Almond Genome Research Articles

Genome-Wide Analysis of DREB Family Genes and Characterization of Cold Stress Responses in the Woody Plant Prunus nana.

Dehydration response element binding factor (DREB) is a family of plant-specific transcription factors, whose members participate in the regulation of plant responses to various abiotic stresses. Prunus nana, also known as the wild almond, is a member of the Rosaceae family that is rare and found to grow in the wild in China. These wild almond trees are found in hilly regions in northern Xinjiang, and exhibit greater drought and cold stress resistance than cultivated almond varieties. However, the response of P. nana DREBs (PnaDREBs) under low temperature stress is still unclear. In this study, 46 DREB genes were identified in the wild almond genome, with this number being slightly lower than that in the sweet almond (Prunus dulcis cultivar 'Nonpareil'). These DREB genes in wild almond were separated into two classes. All PnaDREB genes were located on six chromosomes. PnaDREB proteins that were classified in the same groups contained specific shared motifs, and promoter analyses revealed that PnaDREB genes harbored a range of stress-responsive elements associated with drought, low-temperature stress, light responsivity, and hormone-responsive cis-regulatory elements within their promoter regions. MicroRNA target site prediction analyses also suggested that 79 miRNAs may regulate the expression of 40 of these PnaDREB genes, with PnaDREB2. To examine if these identified PnaDREB genes responded to low temperature stress, 15 of these genes were selected including seven homologous to Arabidopsis C-repeat binding factor (CBFs), and their expression was assessed following incubation for 2 h at 25 °C, 5 °C, 0 °C, -5 °C, or -10 °C. In summary, this analysis provides an overview of the P. nana PnaDREB gene family and provides a foundation for further studies of the ability of different PnaDREB genes to regulate cold stress responses in almond plants.

Genome-Wide Analysis of the Almond AP2/ERF Superfamily and Its Functional Prediction during Dormancy in Response to Freezing Stress.

Simple SummaryThe ethylene-responsive element (AP2/ERF) is one of the key and conserved transcription factors (TFs) in plants, and it plays a crucial role in regulating plant growth, development, and stress response. The cultivated almond in Xinjiang is often affected by short-term ultralow temperature freezing stress during the winter dormancy period, resulting in the death of large-scale almond plants. In this study, we conducted the first genome-wide analysis of the PdAP2/ERF family in almond, including protein physicochemical properties, phylogenetic relationships, motif types, gene structures, gene replication types, collinearity relationships, and cis-element types in promoter regions. We further analyzed the expression patterns of the PdAP2/ERF gene in different tissues of almond and under freezing stress at different temperatures in annual dormant branches using transcriptome data. In addition, we also analyzed the expression levels of 13 PdAP2/ERF genes in four tissues of almond and in annual dormant branches treated with freezing stress at different temperatures using fluorescence quantitative technology. This study laid the foundation for further exploring the function of the PdAP2/ERF gene in almond.The AP2/ERF transcription factor family is one of the largest transcription factor families in plants and plays an important role in regulating plant growth and development and the response to biotic and abiotic stresses. However, there is no report on the AP2/ERF gene family in almond (Prunus dulcis). In this study, a total of 136 PdAP2/ERF genes were identified from the almond genome, and their protein physicochemical properties were analyzed. The PdAP2/ERF members were divided into five subgroups: AP2, RAV, ERF, DREB, and Soloist. The PdAP2/ERF members in each subgroup had conserved motif types and exon/intron numbers. PdAP2/ERFS members are distributed on eight chromosomes, with 22 pairs of segmental duplications and 28 pairs of tandem duplications. We further explored the colinear relationship between almond and Arabidopsis thaliana, Oryza sativa, Malus domestica, and Prunus persica AP2/ERF genes and their evolution. The results of cis-acting elements showed that PdAP2/ERF members are widely involved in various processes, such as growth and development, hormone regulation, and stress response. The results based on transcriptome expression patterns showed that PdAP2/ERF genes had significant tissue-specific expression characteristics and were involved in the response of annual dormant branches of almond to low-temperature freezing stress. In addition, the fluorescence quantitative relative expression results of 13 representative PdAP2/ERF genes in four tissues of ‘Wanfeng’ almond and under six low-temperature freezing treatments of annual dormant branches were consistent with the transcriptome results. It is worth noting that the fluorescence quantitative expression level showed that the PdERF24 gene was extremely significant at −30 °C, suggesting that this gene may play an important role in the response of almond dormancy to ultralow temperature freezing stress. Finally, we identified 7424 and 6971 target genes based on AP2 and ERF/DREB DNA-binding sites, respectively. The GO and KEGG enrichment results showed that these target genes play important roles in protein function and multiple pathways. In summary, we conducted bioinformatics and expression pattern studies on PdAP2/ERF genes, including 13 PdAP2/ERF genes, and performed fluorescence quantitative analysis of annual dormant shoots under different low-temperature freezing stress treatments to understand the tolerance of almond dormancy to freezing stress and suggest future improvements.

Identification of Putative Markers of Non-infectious Bud Failure in Almond [Prunus dulcis (Mill.) D.A. Webb] Through Genome Wide DNA Methylation Profiling and Gene Expression Analysis in an Almond × Peach Hybrid Population.

Almond [Prunus dulcis (Mill.) D.A. Webb] is an economically important nut crop susceptible to the genetic disorder, Non-infectious Bud Failure (NBF). Despite the severity of exhibition in several prominent almond cultivars, no causal mechanism has been identified underlying NBF development. The disorder is hypothesized to be associated with differential DNA methylation patterns based on patterns of inheritance (i.e., via sexual reproduction and clonal propagation) and previous work profiling methylation in affected trees. Peach (Prunus persica L. Batsch) is a closely related species that readily hybridizes with almond; however, peach is not known to exhibit NBF. A cross between an NBF-exhibiting ‘Carmel’ cultivar and early flowering peach (‘40A17’) produced an F1 where ∼50% of progeny showed signs of NBF, including canopy die-back, erratic branching patterns (known as “crazy-top”), and rough bark. In this study, whole-genome DNA methylation profiles were generated for three F1 progenies exhibiting NBF and three progenies considered NBF-free. Subsequent alignment to both the almond and peach reference genomes showed an increase in genome-wide methylation levels in NBF hybrids in CG and CHG contexts compared to no-NBF hybrids when aligned to the almond genome but no difference in methylation levels when aligned to the peach genome. Significantly differentially methylated regions (DMRs) were identified by comparing methylation levels across the genome between NBF- and no-NBF hybrids in each methylation context. In total, 115,635 DMRs were identified based on alignment to the almond reference genome, and 126,800 DMRs were identified based on alignment to the peach reference genome. Nearby genes were identified as associated with the 39 most significant DMRs occurring either in the almond or peach alignments alone or occurring in both the almond and peach alignments. These DMR-associated genes include several uncharacterized proteins and transposable elements. Quantitative PCR was also performed to analyze the gene expression patterns of these identified gene targets to determine patterns of differential expression associated with differential DNA methylation. These DMR-associated genes, particularly those showing corresponding patterns of differential gene expression, represent key targets for almond breeding for future cultivars and mitigating the effects of NBF-exhibition in currently affected cultivars.

Gene Expression Analysis in Cold Stress Conditions Reveals BBX20 and CLO as Potential Biomarkers for Cold Tolerance in Almond

Late spring frosts can become one of the limiting factors for the expansion of cultivation area towards a harsher climate for the almond [Prunus amygdalus Batsch syn P. dulcis (Mill.) D.A. Webb] crop as spring frost can damage up to 90% of the harvest. In order to identify key genes favoring cold tolerance in almonds, branches from three late-blooming genotypes: ‘Guara’, ‘Soleta’ and ‘Belona’ were exposed at −4 °C during 24 h in a constant climate chamber. Phenotype analysis showed that ‘Guara’ and ‘Soleta’ had a greater acclimation capacity to cold than ‘Belona’. The qRT-PCR BioMark System technology was used to monitor the relative expression of 30 candidate genes with a potential relation to cold response, which are either involved in the ICE-CBF-COR pathway or the independent CBF pathway, and also genes not yet characterized or with unknown function in almond genome. Differences in the gene expression profiles were found among the three studied genotypes and the three time-points of cold exposure (0, 2 and 24 h). BBX20 and CLO genes behaved as differentiator genes between tolerant and susceptible genotypes in cold stress response in almond pistils. In addition, the differences of expression among the tolerant genotypes suggested the intervention of different mechanisms responding to cold stress in almonds.

How almonds became sweet

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. - submission by Raquel Sánchez-Pérez, Birger Lindberg Møller

Transposons played a major role in the diversification between the closely related almond and peach genomes: results from the almond genome sequence.

SummaryWe sequenced the genome of the highly heterozygous almond Prunus dulcis cv. Texas combining short‐ and long‐read sequencing. We obtained a genome assembly totaling 227.6 Mb of the estimated almond genome size of 238 Mb, of which 91% is anchored to eight pseudomolecules corresponding to its haploid chromosome complement, and annotated 27 969 protein‐coding genes and 6747 non‐coding transcripts. By phylogenomic comparison with the genomes of 16 additional close and distant species we estimated that almond and peach (Prunus persica) diverged around 5.88 million years ago. These two genomes are highly syntenic and show a high degree of sequence conservation (20 nucleotide substitutions per kb). However, they also exhibit a high number of presence/absence variants, many attributable to the movement of transposable elements (TEs). Transposable elements have generated an important number of presence/absence variants between almond and peach, and we show that the recent history of TE movement seems markedly different between them. Transposable elements may also be at the origin of important phenotypic differences between both species, and in particular for the sweet kernel phenotype, a key agronomic and domestication character for almond. Here we show that in sweet almond cultivars, highly methylated TE insertions surround a gene involved in the biosynthesis of amygdalin, whose reduced expression has been correlated with the sweet almond phenotype. Altogether, our results suggest a key role of TEs in the recent history and diversification of almond and its close relative peach.

How to make almonds palatable

Plant Science The domesticated almond tree has been feeding humans for millennia. Derivation from the wild, bitter, and toxic almond required loss of the cyanogenic diglucoside amygdalin. Sanchez-Perez et al. sequenced the almond genome and analyzed the genomic region responsible for this shift. The key change turned out to be a point mutation in a transcription factor that regulates production of P450 monooxygenases in the biosynthetic pathway for the toxic compound. Science , this issue p. [1095][1] [1]: /lookup/doi/10.1126/science.aav8197

Mutation of a bHLH transcription factor allowed almond domestication.

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.

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

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 β(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.

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

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

IDENTIFICATION OF SIMPLE SEQUENCE REPEAT (SSR) MARKERS LINKED TO FLOWERING TIME IN ALMOND BY BULKED SEGREGANT ANALYSIS (BSA)

In this study, flowering dates were evaluated during two years in a F1 almond progeny of seventy eight seedlings from the cross between the intermediate flowering Italian cultivar ‘Tuono’ and the extra-late flowering Iranian cultivar ‘Shahrood-12’. In addition, a modified-bulk segregant analysis in combination with the application of SSR markers was used to identify molecular markers linked to flowering time. Seventy five nuclear SSR markers spanning the whole almond genome were assayed in the molecular characterization of bulks (extra-early, early, late and extra-late) consisting in several descendants selected from the almond progeny studied. Results showed a quantitative inheritance of flowering date in the almond progeny studied. The seedlings evaluated showed a wide range of flowering dates between both progenitors although some of these descendants were earlier than the early progenitor ‘Tuono’. Two microsatellite loci (CPPCT008 and EPDCU2584) were found to be tightly linked to this important agronomic trait. Finally, we discuss the development of efficient marker-assisted selection strategies applied to almond and other Prunus breeding programs.

Construction of an almond linkage map in an Australian population Nonpareil × Lauranne

BackgroundDespite a high genetic similarity to peach, almonds (Prunus dulcis) have a fleshless fruit and edible kernel, produced as a crop for human consumption. While the release of peach genome v1.0 provides an excellent opportunity for almond genetic and genomic studies, well-assessed segregating populations and the respective saturated genetic linkage maps lay the foundation for such studies to be completed in almond.ResultsUsing an almond intraspecific cross between 'Nonpareil' and 'Lauranne' (N × L), we constructed a moderately saturated map with SSRs, SNPs, ISSRs and RAPDs. The N × L map covered 591.4 cM of the genome with 157 loci. The average marker distance of the map was 4.0 cM. The map displayed high synteny and colinearity with the Prunus T × E reference map in all eight linkage groups (G1-G8). The positions of 14 mapped gene-anchored SNPs corresponded approximately with the positions of homologous sequences in the peach genome v1.0. Analysis of Mendelian segregation ratios showed that 17.9% of markers had significantly skewed genotype ratios at the level of P < 0.05. Due to the large number of skewed markers in the linkage group 7, the potential existence of deleterious gene(s) was assessed in the group. Integrated maps produced by two different mapping methods using JoinMap® 3 were compared, and their high degree of similarity was evident despite the positional inconsistency of a few markers.ConclusionsWe presented a moderately saturated Australian almond map, which is highly syntenic and collinear with the Prunus reference map and peach genome V1.0. Therefore, the well-assessed almond population reported here can be used to investigate the traits of interest under Australian growing conditions, and provides more information on the almond genome for the international community.

Molecular markers for kernel bitterness in almond

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.

Discrimination of SNP genotypes associated with complex haplotypes by high resolution melting analysis in almond: implications for improved marker efficiencies

Developed recently, high resolution melting (HRM) analysis is an efficient, accurate and inexpensive method for distinguishing DNA polymorphisms. HRM has been used to identify mutations in human genes, and to detect SNPs, INDELs and microsatellites in plants. However, its capacity to discriminate DNA variants in the context of complex haplotypes involving INDEL as well as SNP variants has not been examined until now. In this study, we genotyped an almond (Prunus dulcis (Mill.) D. A. Webb, syn. Prunus amygdalus Batsch) pseudo-testcross mapping population that showed segregation of complex haplotypes associated with CYP79D16 promoter sequence. The 175 bp region in question included a 7 bp INDEL and 3 SNPs, and manifested as three different haplotypes in the parents. Thus, with one homozygous and one heterozygous parent, two relevant genotypes were identified in the mapping population. Although the population displayed monomorphism with respect to the INDEL and one of the SNPs, HRM was sufficiently sensitive to distinguish genotypes on the basis of the two informative SNPs, and the resulting data were used to map CYP79D16 to linkage group 6 of the almond genome. Thus the capacity of HRM to resolve genotypes arising from complex haplotypes has been demonstrated, and this has important implications for the design of efficient HRM markers for various genetic applications including mapping, population studies and biodiversity analyses.

Protein analysis during almond embryo development. Identification and characterization of a late embryogenesis abundant protein

The expression pattern of proteins was analysed during late embryogenesis in almond ( Prunus amygdalus) to obtain information on the molecular events leading to the acquisition of the embryo desiccation tolerance. Analysis of the ABA content in developing embryos revealed high levels at 50 DAF with a rapid decrease at the onset of desiccation. Two-dimensional electrophoresis of proteins showed that at least ten different polypeptides were induced by dehydration of the embryos. By immunoscreening of a mature embryo cDNA expression library, the ABA inducible Parab21 cDNA (for P. amygdalus responsive to ABA) encoding for a highly phosphorylated protein was isolated and characterized. Parab21 belongs to the LEA-D11 protein family, and contains all the protein motifs characteristic for the RAB/dehydrin group of proteins. The Parab21 mRNA is expressed during late embryogenesis and is also inducible by water deficit and ABA treatment in vegetative tissues. Southern blotting and mapping data are consistent with a single Parab21 gene in the almond genome although other immunologically-related proteins have been detected suggesting that a complex family of RAB/dehydrin-related genes will be involved in desiccation tolerance in almonds.

Specific mRNA accumulation of a gene coding for an O-methyltransferase in almond ( Prunus amygdalus, Batsch) flower tissues

A cDNA clone, PAB14B, was isolated from an almond flower cDNA library. The 1245 bp nucleotide sequence contains an open reading frame encoding a polypeptide of 356 amino acids which shows sequence similarity to O-methyltransferases (OMTs) from different plant species. The highest identity (49%) was observed with a methyltransferase from pea that catalyses the terminal step in pisatin phytoalexin biosynthesis. Comparison of this almond sequence, with amino acid sequences of described OMTs, identified regions of similarity which probably contribute to the binding site of S-adenosyl- l-methionine. Southern blot analysis suggests that PAB14B may be encoded by more than one gene in the almond genome. Flower-specific expression was shown by RNA blot analysis and the OMT transcript was detected in epidermal and vascular floral tissues by in situ hybridization. The available data suggest that this enzyme may be involved in the flavonoid biosynthesis pathway in almond floral tissues.

Molecular characterization of cDNAs corresponding to genes expressed during almond (Prunus amygdalus Batsch) seed development

A number of different cDNA clones corresponding to the most abundant mRNAs present in immature seeds have been isolated from an almond (Prunus amygdalus cv. Texas) immature seed cDNA library. Those corresponding to proteins involved in storage processes have been further characterized. Two of these cDNAs (PA3BF1 and PA3BE12) code for the almond globulins (prunins), the main family of storage proteins synthesized in seeds during embryogenesis, and another cDNA (PA3BA1) codes for the 15.7 kDa almond oleosin, a protein located on the surface of oil bodies in plant seeds. These cDNAs have been sequenced and their expression during almond fruit development has been studied. Their expression is seed-specific and localized in cotyledons around 100 days after flowering. Both prunin and oleosin genes are present in one or two copies in the almond genome.