Genome-wide identification of members of the Skp1 family in almond (Prunus dulcis), cloning and expression characterization of PsdSSK1.

Skp1 (S-phase kinase-associated protein 1) is the core gene of SCF ubiquitin ligase, which mediates protein degradation, thereby regulating biological processes such as cell cycle progression, transcriptional regulation, and signal transduction. A variety of plant Skp1 gene family studies have been reported. However, the almond Skp1 gene family has not yet been studied. In this study, we identified 18 members of the Prunus dulcis PdSkp1 family that were unevenly distributed across six chromosomes of the almond genome. Phylogenetic tree analysis revealed that the PdSkp1 members can be divided into three groups: I, II, and III. PdSkp1 members in each subfamily have relatively conserved motif types and exon/intron numbers. There were three pairs of fragment duplication genes and one pair of tandem repeat genes, and their functions were highly evolutionarily conserved. Transcriptome data showed that PdSkp1 is expressed in almond flower tissues, and that its expression shows significant change during cross-pollination. Fluorescence quantitative results showed that eight PdSkp1 genes had different expression levels in five tissues of almond, i.e., branches, leaves, flower buds, flesh, and cores. In addition, we cloned a PsdSSK1 gene based on PdSkp1. The cloned PsdSSK1 showed the same protein sequence as PdSkp1-12. Results of qPCR and western blot analysis showed high expression of PsdSSK1 in almond pollen. In conclusion, we report the first clone of the key gene SSK1 that controls self-incompatibility in almonds. Our research lays a foundation for future functional research on PdSkp1 members, especially for exploring the mechanism of almond self-incompatibility. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-023-01278-9.

Genome-Wide Identification and Analysis of the MADS-Box Gene Family in Almond Reveal Its Expression Features in Different Flowering Periods.

The MADS-box gene family is an important family of transcription factors involved in multiple processes, such as plant growth and development, stress, and in particular, flowering time and floral organ development. Almonds are the best-selling nuts in the international fruit trade, accounting for more than 50% of the world's dried fruit trade, and one of the main economic fruit trees in Kashgar, Xinjiang. In addition, almonds contain a variety of nutrients, such as protein and dietary fiber, which can supplement nutrients for people. They also have the functions of nourishing the yin and kidneys, improving eyesight, and strengthening the brain, and they can be applied to various diseases. However, there is no report on the MADS-box gene family in almond (Prunus dulcis). In this study, a total of 67 PdMADS genes distributed across 8 chromosomes were identified from the genome of almond 'Wanfeng'. The PdMADS members were divided into five subgroups-Mα, Mß, Mγ, Mδ, and MIKC-and the members in each subgroup had conserved motif types and exon and intron numbers. The number of exons of PdMADS members ranged from 1 to 20, and the number of introns ranged from 0 to 19. The number of exons and introns of different subfamily members varied greatly. The results of gene duplication analysis showed that the PdMADS members had 16 pairs of segmental duplications and 9 pairs of tandem duplications, so we further explored the relationship between the MADS-box gene members in almond and those in Arabidopsis thaliana, Oryza sativa, Malus domestica, and Prunus persica based on colinear genes and evolutionary selection pressure. The results of the cis-acting elements showed that the PdMADS members were extensively involved in a variety of processes, such as almond growth and development, hormone regulation, and stress response. In addition, the expression patterns of PdMADS members across six floral transcriptome samples from two almond cultivars, 'Wanfeng' and 'Nonpareil', had significant expression differences. Subsequently, the fluorescence quantitative expression levels of the 15 PdMADS genes were highly similar to the transcriptome expression patterns, and the gene expression levels increased in the samples at different flowering stages, indicating that the two almond cultivars expressed different PdMADS genes during the flowering process. It is worth noting that the difference in flowering time between 'Wanfeng' and 'Nonpareil' may be caused by the different expression activities of PdMADS47 and PdMADS16 during the dormancy period, resulting in different processes of vernalization. We identified a total of 13,515 target genes in the genome based on the MIKC DNA-binding sites. 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 the PdMADS gene family and investigated six flowering samples from two almond cultivars, the early-flowering 'Wanfeng' and late-flowering 'Nonpareil', for quantitative expression level identification. These findings lay a foundation for future in-depth studies on the mechanism of PdMADS gene regulation during flowering in different almond cultivars.

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

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 persicaAP2/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.