Pedigree analysis of 220 almond genotypes reveals two world mainstream breeding lines based on only three different cultivars.

Loss of genetic variability is an increasing challenge in tree breeding programs due to the repeated use of a reduced number of founder genotypes. However, in almond, little is known about the genetic variability in current breeding stocks, although several cases of inbreeding depression have been reported. To gain insights into the genetic structure in modern breeding programs worldwide, marker-verified pedigree data of 220 almond cultivars and breeding selections were analyzed. Inbreeding coefficients, pairwise relatedness, and genetic contribution were calculated for these genotypes. The results reveal two mainstream breeding lines based on three cultivars: "Tuono", "Cristomorto", and "Nonpareil". Descendants from "Tuono" or "Cristomorto" number 76 (sharing 34 descendants), while "Nonpareil" has 71 descendants. The mean inbreeding coefficient of the analyzed genotypes was 0.041, with 14 genotypes presenting a high inbreeding coefficient, over 0.250. Breeding programs from France, the USA, and Spain showed inbreeding coefficients of 0.075, 0.070, and 0.037, respectively. According to their genetic contribution, modern cultivars from Israel, France, the USA, Spain, and Australia trace back to a maximum of six main founding genotypes. Among the group of 65 genotypes carrying the Sf allele for self-compatibility, the mean relatedness coefficient was 0.125, with "Tuono" as the main founding genotype (24.7% of total genetic contribution). The results broaden our understanding about the tendencies followed in almond breeding over the last 50 years and will have a large impact into breeding decision-making process worldwide. Increasing current genetic variability is required in almond breeding programs to assure genetic gain and continuing breeding progress.

Genotypic and Environmental Effects on Tocopherol Content in Almond.

Almond is the most important nut species worldwide and almond kernels show the highest levels of tocopherols among all nuts. In almond, tocopherols not only play a substantial role as a healthy food for human consumption, but also in protecting lipids against oxidation and, thus, lengthening the storage time of almond kernels. The main tocopherol homologues detected in almond in decreasing content and biological importance are α-, γ-, δ-, and ß-tocopherol. Tocopherol concentration in almond depends on the genotype and the environment, such as the climatic conditions of the year and the growing management of the orchard. The range of variability for the different tocopherol homologues is of 335-657 mg/kg of almond oil for α-, 2-50 for γ-, and 0.1-22 for ß-tocopherol. Drought and heat have been the most important stresses affecting tocopherol content in almond, with increased levels at higher temperatures and in water deficit conditions. The right cultivar and the most appropriate growing conditions may be selected to obtain crops with effective kernel storage and for the most beneficial effects of almond consumption for human nutrition and health.

Association mapping for kernel phytosterol content in almond.

Almond kernels are a rich source of phytosterols, which are important compounds for human nutrition. The genetic control of phytosterol content has not yet been documented in almond. Association mapping (AM), also known as linkage disequilibrium (LD), was applied to an almond germplasm collection in order to provide new insight into the genetic control of total and individual sterol contents in kernels. Population structure analysis grouped the accessions into two principal groups, the Mediterranean and the non-Mediterranean. There was a strong subpopulation structure with LD decaying with increasing genetic distance, resulting in lower levels of LD between more distant markers. A significant impact of population structure on LD in the almond cultivar groups was observed. The mean r(2) -value for all intra-chromosomal loci pairs was 0.040, whereas, the r(2) for the inter-chromosomal loci pairs was 0.036. For analysis of association between the markers and phenotypic traits five models were tested. The mixed linear model (MLM) approach using co-ancestry values from population structure and kinship estimates (K model) as covariates identified a maximum of 13 significant associations. Most of the associations found appeared to map within the interval where many candidate genes involved in the sterol biosynthesis pathway are predicted in the peach genome. These findings provide a valuable foundation for quality gene identification and molecular marker assisted breeding in almond.

Identification of Genetic Loci Associated with Quality Traits in Almond via Association Mapping.

To design an appropriate association study, we need to understand population structure and the structure of linkage disequilibrium within and among populations as well as in different regions of the genome in an organism. In this study, we have used a total of 98 almond accessions, from five continents located and maintained at the Centro de Investigación y Tecnología Agroalimentaria de Aragón (CITA; Spain), and 40 microsatellite markers. Population structure analysis performed in 'Structure' grouped the accessions into two principal groups; the Mediterranean (Western-Europe) and the non-Mediterranean, with K = 3, being the best fit for our data. There was a strong subpopulation structure with linkage disequilibrium decaying with increasing genetic distance resulting in lower levels of linkage disequilibrium between more distant markers. A significant impact of population structure on linkage disequilibrium in the almond cultivar groups was observed. The mean r2 value for all intra-chromosomal loci pairs was 0.040, whereas, the r2 for the inter-chromosomal loci pairs was 0.036. For analysis of association between the markers and phenotypic traits, five models comprising both general linear models and mixed linear models were selected to test the marker trait associations. The mixed linear model (MLM) approach using co-ancestry values from population structure and kinship estimates (K model) as covariates identified a maximum of 16 significant associations for chemical traits and 12 for physical traits. This study reports for the first time the use of association mapping for determining marker-locus trait associations in a world-wide almond germplasm collection. It is likely that association mapping will have the most immediate and largest impact on the tier of crops such as almond with the greatest economic value.

Methylation of the S f locus in almond is associated with S-RNase loss of function.

Self-compatibility in almond (Prunus dulcis) is attributed to the presence of the S f haplotype, allelic to and dominant over the series of S-alleles controlling self-incompatibility. Some forms of the S f haplotype, however, are phenotypically self-incompatible even though their nucleotide sequences are identical. DNA from leaves and styles from genetically diverse almond samples was cloned and sequenced and then analyzed for changes affecting S f -RNase variants. Epigenetic changes in several cytosine residues were detected in a fragment of 4,700 bp of the 5' upstream region of all self-compatible samples of the S f -RNases, differentiating them from all self-incompatible samples of S f -RNases analyzed. This is the first report of DNA methylation in a Rosaceae species and appears to be strongly associated with inactivation of the S f allele. Results facilitate an understanding of the evolution of self-compatibility/self-incompatibility in almond and other Prunus species, and suggest novel approaches for future crop improvement.

Comparative efficacy of two interventions to discontinue long-term benzodiazepine use: cluster randomised controlled trial in primary care.

BACKGROUND: Benzodiazepines are extensively used in primary care, but their long-term use is associated with adverse health outcomes and dependence. AIMS: To analyse the efficacy of two structured interventions in primary care to enable patients to discontinue long-term benzodiazepine use. METHOD: A multicentre three-arm cluster randomised controlled trial was conducted, with randomisation at general practitioner level (trial registration ISRCTN13024375). A total of 532 patients taking benzodiazepines for at least 6 months participated. After all patients were included, general practitioners were randomly allocated (1:1:1) to usual care, a structured intervention with follow-up visits (SIF) or a structured intervention with written instructions (SIW). The primary end-point was the last month self-declared benzodiazepine discontinuation confirmed by prescription claims at 12 months. RESULTS: At 12 months, 76 of 168 (45%) patients in the SIW group and 86 of 191 (45%) in the SIF group had discontinued benzodiazepine use compared with 26 of 173 (15%) in the control group. After adjusting by cluster, the relative risks for benzodiazepine discontinuation were 3.01 (95% CI 2.03-4.46, P<0.0001) in the SIW and 3.00 (95% CI 2.04-4.40, P<0.0001) in the SIF group. The most frequently reported withdrawal symptoms were insomnia, anxiety and irritability. CONCLUSIONS: Both interventions led to significant reductions in long-term benzodiazepine use in patients without severe comorbidity. A structured intervention with a written individualised stepped-dose reduction is less time-consuming and as effective in primary care as a more complex intervention involving follow-up visits.

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

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

Pollen Tube Growth and Self-Compatibility in Almond.

Although pollen tube growth has been an important criterion for self-compatibility evaluation in almond, there is not a clear-cut separation between positive and negative growth of pollen tubes in the different genotypes. The examination of pollen tube growth after selfing almond seedlings has allowed establishing different levels of compatibility, but not a clear-cut separation between self-compatible (SC) and self-incompatible (SI) genotypes, related to the presence of pseudo-self-compatibility in almond. Consequently, a relationship between pollen tube growth and self-compatibility in almond may be established for evaluating the seedlings in breeding programs.

Molecular modeling of S-RNases involved in almond self-incompatibility.

Gametophytic self-incompatibility (GSI) is a mechanism in flowering plants, to prevent inbreeding and promote outcrossing. GSI is under the control of a specific locus, known as the S-locus, which contains at least two genes, the RNase and the SFB. Active S-RNases in the style are essential for rejection of haploid pollen, when the pollen S-allele matches one of two S-alleles of the diploid pistil. However, the nature of their mutual interactions at genetic and biochemical levels remain unclear. Thus, detailed understanding of the protein structure involved in GSI may help in discovering how the proteins involved in GSI may function and how they fulfill their biological roles. To this end, 3D models of the SC (S(f)) and two SI (S(8) and S(23)) S-RNases of almond were constructed, using comparative modeling tools. The modeled structures consisted of mixed α and ß folds, with six helices and six ß-strands. However, the self-compatible (S(f)) RNase contained an additional extended loop between the conserved domains RC4 and C5, which may be involved in the manifestation of self-compatibility in almond.

Mapping quantitative trait loci for kernel composition in almond.

BACKGROUND: Almond breeding is increasingly taking into account kernel quality as a breeding objective. Information on the parameters to be considered in evaluating almond quality, such as protein and oil content, as well as oleic acid and tocopherol concentration, has been recently compiled. The genetic control of these traits has not yet been studied in almond, although this information would improve the efficiency of almond breeding programs. RESULTS: A map with 56 simple sequence repeat or microsatellite (SSR) markers was constructed for an almond population showing a wide range of variability for the chemical components of the almond kernel. A total of 12 putative quantitative trait loci (QTL) controlling these chemical traits have been detected in this analysis, corresponding to seven genomic regions of the eight almond linkage groups (LG). Some QTL were clustered in the same region or shared the same molecular markers, according to the correlations already found between the chemical traits. The logarithm of the odds (LOD) values for any given trait ranged from 2.12 to 4.87, explaining from 11.0 to 33.1 % of the phenotypic variance of the trait. CONCLUSIONS: The results produced in the study offer the opportunity to include the new genetic information in almond breeding programs. Increases in the positive traits of kernel quality may be looked for simultaneously whenever they are genetically independent, even if they are negatively correlated. We have provided the first genetic framework for the chemical components of the almond kernel, with twelve QTL in agreement with the large number of genes controlling their metabolism.

Tocopherol concentration in almond oil: genetic variation and environmental effects under warm conditions.

The concentration of the different tocopherol homologues in almond kernel oil was determined in 17 almond cultivars grown in two different experimental orchards, in Spain and Morocco. The three main homologues showed a large variability, ranging from 210.9 to 553.4 mg/kg of oil for α-tocopherol, from 4.64 to 14.92 mg/kg for γ-tocopherol, and from 0.2 to 1.02 mg/kg for δ-tocopherol. The year effect was significant, independent of the experimental site, for all homologues and total tocopherol, the values of α-tocopherol, γ-tocopherol, and total tocopherol being higher in 2009 than in 2008, whereas the value of δ-tocopherol was higher in 2008. The location effect was also significant, the values of γ- and δ-tocopherol being higher in Spain than in Morocco, whereas for α-tocopherol the location effect was dependent on the genotype. These effects could not be explained by the temperature differences between sites, but probably other undetermined environmental factors might explain the effect of the location, such as rainfall and irrigation supplementation during fruit growing and ripening.

A modifier locus affecting the expression of the S-RNase gene could be the cause of breakdown of self-incompatibility in almond.

Self-compatibility has become the primary objective of most almond (Prunus amygdalus Batsch) breeding programmes in order to avoid the problems related to the gametophytic self-incompatibility system present in almond. The progeny of the cross 'Vivot' (S(23)S(fa)) x 'Blanquerna' (S(8)S(fi)) was studied because both cultivars share the same S(f) allele but have a different phenotypic expression: active (S(fa)) in 'Vivot' and inactive (S(fi)) in 'Blanquerna'. In addition, the microscopic observation of pollen tube growth after self-pollination over several years showed an unexpected self-incompatible behaviour in most seedlings of this cross. The genotypes of this progeny showed that the S(fi) pollen from 'Blanquerna' was not able to grow down the pistils of 'Vivot' harbouring the S(fa) allele, confirming the active function of this allele against the inactive form of the same allele, S(fi). As self-compatibility was observed in some S(8)S(23) and S(8)S(fa) individuals of this progeny, the S(f) haplotype may not always be linked to the expression and transmission of self-compatibility in almond, suggesting that a modifier locus may be involved in the mechanism of self-incompatibility in plants.

Xenia effects on oil content and fatty acid and tocopherol concentrations in autogamous almond cultivars.

The increasing utilization of self-compatible almond cultivars in solid plantings of a single genotype has raised the question of the effect of the pollen source on the kernel quality of these new autogamous cultivars. Thus, the effect of two different pollen sources, in addition to their own pollen, on the oil content and fatty acid and tocopherol concentrations was studied in four autogamous almond genotypes. The oil content was not affected by the pollination treatment, but self-pollination resulted in significantly higher values for oleic acid. For the tocopherol homologues, the alpha-tocopherol content of the self-pollinated kernels was intermediate between those obtained after cross-pollination with the two foreign pollens, but the self-pollinated kernels had higher values of delta-tocopherol than the cross-pollinated kernels. Thus, the effect of the pollen source was shown to have a clear effect on the fatty acid composition but not on the oil or tocopherol contents of the almond kernels, with an increased quality of the kernels produced after self-pollination because of a higher oleic/linoleic acid ratio.

Variability of oil content and of major fatty acid composition in almond (Prunus amygdalus Batsch) and its relationship with kernel quality.

Oil content and fatty acid composition were determined for two years in the kernel oil of eight cultivars and 47 advanced self-compatible almond genotypes developed in an almond breeding program. Considerable variation between genotypes was found for all parameters. Oil content ranged from 48% to 67% of the total kernel dry weight but was consistent over the two years. Fatty acid composition was also very variable, with significant differences between genotypes, even in genotypes of the same progeny. Oleic acid, ranging from 63% to 78%, and linoleic acid, ranging from 12% to 27%, were the major fatty acids, showing higher values in some selections than in their parents. The large variability observed for all fatty acids and the presence of selections with higher oil and fatty acid contents than the commercial cultivars represents a very promising base to obtain new almond cultivars with oil of higher quality, satisfying the industrial and consumer sectors.

Negative inbreeding effects in tree fruit breeding: self-compatibility transmission in almond.

Inbreeding depression has been observed in most fruit trees, negatively affecting the offspring of related parents. This problem is steadily increasing due to the repeated utilization of parents in breeding programmes. In almond, self-compatibility transmission from 'Tuono' to its offspring remains partially unexplained due to deviations from the expected genotype ratios. In order to test if these deviations could be due to inbreeding, the S-genotypes of the seedlings of four almond families, 'Tuono' (S(1)S(f )) x 'Ferragnès' (S(1)S(3)), 'Tuono' (S(1)S(f)) x 'Ferralise' (S(1)S(3)) and reciprocal crosses were studied. The S-genotype determination of each seedling by separation of stylar S-RNases and by S-allele-specific PCR amplification gave identical results. The ratio of S-genotypes of the family 'Tuono' x 'Ferralise' was the one least adjusted to the expected 1:1 ratio, because the number of self-compatible seedlings (S(f)S(3)) was less than a half the number of self-incompatible ones (S(1)S(3)). A mechanism acting against inbreeding would favour cross-breeding in the following generation to increase heterozygosity. This fact stresses the need to avoid crosses between related parents in fruit breeding programmes.