering time; (2) the analysis of transcriptomes; (3) the analysis of metabolomes by analytical chemistry; (4) epigenetic studies; (5) agronomic practices related to crop productivity under climate change; and (6) decision support tools and modeling.


DOWNLOAD: special issue the molecular mechanism.pdf

Special Issue "Domestication and Transcription Factors Related to Important Traits in Horticultural Crops"

Publication date: 01-08-2020

Authors: Dr. Raquel Sánchez-Pérez (Guest Editor)

Journal: Agronomy

Dear Colleagues, As part of evolution, domestication has enabled wild plants and animals to be cultivated and consumed. This domestication occurred spontaneously or by humans, to adapt to a new environment. As a result, plants have developed new traits, such as bigger branches, bigger and sweeter fruits, etc., in which many transcription factors have a major role. When, where, and how most horticultural crops are domesticated today is being elucidated by the combination of different disciplines: archaeobotany, breeding, biochemistry, bioinformatics, physiology, molecular biology, etc.


Genomic Designing for New Climate-Resilient Almond Varieties

Publication date: 31-03-2020

Authors: Angela S Prudencio, Raquel Sánchez-Pérez, Pedro J Martínez-García, Federico Dicenta, Thomas M Gradziel, Pedro Martínez-Gómez

Journal: Springer, Cham

During the falling temperatures of autumn, temperate tree-crop species, including almond [Prunus dulcis (Miller) Webb], activate a winter survival strategy called endodormancy to protect against unfavorably cold temperatures. Trees cease vegetative growth and form structures called buds to protect enclosed meristems from the unfavorable environmental conditions, including low temperature and desiccation. Chill accumulation allows the progression from flower bud endodormancy stage to flower bud ecodormancy, a type of dormancy regulated by heat accumulation. Environmental stresses including those caused by climate change significantly affect global crop production. Consequently, climate-resilient crops that can withstand an array of climate changes and environmental perturbations will be required to maintain production. In this context, the adaptation of temperate tree-crop species such as almond …


How almonds became sweet

Publication date: 07-02-2020

Authors: Raquel Sánchez-Pérez | Senior Research Scientist Birger Lindberg Møller | Birger Lindberg Møller


How many times have you enjoyed sweet, healthy almonds and, suddenly, all this sweetness became erased by a taste of a single bitter one? Almond genome has enlightened how a single point mutation turned the previously bitter almonds sweet. Salads, vegan milk, yogurt, marzipan - all these products contain healthy sweet almonds. The almond ancestor, which still grows in the wild, carries bitter almonds. Consumption of its bitter kernels can be lethal to us and to wild herbivores. The bitterness comes from the presence of amygdalin, a compound that can release toxic cyanide and that almonds accumulate as a defence against herbivores and pests. The key event enabling us to grow almonds as a crop was the selection of a tree that cannot produce amygdalin and therefore has sweet kernels. Agriculture maintained the sweet kernel (Sk) trait for several thousand years, enabling us to enjoy their delicious taste and generating a business of 7.5 billion US dollars per year. For years, breeders have analysed numerous crosses between sweet and bitter varieties to understand how almonds inherit their taste. They discovered that the Sk trait is controlled by a change in a single gene, however when and where it arose remained unknown. Each individual almond tree carries either sweet or bitter almonds, never a mix of the two. Knowing the gene that gives almonds their sweet taste, we could develop an easy genetic test to select for the sweet kernel taste already at the seedling stage and accelerate the breeding process. We therefore set out to identify the mysterious Sk gene. In our previous research, we discovered the entire recipe the almond uses to make amygdalin. It consists of four steps: the first two steps take place in the seed coat (the thin brown layer that covers the kernel) and the last two in the kernel itself. Specific enzymes are necessary for completing the recipe: their mission is to transform one compound into another. Genes (DNA) encode information about how to make the enzymes (proteins), but before a gene becomes an enzyme it has to temporarily become a messenger (RNA). When we compared the levels of the four messengers in the sweet and bitter varieties, we discovered that no messengers for the two first enzymes were present in the sweet seed coats. The levels of messengers are controlled by another gene encoding formation of a transcription factor. Clue number 1: we were looking for an altered transcription factor. In this study, the sequence of the almond genome - the complete set of all its genes was determined, composed of almost 28 thousand genes, distributed on eight chromosomes. Comparing the sweet and bitter varieties, we narrowed down the position of the Sk gene to an eleven-gene region on chromosome five. Similar to digging in an archaeological cave, we found that five of these genes encoded transcription factors called bHLH 1 through 5. Clue number 2: in the sweet varieties, one of the five bHLH factors carried a unique mutation. We then analyzed the amount of messenger corresponding to each of the five bHLH transcription factors and showed that only those of bHLH1, 2 and 4 were present in the seed coat. To identify which of these were involved in production of amygdalin, we replicated the transition from DNA to protein in a test tube instead of in the intact almond. Only the bHLH2 encoding sequence from the bitter variety was able to produce the messengers for the amygdalin-producing genes. Our main suspect was the transcription factor bHLH2. When we compared the gene sequences encoding the bHLH2 transcription factor in sweet and bitter almond, we discovered that they differed by a single letter. We exchanged that specific letter between the two varieties and saw that it enabled the sweet almond to make amygdalin, and prevented the previously bitter version from doing so. Eureka! We confirmed that the Sweet kernel gene encodes the bHLH2. Thousands of years ago, the mutation in bHLH2 made it possible to introduce almonds into our diet without any toxicity risks. Having identified the gene responsible for sweetness has rendered it possible to set-up an easy DNA test that, already at the almond seedling state, identifies the seedlings, which will develop into almond trees carrying sweet kernels. In the past, removal of almond trees carrying bitter almonds would only be possible after three to four years, when the tree flowered and carried the first set of almonds. And more importantly, with the complete sequence of the almond genome, we now have tools to find other genes responsible for important agronomic traits like flowering time, pest resistance and drought tolerance. This could help to introduce new almond varieties, for example, ones that adapt better to the effects of climate change.


Almond [Prunus dulcis (Miller) D.A. Webb] Breeding

Publication date: 02-01-2020

Authors: Pedro J Martínez-García, Angela S Prudencio, Raquel Sánchez-Pérez, Felipe Pérez De Los Cobos, Ossama Kodad, Hassouna Gouta, Manuel Rubio, Pedro Martínez-Gómez

Journal: Springer, Cham

Almond [Prunus dulcis (Miller) D.A. Webb] presents clear challenges within plant breeding given its plurennial woody character, long juvenile period and multiplication by grafting. These challenges make the improvement process generally long and tedious. Therefore, it is necessary to have the most current information on developing new cultivars that currently take 12 years. Additionally, the breeder has to consider diverse internal (genetic background of the existing material, actual and the new methodologies) and external factors (consumer preferences, biotic and abiotic factors) to ensure the success of a new cultivar. The degree of knowledge of these aspects determines the quality of the prediction and success in the design of new almond cultivars. Although the size of the breeding population can be unlimited, the management, phenotyping and selection of these seedlings are major limiting factors. High …


Mutation of a bHLH transcription factor allowed almond domestication

Publication date: 14-06-2019

Authors: Sánchez-Pérez R*, Pavan S, Mazzeo R, Moldovan C, Aiese R, Del Cueto J, Ricciardi F, Lotti C, Ricciardi L, Dicenta F, López-Marqués R, Møller BL

Journal: Science

Wild almond species accumulate the bitter and toxic cyanogenic diglucoside amygdalin. Almond domestication was enabled by the selection of genotypes harboring sweet kernels. We report the completion of the almond reference genome. Map-based cloning using an F1 population segregating for kernel taste led to the identification of a 46-kilobase gene cluster encoding five basic helix-loop-helix transcription factors, bHLH1 to bHLH5. Functional characterization demonstrated that bHLH2 controls transcription of the P450 monooxygenase–encoding genes PdCYP79D16 and PdCYP71AN24, which are involved in the amygdalin biosynthetic pathway. A nonsynonymous point mutation (Leu to Phe) in the dimerization domain of bHLH2 prevents transcription of the two cytochrome P450 genes, resulting in the sweet kernel trait.


From Functional Genomics to Biotechnology in Ornamental Plants

Publication date: 16-04-2019

Authors: Swee-Suak Ko, Akira Kanno, Raquel Sánchez-Pérez, Hsin-Hung Yeh, Annette Hohe, Mariana Mondragón-Palomino

Journal: Frontiers in plant science

“Among roses there are many differences, in the number of petals, in roughness, in beauty of color, and in sweetness of scent. Most have five petals, but some have twelve or twenty, and some a great many more than these; for there are some, they say, which are even called ‘hundred-petalled.”’(Theophrastus, ca. 350 BC) Ancient civilizations around the world already selected, cultivated and exchanged plants because of their distinctive or unusual flowers. This interest motivated, for instance, the domestication of Dahlia species by the Aztecs (Treviño de Castro et al., 2007) as well as the breeding of peonies in Imperial China and tulips in the Ottoman Empire (Kingsbury, 2009). Ornamental plant breeding experienced significant advances during the seventeenth and eighteenth centuries after the mechanisms of plant reproduction and hybridization were understood and commercial routes exchanged plants and their products around the globe. Scientific advances and international floriculture trade also drive the most recent breakthrough in ornamental plant sciences: The complete sequencing of the genomes of Rosa chinensis, Aquilegia coerulea, Petunia as well as Phalaenopsis and several other orchids (Cai et al., 2015; Bombarely et al., 2016; Chao et al., 2018; Filiault et al., 2018; Raymond et al., 2018), facilitate both basic scientific research and motivate the development of a biotechnological tool-kit for functional genomics. In this Research Topic, we present a primer into the changing face of this area and its new applications to floriculture in the twenty-first century.



Polymorphisms in cyanogenic glucoside and cyano-aminoacid content in natural accessions of common vetch (Vicia sativa.L) and selection for improved agronomic performance

Publication date: 08-03-2019

Authors: Aouida M, Rook F, Öchsner AB, Sánchez-Pérez R, Abid G, Fauconnier ML, Jebara M*.

Journal: Plant Breeding

Common vetch (Vicia sativa L.) is an important annual forage legume. It is used as a cover crop, green manure, pasture legume and for silage and hay production. Its seeds can be used as a source of highly digestible protein and minerals in animal diets. However, their utilization as a feedstuff for monogastric animals is hindered by the fact that the seeds contain cyanogenic antinutritional factors that reduce their palatability. An effective utilization of V. sativa seeds as a successful monogastric feed stuff requires selection for higher protein availability and minimization of the cyanogenic antinutritional factors content. In this study, we selected one natural accession named Mjez Ibeb, from a collection of 25 accessions and cultivars, based on its superior agronomic performance and its naturally occurring genetic variation for cyanogenic traits. We investigated the genetic variation that exists for the cyanogenesis trait in more detail and analysed the seeds of 133 lines derived from accession Mjez lbeb. Of these, 40 naturally polymorphic lines that showed deficiencies in cyanogenesis and cyano?amino acid content, were subsequently selected for detailed chemical analysis. Cyanogenic glucosides and cyano?amino acid concentrations varied widely in the 40 lines. Multivariate analysis was performed and three lines (L16, L21, L18) with low content of cyanogenic compounds were identified.


Penta and Makako: two extra-late flowering self-compatible almond cultivars from CEBAS-CSIC

Publication date: 02-11-2018

Authors: Dicenta F, Cremades T, Martínez-García PJ, Martínez-Gómez P, Ortega E, Rubio M, Sánchez-Pérez R, López-Alcolea J, Egea J.

Journal: HortScience

The Centro de Edafología y Biología Aplicada del Segura–Consejo Superior de Investigaciones Científicas (CEBAS-CSIC) Almond Breeding Program began in 1971, when the germplasm collection was established, with the aim of obtaining new self-compatible and late-flowering cultivars. The first crosses were carried out in 1985. Late flowering decreases the risk of late frosts coinciding with flowering or fruit formation, thus avoiding crop losses. Self-compatibility enables a cultivar to produce fruit after pollination with its own pollen, which has enormous advantages for the grower compared with traditional self-incompatible cultivars (Dicenta et al., 2002). Self-compatibility allows the grower to cultivate only one cultivar in the orchard, not occupying space with the pollinizer. Pollination and fruit set is more efficient because all trees flower simultaneously, and each flower is able to pollinate itself. Therefore, beehives are not compulsory, although are highly recommended. Furthermore, poor weather conditions (low temperatures, wind) affect fruit set to a lesser extent. Finally, harvesting can be carried out at the same time, and the storage of a single cultivar is easier.


Elucidation of the Amygdalin Pathway Reveals the Metabolic Basis of Bitter and Sweet Almonds (Prunus dulcis)

Publication date: 01-11-2018

Authors: Thodberg S, Del Cueto J, Mazzeo R, Pavan S, Lotti C, Dicenta F, Neilson EHJ, Møller BL, Sánchez-Pérez R*.

Journal: Plant Physiology

Almond (Prunus dulcis) is the principal Prunus species in which the consumed and thus commercially important part of the fruit is the kernel. As a result of continued selection, the vast majority of almonds have a nonbitter kernel. However, in the field, there are trees carrying bitter kernels, which are toxic to humans and, consequently, need to be removed. The toxicity of bitter almonds is caused by the accumulation of the cyanogenic diglucoside amygdalin, which releases toxic hydrogen cyanide upon hydrolysis. In this study, we identified and characterized the enzymes involved in the amygdalin biosynthetic pathway: PdCYP79D16 and PdCYP71AN24 as the cytochrome P450 (CYP) enzymes catalyzing phenylalanine-to-mandelonitrile conversion, PdUGT94AF3 as an additional monoglucosyl transferase (UGT) catalyzing prunasin formation, and PdUGT94AF1 and PdUGT94AF2 as the two enzymes catalyzing amygdalin formation from prunasin. This was accomplished by constructing a sequence database containing UGTs known, or predicted, to catalyze a ?(1?6)-O-glycosylation reaction and a Basic Local Alignment Search Tool search of the draft version of the almond genome versus these sequences. Functional characterization of candidate genes was achieved by transient expression in Nicotiana benthamiana. Reverse transcription quantitative polymerase chain reaction demonstrated that the expression of PdCYP79D16 and PdCYP71AN24 was not detectable or only reached minute levels in the sweet almond genotype during fruit development, while it was high and consistent in the bitter genotype. Therefore, the basis for the sweet kernel phenotype is a lack of expression of the genes encoding the two CYPs catalyzing the first steps in amygdalin biosynthesis.


DOWNLOAD: 35.Elucidation of the amygdalin pathway.pdf

Synteny-Based Development of CAPS Markers Linked to the Sweet kernel LOCUS, Controlling Amygdalin Accumulation in Almond (Prunus dulcis (Mill.) D. A. Webb)

Publication date: 31-07-2018

Authors: Ricciardi F, Del Cueto J, Bardaro N, Mazzeo R, Ricciardi L, Dicenta F, Sánchez-Pérez R*, Pavan S*, Lotti C.

Journal: Genes

The bitterness and toxicity of wild-type seeds of Prunoideae is due to the cyanogenic glucoside amygdalin. In cultivated almond (Prunus dulcis (Mill.) D.A. Webb), a dominant mutation at the Sk locus prevents amygdalin accumulation and thus results in edible sweet kernels. Here, we exploited sequence similarity and synteny between the genomes of almond and peach (Prunus persica (L.) Batsch) to identify cleaved amplified polymorphic sequence (CAPS) molecular markers linked to the Sk locus. A segregant F1 population was used to map these markers on the Sk genomic region, together with Sk-linked simple sequence repeat (SSR) markers previously described. Molecular fingerprinting of a cultivar collection indicated the possibility to use CAPS polymorphisms identified in this study in breeding programs arising from different parental combinations. Overall, we highlight a set of codominant markers useful for early selection of sweet kernel genotypes, an aspect of primary importance in almond breeding. In addition, by showing collinearity between the physical map of peach and the genetic map of almond with respect to the Sk genomic region, we provide valuable information for further marker development and Sk positional cloning.


B-Glucosidase activity in almond seeds

Publication date: 01-05-2018

Authors: Del Cueto J, Møller BL, Dicenta F, Sánchez-Pérez R*.

Journal: Plant Physiology and Biochemistry

Almond bitterness is the most important trait for breeding programs since bitter-kernelled seedlings are usually discarded. Amygdalin and its precursor prunasin are hydrolyzed by specific enzymes called ?-glucosidases. In order to better understand the genetic control of almond bitterness, some studies have shown differences in the location of prunasin hydrolases (PH, the ?-glucosidase that degrades prunasin) in sweet and bitter genotypes. The aim of this work was to isolate and characterize different PHs in sweet- and bitter-kernelled almonds to determine whether differences in their genomic or protein sequences are responsible for the sweet or bitter taste of their seeds. RNA was extracted from the tegument, nucellus and cotyledon of one sweet (Lauranne) and two bitter (D05–187 and S3067) almond genotypes throughout fruit ripening. Sequences of nine positive Phs were then obtained from all of the genotypes by RT-PCR and cloning. These clones, from mid ripening stage, were expressed in a heterologous system in tobacco plants by agroinfiltration. The PH activity was detected using the Feigl-Anger method and quantifying the hydrogen cyanide released with prunasin as substrate. Furthermore, ?-glucosidase activity was detected by Fast Blue BB salt and Umbelliferyl method. Differences at the sequence level (SNPs) and in the activity assays were detected, although no correlation with bitterness was found.


'Makako': a new extra-late flowering self-compatible cultivar from CEBAS-CSIC

Publication date: 05-11-2017

Authors: F Dicenta, J Egea, T Cremades, P Martínez-Gómez, E Ortega, M Rubio, R Sánchez-Pérez, PJ Martínez-García

Journal: VII International Symposium on Almonds and Pistachios 1219

Late flowering, to avoid the late frosts, is one of the main objectives of the Almond Breeding Program of CEBAS-CSIC. The late flowering self-compatible ‘Antoñeta’and ‘Marta’were released long time ago, and they are nowadays broadly cultivated. Afterwards, we released the extra-late self-compatible ‘Penta’and the ultra-late self-compatible ‘Tardona’, the latest flowering almond cultivar ever released. These cultivars are enabling the culture of almonds in cold areas, where the rest of cultivars cannot be cultivated because of frost. The new self-compatible extra-late flowering ‘Makako’is very productive like ‘Penta’, but it flowers a few days before and it ripens a week later, enabling the harvest of matures fruits in large plantations. Furthermore,‘Makako’is more vigorous than ‘Penta’and it has a larger kernel. In our opinion, the combination of ‘Penta’with ‘Makako’is an excellent choice for the new broad plantations in …


Transcriptome and metabolite changes during hydrogen cyanamide-induced floral bud break in sweet cherry

Publication date: 17-07-2017

Authors: Ionescu IA, Lopez-Ortega G, Burow M, Bayo-Canha A, Junge A, Gericke O, Møller BL, and Sánchez-Pérez R*.

Journal: Frontiers in Plant Science

Release of bud dormancy in perennial woody plants is a temperature-dependent process and thus flowering in these species is heavily affected by climate change. The lack of cold winters in temperate growing regions often results in reduced flowering and low fruit yields. This is likely to decrease the availability of fruits and nuts of the Prunus spp. in the near future. In order to maintain high yields, it is crucial to gain detailed knowledge on the molecular mechanisms controlling the release of bud dormancy. Here, we studied these mechanisms using sweet cherry (Prunus avium L.), a crop where the agrochemical hydrogen cyanamide (HC) is routinely used to compensate for the lack of cold winter temperatures and to induce flower opening. In this work, dormant flower buds were sprayed with hydrogen cyanamide followed by deep RNA sequencing, identifying three main expression patterns in response to HC. These transcript level results were validated by quantitative real time polymerase chain reaction and supported further by phytohormone profiling (ABA, SA, IAA, CK, ethylene, JA). Using these approaches, we identified the most up-regulated pathways: the cytokinin pathway, as well as the jasmonate and the hydrogen cyanide pathway. Our results strongly suggest an inductive effect of these metabolites in bud dormancy release and provide a stepping stone for the characterization of key genes in bud dormancy release.


Bottom-up elucidation of glycosidic bond stereochemistry

Publication date: 28-05-2017

Authors: Gray C, Schindler B, Migas L, Picmanova M, Allouche A-R, Green A, Mandal S, Motawia M, Sánchez-Pérez R, Bjarnholt N, Møller BL, Rijs A, Barran P, Compagnon I, Eyers C, Flitsch S

Journal: Analytical Chemistry

The lack of robust, high-throughput, and sensitive analytical strategies that can conclusively map the structure of glycans has significantly hampered progress in fundamental and applied aspects of glycoscience. Resolution of the anomeric ?/? glycan linkage within oligosaccharides remains a particular challenge. Here, we show that “memory” of anomeric configuration is retained following gas-phase glycosidic bond fragmentation during tandem mass spectrometry (MS2). These findings allow for integration of MS2 with ion mobility spectrometry (IM-MS2) and lead to a strategy to distinguish ?- and ?-linkages within natural underivatized carbohydrates. We have applied this fragment-based hyphenated MS technology to oligosaccharide standards and to de novo sequencing of purified plant metabolite glycoconjugates, showing that the anomeric signature is also observable in fragments derived from larger glycans. The discovery of the unexpected anomeric memory effect is further supported by IR-MS action spectroscopy and ab initio calculations. Quantum mechanical calculations provide candidate geometries for the distinct anomeric fragment ions, in turn shedding light on gas-phase dissociation mechanisms of glycosidic linkages.


Cyanogenic Glucosides and Derivatives in Almond and Sweet Cherry Flower Buds from Dormancy to Flowering

Publication date: 19-05-2017

Authors: Del Cueto J, Ionescu IA, Picmanova M, Gericke O, Motawia MS, Olsen CE, Campoy JA, Dicenta F, Moller BL, Sánchez-Pérez, R*

Journal: Frontiers in Plant Science

Almond and sweet cherry are two economically important species of the Prunus genus. They both produce the cyanogenic glucosides prunasin and amygdalin. As part of a two-component defense system, prunasin and amygdalin release toxic hydrogen cyanide upon cell disruption. In this study, we investigated the potential role within prunasin and amygdalin and some of its derivatives in endodormancy release of these two Prunus species. The content of prunasin and of endogenous prunasin turnover products in the course of flower development was examined in five almond cultivars – differing from very early to extra-late in flowering time – and in one sweet early cherry cultivar. In all cultivars, prunasin began to accumulate in the flower buds shortly after dormancy release and the levels dropped again just before flowering time. In almond and sweet cherry, the turnover of prunasin coincided with increased levels of prunasin amide whereas prunasin anitrile pentoside and ?-D-glucose-1-benzoate were abundant in almond and cherry flower buds at certain developmental stages. These findings indicate a role for the turnover of cyanogenic glucosides in controlling flower development in Prunus species.


Chemical control of flowering time.

Publication date: 10-12-2016

Authors: Ionescu IA, Møller BL, Sánchez-Pérez R*.

Journal: Journal of Experimental Botany

Flowering at the right time is of great importance; it secures seed production and therefore species survival and crop yield. In addition to the genetic network controlling flowering time, there are a number of much less studied metabolites and exogenously applied chemicals that may influence the transition to flowering as well as flower opening. Increased emphasis on research within this area has the potential to counteract the negative effects of global warming on flowering time, especially in perennial crop plants. Perennial crops have a requirement for winter chill, but winters become increasingly warm in temperate regions. This has dramatic effects on crop yield. Different strategies are therefore being developed to engineer flowering time to match local growing conditions. The majority of these efforts are within plant breeding, which benefits from a substantial amount of knowledge on the genetic aspects of flowering time regulation in annuals, but less so in perennials. An alternative to plant breeding approaches is to engineer flowering time chemically via the external application of flower-inducing compounds. This review discusses a variety of exogenously applied compounds used in fruit farming to date, as well as endogenous growth substances and metabolites that can influence flowering time of annuals and perennials.


The origin of the self-compatible almond ‘Guara’

Publication date: 14-12-2015

Authors: Dicenta F, Sánchez-Pérez R, Rubio M, Egea J, Batlle I, Miarnau X, Palascino M, Lipari E, Confolent C, Martínez-Gómez P, Duval H.

Journal: Scientia Horticulturae

‘Guara’ has been the most planted almond cultivar in Spain in recent years. The introducers of this cultivar reported its origin as unknown and suggested that it is related to the Italian almond cultivar ‘Tuono’. Indeed, the experience of farmers and researchers has revealed strong similarities between ‘Guara’ and ‘Tuono’. In order to compare the identity of the two cultivars, their genetic profiles (fingerprints) were determined with a set of 12 SSR markers used to analyse the INRA clones of ‘Guara’ and ‘Tuono’ as well as two clones considered synonymous with ‘Tuono’, namely ‘Supernova’ and ‘Mazzetto’. A supplementary set of 23 SSRs was also analysed in ‘Guara’ and ‘Tuono’ samples from different reference collections of CEBAS-CSIC (Murcia, Spain), INRA (Avignon, France), IRTA (Constantí, Spain), and the University of Bari (Bari, Italy). The results confirmed that ‘Guara’ and ‘Tuono’ present identical DNA fingerprints for the 35 SSRs analysed. This genotypic information together with the similar characteristics of the two cultivars demonstrates that Spanish ‘Guara’ is actually the same cultivar as the Italian ‘Tuono’.


A recycling pathway for cyanogenic glycosides evidenced by the comparative metabolic profiling in three cyanogenic plant species

Publication date: 11-06-2015

Authors: Picmanova M, Neilson EH, Motawia MS, Olsen CE, Agerbirk N, Gray CJ, Flitsch S, Meier S, Silvestro D, Jorgensen K, Sánchez-Pérez R, Møller BL, Bjarnholt N.

Journal: Biochemical Journal

Cyanogenic glycosides are phytoanticipins involved in plant defence against herbivores by virtue of their ability to release toxic hydrogen cyanide (HCN) upon tissue disruption. In addition, endogenous turnover of cyanogenic glycosides without the liberation of HCN may offer plants an important source of reduced nitrogen at specific developmental stages. To investigate the presence of putative turnover products of cyanogenic glycosides, comparative metabolic profiling using LC–MS/MS and high resolution MS (HR–MS) complemented by ion-mobility MS was carried out in three cyanogenic plant species: cassava, almond and sorghum. In total, the endogenous formation of 36 different chemical structures related to the cyanogenic glucosides linamarin, lotaustralin, prunasin, amygdalin and dhurrin was discovered, including di- and tri-glycosides derived from these compounds. The relative abundance of the compounds was assessed in different tissues and developmental stages. Based on results common to the three phylogenetically unrelated species, a potential recycling endogenous turnover pathway for cyanogenic glycosides is described in which reduced nitrogen and carbon are recovered for primary metabolism without the liberation of free HCN. Glycosides of amides, carboxylic acids and ‘anitriles’ derived from cyanogenic glycosides appear as common intermediates in this pathway and may also have individual functions in the plant. The recycling of cyanogenic glycosides and the biological significance of the presence of the turnover products in cyanogenic plants open entirely new insights into the multiplicity of biological roles cyanogenic glycosides may play in plants.


Recent advancements to study flowering time in almond and other Prunus species.

Publication date: 11-07-2014

Authors: Sánchez-Pérez R*, Del Cueto J, Dicenta F, Martinez-Gomez P.

Journal: Frontiers in Plant Science

Flowering time is an important agronomic trait in almond since it is decisive to avoid the late frosts that affect production in early flowering cultivars. Evaluation of this complex trait is a long process because of the prolonged juvenile period of trees and the influence of environmental conditions affecting gene expression year by year. Consequently, flowering time has to be studied for several years to have statistical significant results. This trait is the result of the interaction between chilling and heat requirements. Flowering time is a polygenic trait with high heritability, although a major gene Late blooming (Lb) was described in “Tardy Nonpareil.” Molecular studies at DNA level confirmed this polygenic nature identifying several genome regions (Quantitative Trait Loci, QTL) involved. Studies about regulation of gene expression are scarcer although several transcription factors have been described as responsible for flowering time. From the metabolomic point of view, the integrated analysis of the mechanisms of accumulation of cyanogenic glucosides and flowering regulation through transcription factors open new possibilities in the analysis of this complex trait in almond and in other Prunus species (apricot, cherry, peach, plum). New opportunities are arising from the integration of recent advancements including phenotypic, genetic, genomic, transcriptomic, and metabolomics studies from the beginning of dormancy until flowering.


Co-occurrence of cyanogenic glucosides and their derivatives as a common feature in metabolic profiles of almond and cassava.

Publication date: 01-01-2014

Authors: Picmanová M, Nealson EH, Motawie MS, Sánchez-Pérez R, Olsen CE, Møller BL, Jørgensen K, Bjarnholt N.

Journal: Planta Medica


Quantitative Trait Loci (QTL) and Mendelian Trait Loci (MTL) Analysis in Prunus: a Breeding Perspective and Beyond

Publication date: 07-09-2013

Authors: Salazar JA, Ruiz D, Campoy JA, Sánchez-Pérez R, Crisosto CH, Martinez-Garcia PJ, Blenda A, Jung S, Main D, Martinez-Gomez P, Rubio M.

Journal: Plant Molecular Biology Reporter

Trait loci analysis, a classic procedure in quantitative (quantitative trait loci, QTL) and qualitative (Mendelian trait loci, MTL) genetics, continues to be the most important approach in studies of gene labeling in Prunus species from the Rosaceae family. Since 2011, the number of published Prunus QTLs and MTLs has doubled. With increased genomic resources, such as whole genome sequences and high-density genotyping platforms, trait loci analysis can be more readily converted to markers that can be directly utilized in marker-assisted breeding. To provide this important resource to the community and to integrate it with other genomic, genetic, and breeding data, a global review of the QTLs and MTLs linked to agronomic traits in Prunus has been performed and the data made available in the Genome Database for Rosaceae. We describe detailed information on 760 main QTLs and MTLs linked to a total of 110 agronomic traits related to tree development, pest and disease resistance, flowering, ripening, and fruit and seed quality. Access to these trait loci enables the application of this information in the post-genomic era, characterized by the availability of a high-quality peach reference genome and new high-throughput DNA and RNA analysis technologies.


Comparative Genomics Analysis in Prunoideae to Identify Biologically Relevant Polymorphisms

Publication date: 13-06-2013

Authors: Koepke T, Schaeffer S, Harper A, Dicenta F, Edwards M, Henry RJ, Møller BL, Meisel L, Oraguzie N, Silva H, Sánchez-Pérez R*, Dhingra A*.

Journal: Plant Biotechnology Journal

Prunus is an economically important genus with a wide range of physiological and biological variability. Using the peach genome as a reference, sequencing reads from four almond accessions and one sweet cherry cultivar were used for comparative analysis of these three Prunus species. Reference mapping enabled the identification of many biological relevant polymorphisms within the individuals. Examining the depth of the polymorphisms and the overall scaffold coverage, we identified many potentially interesting regions including hundreds of small scaffolds with no coverage from any individual. Non-sense mutations account for about 70 000 of the 13 million identified single nucleotide polymorphisms (SNPs). Blast2GO analyses on these non-sense SNPs revealed several interesting results. First, non-sense SNPs were not evenly distributed across all gene ontology terms. Specifically, in comparison with peach, sweet cherry is found to have non-sense SNPs in two 1-aminocyclopropane-1-carboxylate synthase (ACS) genes and two 1-aminocyclopropane-1-carboxylate oxidase (ACO) genes. These polymorphisms may be at the root of the nonclimacteric ripening of sweet cherry. A set of candidate genes associated with bitterness in almond were identified by comparing sweet and bitter almond sequences. To the best of our knowledge, this is the first report in plants of non-sense SNP abundance in a genus being linked to specific GO terms.


Influence of the pollinizer in the amygdalin content of almonds

Publication date: 18-05-2012

Authors: Sánchez-Pérez R*, Arrázola G, Martín ML, Grané N and Dicenta F

Journal: Scientia Horticulturae

Bitter kernel taste in almonds is a monogenic trait, being bitter recessive. Bitterness depends on the content of the cyanogenic glucoside amygdalin, whose concentration can be more than 1000 times higher in bitter kernels than in sweet ones. Although previous studies showed that the bitter taste of almonds was not influenced by the pollinizer, nothing is known about the influence of the pollinizer on the amygdalin content of the kernel. In order to study the possible effect of the pollinizer on the content of amygdalin, we analyzed seeds from four cultivars: Del Cid (SkSk: sweet), Marcona (Sksk: sweet), Garrigues (Sksk: slightly bitter) and S3067 (sksk: bitter), which were crossed with four different pollinizers: Ramillete (SkSk: sweet), Atocha (Sksk: sweet), S3065 (Sksk: slightly bitter) and S3067. Results showed that when a sweet pollinizer was involved, the content of amygdalin could be reduced by up to 21%, compared to the bitter × bitter crosses.


Prunasin hydrolases localization during fruit development in sweet and bitter almonds

Publication date: 21-02-2012

Authors: Sánchez-Pérez R, Saez F, Jonas Borch-Jensen, Dicenta F, Møller BL, Jørgensen K.

Journal: Plant Physiology

Amygdalin is a cyanogenic diglucoside and constitutes the bitter component in bitter almond (Prunus dulcis). Amygdalin concentration increases in the course of fruit formation. The monoglucoside prunasin is the precursor of amygdalin. Prunasin may be degraded to hydrogen cyanide, glucose, and benzaldehyde by the action of the ?-glucosidase prunasin hydrolase (PH) and mandelonitirile lyase or be glucosylated to form amygdalin. The tissue and cellular localization of PHs was determined during fruit development in two sweet and two bitter almond cultivars using a specific antibody toward PHs. Confocal studies on sections of tegument, nucellus, endosperm, and embryo showed that the localization of the PH proteins is dependent on the stage of fruit development, shifting between apoplast and symplast in opposite patterns in sweet and bitter cultivars. Two different PH genes, Ph691 and Ph692, have been identified in a sweet and a bitter almond cultivar. Both cDNAs are 86% identical on the nucleotide level, and their encoded proteins are 79% identical to each other. In addition, Ph691 and Ph692 display 92% and 86% nucleotide identity to Ph1 from black cherry (Prunus serotina). Both proteins were predicted to contain an amino-terminal signal peptide, with the size of 26 amino acid residues for PH691 and 22 residues for PH692. The PH activity and the localization of the respective proteins in vivo differ between cultivars. This implies that there might be different concentrations of prunasin available in the seed for amygdalin synthesis and that these differences may determine whether the mature almond develops into bitter or sweet.


Clarifying omics concepts, challenges and opportunities for Prunus breeders in the post-genomic era.

Publication date: 06-02-2012

Authors: Martínez-Gómez P, Sánchez-Pérez R, Rubio M

Journal: OMICS: A Journal of Integrative Biology

The recent sequencing of the complete genome of the peach, together with the availability of new high-throughput genome, transcriptome, proteome, and metabolome analysis technologies, offers new possibilities for Prunus breeders in what has been described as the postgenomic era. In this context, new biological challenges and opportunities for the application of these technologies in the development of efficient marker-assisted selection strategies in Prunus breeding include genome resequencing using DNA-Seq, the study of RNA regulation at transcriptional and posttranscriptional levels using tilling microarray and RNA-Seq, protein and metabolite identification and annotation, and standardization of phenotype evaluation. Additional biological opportunities include the high level of synteny among Prunus genomes. Finally, the existence of biases presents another important biological challenge in attaining knowledge from these new high-throughput omics disciplines. On the other hand, from the philosophical point of view, we are facing a revolution in the use of new high-throughput analysis techniques that may mean a scientific paradigm shift in Prunus genetics and genomics theories. The evaluation of scientific progress is another important question in this postgenomic context. Finally, the incommensurability of omics theories in the new high-throughput analysis context presents an additional philosophical challenge.


Inheritance of chilling and heat requirements and QTLs analysis in almond

Publication date: 30-11-2011

Authors: Sánchez-Pérez R*, Dicenta F, Martínez-Gómez P.

Journal: Tree Genetics & Genomes

The chilling and heat requirements and flowering time were studied, for 2 years, in an almond progeny from the cross between the late-flowering French selection “R1000” and the early-flowering Spanish “Desmayo Largueta”. These three temperature-dependent traits showed quantitative inheritance, although for chilling requirements and flowering time a major gene could be involved, modified by other minor genes. The results indicate that flowering time is mainly a consequence of the chilling requirements; heat requirements having a smaller effect. In agreement with the genetic findings, a significant Quantitative Trait Loci (QTL) for chilling requirements was found in G4 together with other minor QTLs in G1, G3, and G7. For heat requirements, two QTLs in G2 and G7 were identified. The results also show the high influence of temperature in the expression of the three traits and their QTL analyses. In addition, QTL analysis for flowering time allowed the identification of one significant QTL in linkage group 4 (G4) that explained most of the phenotypic variation together with other minor QTLs located in G1, G6, and G7.


Molecular markers for kernel bitterness in almond

Publication date: 13-10-2009

Authors: Sánchez-Pérez R*, Howad W, Garcia-Mas J, Arús P, Martínez-Gómez P and Dicenta F.

Journal: Tree Genetics & Genomes

Upon crushing, amygdalin present in bitter almonds is hydrolysed to benzaldehyde, which gives a bitter flavour, and to cyanide, which is toxic. Bitterness is attributable to the recessive allele of the Sweet kernel (Sk/sk) gene and is selected against in breeding programmes. Almond has a long intergeneration period due to its long juvenile phase, so breeders must wait 3 or 4 years to evaluate fruit traits in the field. For this reason, it is important to develop molecular markers to distinguish between sweet and bitter genotypes. The Sk gene is known to map to linkage group five (G5) of the almond genome, but its function is still undefined. Candidate genes involved in the amygdalin pathway have been mapped, but none of them were located to G5. We have saturated G5 with additional Simple Sequence Repeats (SSRs) using the progeny from the cross “R1000”?×?“Desmayo Largueta” and found six SSRs (UDA-045, EPDCU2584, CPDCT028, BPPCT037, PceGA025, and CPDCT016) closely linked to the Sk locus. The genotypes of four of these SSRs flanking the Sk locus, in a number of parents and a few seedlings of the CEBAS-CSIC almond breeding programme, allowed us to estimate the haplotypes of the parents, identifying the marker alleles adequate for an early and highly efficient selection against bitter genotypes. This analysis has established the usefulness of SSRs for screening populations of fruit trees such as almond by an easy, polymerase chain reaction-based method.


Tissue and cellular localization of individual ?-glycosidases using a substrate-specific sugar reducing assay.

Publication date: 13-08-2009

Authors: Sánchez-Pérez R*, Jørgensen K, Motawia MS, Dicenta F and Møller BL.

Journal: Plant Journal

Traditional methods to localize beta-glycosidase activity in tissue sections have been based on incubation with the general substrate 6-bromo-2-naphthyl-beta-d-glucopyranoside. When hydrolysed in the presence of salt zinc compounds, 6-bromo-2-naphthyl-beta-d-glucopyranoside affords the formation of an insoluble coloured product. This technique does not distinguish between different beta-glycosidases present in the tissue. To be able to monitor the occurrence of individual beta-glycosidases in different tissues and cell types, we have developed a versatile histochemical method that can be used for localization of any beta-glycosidase that upon incubation with its specific substrate releases a reducing sugar. Experimentally, the method is based on hydrolysis of the specific substrate followed by oxidation of the sugar released by a tetrazolium salt (2,3,5-triphenyltetrazolium chloride) that forms a red insoluble product when reduced. The applicability of the method was demonstrated by tissue and cellular localization of two beta-glucosidases, amygdalin hydrolase and prunasin hydrolase, in different tissues and cell types of almond. In those cases where the analysed tissue had a high content of reducing sugars, this resulted in strong staining of the background. This interfering staining of the background was avoided by prior incubation with sodium borohydride. The specificity of the devised method was demonstrated in a parallel localization study using a specific antibody towards prunasin hydrolase.


B-glucosidases as detonators of Plant Chemical Defense

Publication date: 01-06-2008

Authors: Morant AV, Jørgensen K, Paquette SM, Sánchez-Pérez R, Møller BL, and Bak S.

Journal: Phytochemistry

Some plant secondary metabolites are classified as phytoanticipins. When plant tissue in which they are present is disrupted, the phytoanticipins are bio-activated by the action of beta-glucosidases. These binary systems--two sets of components that when separated are relatively inert--provide plants with an immediate chemical defense against protruding herbivores and pathogens. This review provides an update on our knowledge of the beta-glucosidases involved in activation of the four major classes of phytoanticipins: cyanogenic glucosides, benzoxazinoid glucosides, avenacosides and glucosinolates. New aspects of the role of specific proteins that either control oligomerization of the beta-glucosidases or modulate their product specificity are discussed in an evolutionary perspective.


Fruit development in almond for fresh consumption

Publication date: 01-04-2008

Authors: Martínez-Gómez P, Sánchez-Pérez R and Dicenta, F

Journal: Journal of the American Pomological Society

Commercialization of fresh almond [Prunus dulcis (Mill.) D.A. Webb] seed is a potential opportunity for diversification of the almond market. Characteristics required for the commercialization of this product include early market availability, suitable seed size and high seed/fruit weight ratio. In order to determine the effect of flowering and ripening dates on fresh market quality, fruit and seed development were studied in four almond cultivars: ‘Ramillete’ (early flowering and ripening), ‘Desmayo Largueta’ (early flowering and late ripening), ‘Antoñeta’ (late flowering and early ripening), and ‘Wawona’ (late flowering and ripening). Data were collected for time from flowering to ripening, fruit and seed length, seed fresh and dry weight, the seed/fruit weight ratio, and endocarp hardness. Results confirm a rapid growth of fruit in March and April. Accelerated cotyledon development was observed in April and May, while the hardening of the endocarp occurred gradually until ripening (mesocarp split). In general, the development of fruit and seed traits was similar for the four almond cultivars, regardless of their flowering and ripening date. However, the growth of cotyledons depended on both flowering and ripening dates, with early flowering and ripening cultivars appearing most suitable for the fresh almond market.


Bitterness in almond.

Publication date: 01-03-2008

Authors: Sánchez-Pérez R, Jørgensen K, Olsen CE, Dicenta F and Møller BL

Journal: Plant Physiology

Bitterness in almond (Prunus dulcis) is determined by the content of the cyanogenic diglucoside amygdalin. The ability to synthesize and degrade prunasin and amygdalin in the almond kernel was studied throughout the growth season using four different genotypes for bitterness. Liquid chromatography-mass spectrometry analyses showed a specific developmentally dependent accumulation of prunasin in the tegument of the bitter genotype. The prunasin level decreased concomitant with the initiation of amygdalin accumulation in the cotyledons of the bitter genotype. By administration of radiolabeled phenylalanine, the tegument was identified as a specific site of synthesis of prunasin in all four genotypes. A major difference between sweet and bitter genotypes was observed upon staining of thin sections of teguments and cotyledons for ?-glucosidase activity using Fast Blue BB salt. In the sweet genotype, the inner epidermis in the tegument facing the nucellus was rich in cytoplasmic and vacuolar localized ?-glucosidase activity, whereas in the bitter cultivar, the ?-glucosidase activity in this cell layer was low. These combined data show that in the bitter genotype, prunasin synthesized in the tegument is transported into the cotyledon via the transfer cells and converted into amygdalin in the developing almond seed, whereas in the sweet genotype, amygdalin formation is prevented because the prunasin is degraded upon passage of the ?-glucosidase-rich cell layer in the inner epidermis of the tegument. The prunasin turnover may offer a buffer supply of ammonia, aspartic acid, and asparagine enabling the plants to balance the supply of nitrogen to the developing cotyledons.


Mapping major genes and quantitative trait loci controlling agronomic traits in almond

Publication date: 08-05-2007

Authors: Sánchez-Pérez R, Howad W, Dicenta F, Arús P and Martínez-Gómez P.

Journal: Plant Breeding

Six tree traits (self?compatibility, blooming date, blooming density, productivity, leafing date and ripening time) and five pomological traits (kernel taste, in?shell weight, shell hardness, kernel weight and double kernel) were studied in an F1 almond progeny of 167 seedlings from the cross between the French cultivar ‘R1000’ and the Spanish cultivar ‘Desmayo Largueta’. In addition, a set of 135 codominant microsatellites or simple?sequence repeat (SSR) markers developed from peach, cherry and almond were used for the molecular characterization of the progeny. A genetic linkage map was constructed with 56 of these SSRs. Cosegregation analysis allowed the identification of the map positions of two major genes to be confirmed for kernel taste (Sk) in linkage group five (G5) and for self?incompatibility (S) in G6. QTLs mapped include two for leafing date (Lf?Q1 and Lf?Q2) in G1 and G4, one for shell hardness (D?Q) in G2, one each for double kernel (Dk?Q) and productivity (P?Q) in G4, one for blooming date (Lb?Q) in G4, two for kernel weight (Kw?Q1 and Kw?Q2) in G1 and G4, and two for in?shell weight (Shw?Q1 and Shw?Q2) in G1 and G2. Four SSR loci (BPPCT011, UDP96?013, UDP96?003 and PceGA025) were linked to the important agronomic traits of leafing date, shell hardness, blooming date and kernel taste. Finally, the development of efficient marker?assisted selection strategies applied to almond and other Prunus breeding programmes was also discussed.


Inheritance and relationships of important agronomic traits in almond.

Publication date: 12-01-2007

Authors: Sánchez-Pérez R, Ortega E, Duval H, Martínez-Gómez P and Dicenta, F

Journal: Euphytica

The inheritance of 16 important agronomic traits and its relationship were studied for four years in a population of 167 almond [P. dulcis (Mill.) D.A. Webb] seedlings obtained from a cross between the French selection ‘R1000’ (‘Tardy Nonpareil’ × ‘Tuono’) and the Spanish cultivar ‘Desmayo Largueta’. For some traits (blooming and leafing date) descendants segregated between the value of both progenitors, meanwhile for others the mean of the offspring was lower (bloom density, productivity and ripening date) or higher (in-shell/kernel ratio and double kernels). As expected, kernel bitterness and self-incompatibility behaved as monogenic traits. Some important correlations between traits were detected. The implications of the transmission and the correlation of these traits in the breeding programmes are discussed.


Cyanogenic glycosides; a case study for evolution and application of cytochromes P450.

Publication date: 15-11-2006

Authors: Bak S, Paquette SM, Morant M, Rasmussen AV, Saito S, Bjarnholt N, Zagrobelny M, Jørgensen K, Hamann T, Osmani S, Simonsen HT, Sánchez-Pérez R, van Hesswijck TB, Jørgensen B y Møller BL.

Journal: Phytochemistry Reviews

Cyanogenic glycosides are ancient biomolecules found in more than 2,650 higher plant species as well as in a few arthropod species. Cyanogenic glycosides are amino acid-derived ?-glycosides of ?-hydroxynitriles. In analogy to cyanogenic plants, cyanogenic arthropods may use cyanogenic glycosides as defence compounds. Many of these arthropod species have been shown to de novo synthesize cyanogenic glycosides by biochemical pathways that involve identical intermediates to those known from plants, while the ability to sequester cyanogenic glycosides appears to be restricted to Lepidopteran species. In plants, two atypical multifunctional cytochromes P450 and a soluble family 1 glycosyltransferase form a metabolon to facilitate channelling of the otherwise toxic and reactive intermediates to the end product in the pathway, the cyanogenic glycoside. The glucosinolate pathway present in Brassicales and the pathway for cyanoalk(en)yl glucoside synthesis such as rhodiocyanosides A and D in Lotus japonicus exemplify how cytochromes P450 in the course of evolution may be recruited for novel pathways. The use of metabolic engineering using cytochromes P450 involved in biosynthesis of cyanogenic glycosides allows for the generation of acyanogenic cassava plants or cyanogenic Arabidopsis thaliana plants as well as L. japonicus and A. thaliana plants with altered cyanogenic, cyanoalkenyl or glucosinolate profiles.


Comparison of SSR polymorphisms using automated capillary sequencers, and polyacrylamide and agarose gel electrophoresis: implications for the assessment of genetic diversity and relatedness in almond.

Publication date: 08-05-2006

Authors: Sánchez-Pérez R, Ballester J, Dicenta F, Arús P and Martínez-Gómez P.

Journal: Scientia Horticulturae

In this work we have performed a comparative study of the utilization of three electrophoresis separation methods for the analysis of peach SSR polymorphism in almond and its implications in the assessment of genetic diversity and relatedness. Automated SSR detection and polyacrylamide gel electrophoresis were the most efficient methods and would be able to resolve allelic variation at a finer scale than the electrophoresis in MetaPhor® agarose. Moreover, automated electrophoresis detection is much more expensive in terms of cost of equipment and cost of the analysis. In addition, SSR detection using polyacrylamide gel electrophoresis showed similar results than automated sequencing, although it is more time-consuming and toxic than electrophoresis in agarose gels. Discrepancies among polyacrylamide and automated capillary, and MetaPhor® were observed when differences in SSR polymorphisms were between 1 and 5 bp. While the use of MetaPhor® agarose appears less indicated for genotype characterization, this technique may be the most convenient in other applications, i.e. mapping of population, due to its lower cost and easier routine application. The utilization MetaPhor® agarose produced a slightly different clustering of genotypes in spite of the big differences observed in the DNA fingerprinting. However, bootstrap values using MetaPhor® agarose were lower indicating a lower accuracy of this SSR polymorphism analysis method.


Level and transmission of genetic heterozygosity in apricot, explored by simple sequence repeat markers.

Publication date: 24-06-2005

Authors: Sánchez-Pérez R, Martínez-Gómez P, Dicenta F, Egea J and Ruiz D

Journal: Genetic Resources and Crop Evolution

In this study, 17 peach simple sequence repeat (SSR) sequences were used in the exploration of the genetic heterozygosity level of several apricot cultivars from Spain, France, Greece, and the USA, and 23 descendants. The genotypes can be classified in three groups as a function of their genetic heterozygosity (1) local cultivars from Murcia (Spain) (‘Gitanos’ and ‘Pepito del Rubio’) and several descendants from crosses among these cultivars, with very low genetic heterozygosities (less than 0.30); (2) cultivars from France and Spain (‘Moniquí’, ‘Currot’ and ‘Bergeron’) and several descendants, with intermediate levels of genetic heterozygosity (around 0.45); and (3) cultivars ‘Orange Red’ and ‘Goldrich’ from North America and ‘Lito’ from Greece, with the remaining descendants, having genetic heterozygosities higher than 0.50. The results showed the high increase of genetic heterozygosity in the case of descendants from complementary crosses. The use of cultivars from North America could increase greatly the genetic heterozygosity in the Spanish apricot breeding programs, enlarging the genetic variability of the local cultivars. On the other hand, in the case of transgressive crosses among local Spanish cultivars, the increase of genetic heterozygosity was much lower.


Improved technique for counting chromosomes in almond

Publication date: 30-05-2005

Authors: Martínez-Gómez P, Sánchez-Pérez R, Vaknin Y, Dicenta F y Gradziel TM

Journal: Scientia Horticulturae

n almond [Prunus dulcis (Mill.) D.A. Webb], there are few chromosome studies. In this work, different methods described for slide preparation and staining of chromosomes in other Prunus species have been evaluated using almond root tips. By modifying and updating traditional methods, an improved technique for counting chromosomes has been developed. Root tips were first placed in cold water for 4 h at 0 °C and treated with 0.2% colchicine for 3 h at 5 °C. They were then fixed for 24 h at 5 °C in methanol, propionic acid, chloroform (6:3:2), and stored in 70% ethanol. This optimized protocol allowed the identification of chromosomes in the different stages of mitosis late prophase being the best stage for chromosome counting.


Application SSR markers in apricot breeding: molecular characterization, protection, and genetic relationships.

Publication date: 30-01-2005

Authors: Sánchez-Pérez R, Ruiz D, Dicenta F, Egea J and Martínez-Gómez P

Journal: Scientia Horticulturae

Seventeen peach simple sequence repeat (SSR) markers have been used in the molecular characterization of 8 apricot (Prunus armeniaca L.) cultivars from Spain, North America, France, and Greece; a new breeding line from the apricot breeding program of INRA (Avignon, France); and 13 breeding lines and 3 new releases from the apricot breeding program of CEBAS-CSIC (Murcia, Spain). DNA fingerprints have been developed establishing the genetic relatedness among cultivars, new releases, and breeding lines. Amplification of SSR loci was obtained for all 17 primer pairs and 14 of them produced polymorphic amplification. The number of presumed alleles revealed by the SSR analysis ranged from one to nine, although most primers revealed three alleles or more. The mean number of alleles per locus was 4.1. Results allowed the molecular identification of all the apricot genotypes assayed. Apricot genotypes clustered into seven principal groups in accordance with their origin and pedigree. The implications of these results for apricot breeding programs in terms of protection of new release and design of new crosses are also discussed.


Identification of S-alleles in almond using multiplex-PCR.

Publication date: 01-08-2004

Authors: Sánchez-Pérez R, Dicenta F and Martínez-Gómez P.

Journal: Euphytica

The S-genotypes of eight almond (Prunus dulcis Miller (D.A. Webb)) cultivars from different geographical origins and of nine new selections from the CEBAS-CSIC (Murcia, Spain) breeding program were determined using single and multiplex PCR with different sets of specific oligonucleotide primers. The results of PCR using the AS1II- and AmyC5R-specific primers showed amplification in a single reaction of 10 different self-incompatibility alleles and of the self-compatibility allele S f. However, the amplified fragments of the S f allele were of similar sizes to those amplified from the S 3 self-incompatibility allele. For this reason, a specific PCR primer CEBASf was designed from the intron sequence of S f. A multiplex-PCR reaction using the AS1II, CEBASf and AmyC5R primers permitted unequivocal identification of the 10 self-incompatibility alleles and of the self-compatibility allele. Multiplex PCR opens the possibility to identify new S-alleles using different sets of primers. The applications of these PCR markers in the almond-breeding programs are discussed.


Rescue of multiple embryos in almond through in vivo micrografts.

Publication date: 01-01-2004

Authors: Martínez-Gómez P, Sánchez-Pérez R, Dicenta F y Gradziel, T. M.

Journal: Journal of Food, Agriculture & Environment

Multiple embryos (two or three embryos within the same seedcoat) occur spontaneously in certain almond [Prunus dulcis (Mill.) D.A. Webb] cultivars including ‘Nonpareil’ and ‘Mission’. Seedlings from the same multiple-seeded ovule are frequently viable, though often, one of the seedlings shows weak growth and develops poorly (aberrant seedlings). These aberrant seedlings have recently been identified as aneuploids, and so are useful for genetic studies of almond. In this work, we examine the success of in vivo micrografting for the early propagation of this weak material. Variables evaluated included genotype (aneuploid or diploid), type of micro-scion and growth stage of the rootstock. Micro-scions tested consisted of small (3 mm) micro-wedges from sprouted apical buds from two-weeks-old seedlings. In addition, both direct and inverse approach micrografts were assayed. Results showed a lower success for micrograft of aberrant seedlings (aneuploids) (5%) relative to normal seedlings (diploids) (42.5%). Inverse approach micrografts onto very young rootstocks showed the best results for aberrant seedlings (aneuploid) with a 15% success rate. Significant differences were observed in the growth of micrografted aberrant seedlings (aneuploid) as compared to seedlings growing on their own roots.


Characterization of gdp1 as encoding a GDPase in the fission yeast Schizosaccharomyces pombe.

Publication date: 01-11-2003

Authors: Sánchez-Pérez R, Franco A, Gacto M, Notario V and Cansado J.

Journal: Fems Microbiology Letters

We have isolated the gdp1+ gene from Schizosaccharomyces pombe coding for a membrane protein with guanosine diphosphatase (GDPase) activity, which is highly homologous to Golgi GDPases isolated from other yeast species. The gdp1+ product, Gdp1p, displays both GDPase and uridine diphosphatase (UDPase) activities in vitro, with a strong dependence for calcium and manganese cations. The observation of a defect in N-glycosylation of invertase in S. pombe?gdp1 cells together with the ability of gdp1+ to functionally complement the defective O-mannosylation of chitinase in Saccharomyces cerevisiae cells disrupted in the GDA1 gene (gdp1+ homolog), suggests a main role for Gdp1p in protein glycosylation in fission yeast.


New approach to Prunus tree crop breeding.

Publication date: 20-12-2002

Authors: Martínez-Gómez P, Sozzi GO, Sánchez-Pérez R, Rubio M and Gradziel TM.

Journal: Journal of Food, Agriculture & Environment

New strategies for Prunus improvement, including germplasm and molecular marker development and improved propagationtechniques, are described. In germplasm improvement, the introduction of genes from related Prunus species conferring severaltraits including self-compatibility, growth habit, drought resistance, and kernel quality are being pursued. Twin seeds (twoembryos within the same seedcoat) have produced seedlings useful for genetic studies. Promising propagation methods includein-vitro techniques for the evaluation of plant material and in-vivo micrograft techniques that allow the early propagation ofhigh-risk genotypes. In addition, the growth of seedlings in controlled environments, including the induction of an artificial restperiod in cold chambers, provides a useful strategy for obtaining vigorously growing plants year round. Molecular markershave also become an essential tool in Prunus breeding studies. Different types of molecular markers, including isoenzymes,RFLPs, RAPDs, AFLPs and SSRs, have been employed for the genetic characterization of germplasm, the establishment ofgenetic relationships between cultivars and species, and the construction of genetic maps. Methodologies for the analysis ofmarker-assisted selection include the use of mapping populations segregating for desired characters and bulk segregant analysis.


Characterization of multiple embryos in almond.

Publication date: 02-09-2002

Authors: Martínez-Gómez P, Sánchez-Pérez R, Dicenta F and Gradziel TM.

Journal: Spanish Journal of Agricultural Research

La aparición de embriones múltiples dentro del mismo tegumento es un fenómeno espontáneo que ocurre en semi-llas de algunas variedades de almendro [Prunus dulcis(Mill.) D.A. Webb] como ‘Nonpareil’ o ‘Mission’. Las plán-tulas procedentes de estas semillas poliembriónicas son viables, aunque a menudo muestran un crecimiento más dé-bil y retrasado. Estas plántulas enanas han sido caracterizadas como haploides en trabajos previos. En este trabajohemos estudiado 450 plántulas procedentes de semillas poliembriónicas de la variedad californiana ‘Nonpareil’ y 100 plántulas procedentes de semillas poliembriónicas de las selecciones españolas ‘D97396’ y ‘D97407’, obser-vando su germinación y posterior crecimiento. Estas plántulas han sido caracterizadas molecularmente mediante mar-cadores isoenzimáticos y microsatélites. Además, hemos identificado los cariotipos mitóticos en los ápices radicula-res. El porcentaje de semillas con embriones múltiples donde una de las plantas procedente de uno de estos embrionesmostró un crecimiento aberrante fue del 25%, con un índice de mortalidad de las plántulas aberrantes procedentes deestos embriones del 90%. Se ha observado un origen sexual de estos embriones múltiples. En algunos casos estos dosembriones son genéticamente iguales y dan lugar a dos plantas idénticas, mientras que en el caso de semillas con em-briones aberrantes el embrión secundario pierde parte de la dotación genómica, dando lugar a plantas aneuploidesjunto a las plantas diploides procedentes del embrión primario. El uso y utilización de este material en los programasde mejora genética del almendro se discute también en este trabajo



Special Issue "Prunus Dormancy and Breeding"

Publication date: 05-08-2022

Authors: Guest Editor: Dr. Jose A. Egea; Dra. Raquel Sánchez-Pérez ; Dr. David Ruiz.

Journal: Plants



Seminario "To be bitter or to be sweet: Defense versus domestication"

Fecha: 27-10-2023 Instituto de Biología Molecular y Celular de Plantas de Valencia (Ibmcp) Raquel Sánchez Pérez - CEBAS /CSIC / Spain

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The XVI Eucarpia Symposium on Fruit Breeding and Genetics . September 11–16, 2023, Dresden-Pillnitz

The XVI Eucarpia Symposium on Fruit Breeding and Genetics will be held under the umbrella of Eucarpia and the International Society for Horticultural Sciences in Dresden-Pillnitz from 11 to 16 September 2023. This symposium brings together scientists working on topics related to fruit breeding and breeding research on temperate fruit crops and the conservation and use of fruit genetic resources.

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11th International Rosaceae Genomics Conference (March 2023 · Nelson, New Zealand · Organiser)

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