Federico Dicenta

Bitterness in Almonds

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 β-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 β-glucosidase activity, whereas in the bitter cultivar, the β-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 β-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.

A stylar ribonuclease assay to detect self-compatible seedlings in almond progenies

Abstract Six almond progenies, each the product of a cross between a self-compatible and a self-incompatible parent, were analysed for stylar ribonucleases. Proteins were extracted and separated using non-equilibrium pH gradient electrofocusing (NEPHGE), and the gels were stained for ribonuclease activity. Most seedlings showed either two principal bands, interpreted as corresponding to two incompatibility alleles, or a single band. The seedlings were also bagged in the field at flowering time to determine fruit set after selfing, and some were also examined for the growth of pollen-tubes in selfed styles using UV fluorescence microscopy. With very few exceptions, those seedlings showing single-banded zymograms were found to be self-compatible according to field and microscope studies, and those with two bands were found to be self-incompatible. We conclude that the allele for self-compatibility in almond does not code for ribonuclease activity and that the ribonuclease isoenzyme assay is a convenient technique for predicting self-compatibility in segregating progenies. A novel band in two derivatives of ’Ferrastar’ was ascribed to a new incompatibility allele, S10.

QTL analysis of resistance to sharka disease in the apricot ( Prunus armeniaca L.) ‘Polonais’ × ‘Stark Early Orange’ F1 progeny

Different hypotheses on the genetic control of the resistance to the plum pox virus (PPV) have been reported in apricot, but there was a lack of agreement about the number of loci involved. In recent years, apricot genetic maps have been constructed from progenies derived from ‘Stark Early Orange’ or ‘Goldrich’, two main sources of resistance, three of these including the mapping of the PPV resistance loci. As the location of the locus was not precisely established, we mapped the PPV resistance loci using interval mapping (IM), composite interval mapping (CIM), and the Kruskal–Wallis non-parametric test in the F1 progeny derived from a cross between the susceptible cv. ‘Polonais’ and ’Stark Early Orange’. Four genomic regions were identified as being involved in PPV resistance. One of these mapped to the upper region of linkage group 1 of ‘Stark Early Orange’, and accounted for 56% of the phenotypic variation. Its location was similar to the one previously identified in ‘Goldrich’ and Prunus davidiana. In addition, a gene strongly associated to these major quantitative trait loci (QTL) was found to be related to PPV infection. Two putative QTLs were detected on linkage groups 3 of ‘Polonais’ and 5 of both ‘Polonais’ and ‘Stark Early Orange’ with both parametric and non-parametric methods at logarithm of odds (LOD) scores slightly above the detection threshold. The last QTL was only detected in the early stage of the infection. PPV resistance is, thus, controlled by a major dominant factor located on linkage group 1. The hypothesis of recessive factors with lower effect is discussed.

MECHANISMS OF DORMANCY IN SEEDS OF PEACH (PRUNUS PERSICA (L.) BATSCH) CV. GF305

Two mechanisms of dormancy were found in peach cultivar GF305, which is frequently used for indexing fruit viruses. Seed coat dormancy manifested itself in inhibition of germination, and embryo dormancy was expressed in reduced growth of germinated plants. The seed coat dormancy was overcome by removal of the seed coat or by stratification for 2 weeks at 7°C followed by removal of the endocarp. Embryo dormancy was overcome by stratification periods around 12 weeks. No effect of the seed coat on embryo dormancy was observed.

Inheritance of self-compatibility in almond: breeding strategies to assure self-compatibility in the progeny

Abstract To assure self-compatibility in the progenies, three different crosses were conducted for the first time in an almond breeding programme: self-pollination (266 descendants from 30 families), crosses between parents sharing an S-allele (108 descendants from five families) and crosses with homozygous self-compatible parents (62 descendants from five families). Depending on the cross, self-compatibility in the progenies was determined by observing pollen tube growth (by means of fluorescence microscopy), stylar S-RNases analysis or allele-specific PCR. The results obtained fit with the accepted hypothesis of inheritance of self-compatibility and the three crossing strategies used ensured 100% of self-compatible descendants. These strategies increase the efficiency of the breeding programme and avoid the laborious task of evaluating this characteristic. From the breeding point of view, self-fertilisation and crosses between relatives tend to produce inbreeding. Furthermore, these methods reduce the possibilities of choosing the parental combination. The use of homozygous self-compatible parents does not have any of these disadvantages. As far as we know, this is the first time that allele-specific PCR has been used for early selection of self-compatible seedlings. The advantages and disadvantages of the three methodologies used to determine self-compatibility are discussed.

Pollen tube dynamics following half- and fully-compatible pollinations in self-compatible almond cultivars

Abstract. Pollen tube growth dynamics along the style at different times after self- and cross-pollination of four heterozygous self-compatible (SfSx) almond cultivars were studied by means of fluorescence microscopy. Results showed a reduction of pollen tube number along the style following both self-pollination (half-compatible reaction) and cross-pollination (fully-compatible). An important decrease in the percentage of pollen tubes present took place in the upper section of the style in both crosses. As expected in a gametophytic incompatibility system, a higher number of pollen tubes were stopped along the style in the case of the half-compatible crosses; however, the percentage of pollen tubes reaching the base of the style was similar in both cases. These results reveal the existence of other mechanisms controlling pollen tube growth along the style.

Inheritance of the kernel flavour in almond

In this study, the sweet, bitter or slightly bitter flavour of the kernel of 1969 almond trees was determined over a 2-year period; the trees are seedlings of 51 families deriving from crosses of 12 varieties of almond. It is proven that this trait is controlled by just one gene, with bitterness being due to the recessive allele. Trees with slightly bitter kernels are heterozygous forms, in which the bitter flavour is scarcely expressed due to an alteration of the total dominance system. In addition, the varieties used as parents have been classified as homozygous or heterozygous for this trait.

Identification of S-alleles in almond using multiplex PCR

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 Sf. However, the amplified fragments of the Sf allele were of similar sizes to those amplified from the S3 self-incompatibility allele. For this reason, a specific PCR primer CEBASf was designed from the intron sequence of Sf. 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.

Genotype x environment interaction in QTL analysis of an intervarietal almond cross by means of genetic markers.

Besides QTL location and the estimation of gene effects, QTL analysis based on genetic markers could be used to comprehensively investigate quantitative trait-related phenomena such as pleiotropy, gene interactions, heterosis, and genotype-by-environment interaction (G x E). Given that the G x E interaction is of great relevance in tree improvement, the objective of the research presented here was to study the effect of years on QTL detection for 15 quantitative traits by means of isozymatic markers in a large progeny group of an intervarietal cross of almond. At least 17 putative QTLs were detected, 3 of which had alleles with opposite effects to those predicted from the parental genotypes. Only 3 QTLs behaved homogeneously over the years. Three possible causes are discussed in relation to this lack of stability: the power of the test statistic being used, the low contribution of the QTL to the genetic variation of the trait, and a differential gene expression dependent on the year (G x E). Most cases showing lack of stability involved traits whose heritability estimates change drastically from year to year and/or whose correlation coefficients between years are low, suggesting the presence of G x E as the most likely cause. A marker-assisted selection scheme to improve late flowering and short flowering duration is suggested for an early and wide screening of the progeny.

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

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.