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Background The pomegranate is cultivated across a wide range of geographical regions worldwide for ornamental purposes, fruit aril for the food industry, and juice production. The pomegranate genome is reported to consist of 2n = 16 chromosomes in some cultivars and 2n = 18 in others. Clustering assembled contigs using Hi-C technology resulted in eight pseudochromosomes, alternatively, anchoring these contigs onto a genetic map produced nine pseudochromosomes. The origin of the domesticated pomegranate is unknown. Previous studies have suggested that the variant Daru, which is grown in the region of India and Nepal, is the origin of the wild stock. Results Here we explored the phylogeny, genetic structure, and substitution pattern of P. granatum . Phylogenetic analysis of Punica granatum accessions revealed that Daru seedlings constitute a distinct clade, which is a sister to the clade of Asian accessions, rather than an outgroup. No evidence of introgression was observed between Daru and cultivated accessions. Analysis of the non-synonymous to synonymous substitutions ratio in genic regions outlines a similar profile of the Daru and the domesticated pomegranates with overall positive selection, while the wild type P. protopunica illustrated a different profile with positive selection only in some regions. Domesticated pomegranate variants show low heterozygosity and nucleotide diversity, particularly on chromosome 1. Conclusions Our findings suggest that differences in chromosome numbers may be associated with the formation of chromosome quadrivalents and their impact on the arrangement of contigs into pseudochromosomes. The profile of Kn to Ks ratio in domesticated pomegranates likely reflects positive selection. Genetic profiling of Daru, domesticated P. granatum , and wild-type P. protopunica revealed that Daru more closely resembles domesticated plants, challenging the notion that it represents the wild pomegranate.

Pomegranate (Punica granatum L.) which belongs to family Lythraceae, is one of the most important fruit crops of many tropical and subtropical regions. A high variability in fruit color is observed among different pomegranate accessions, which arises from the qualitative and quantitative differences in anthocyanins. However, the mechanism of fruit color variation is still not fully elucidated. In the present study, we investigated the red color mutation between a red-skinned pomegranate ‘Hongbaoshi’ and a purple-red-skinned cultivar ‘Moshiliu’, by using transcriptomic and metabolomic approaches. A total of 51 anthocyanins were identified from fruit peels, among which 3-glucoside and 3,5-diglucoside of cyanidin (Cy), delphinidin (Dp), and pelargonidin (Pg) were dominant. High proportion of Pg in early stages of ‘Hongbaoshi’ but high Dp in late stages of ‘Moshiliu’ were characterized. The unique high levels of Cy and Dp anthocyanins accumulating from early developmental stages accounted for the purple-red phenotype of ‘Moshiliu’. Transcriptomic analysis revealed an early down-regulated and late up-regulated of anthocyanin-related structure genes in ‘Moshiliu’ compared with ‘Hongbaoshi’. Alao, ANR was specially expressed in ‘Hongbaoshi’, with extremely low expression levels in ‘Moshiliu’. For transcription factors R2R3-MYB, the profiles demonstrated a much higher transcription levels of three subgroup (SG) 5 MYBs and a sharp decrease in expression of SG6 MYB LOC116202527 in high-anthocyanin ‘Moshiliu’. SG4 MYBs exhibited two entirely different patterns, LOC116203744 and LOC116212505 were down-regulated whereas LOC116205515 and LOC116212778 were up-regulated in ‘Moshiliu’ pomegranate. The results indicate that specific SG members of the MYB family might promote the peel coloration in different manners and play important roles in color mutation in pomegranate.Key messageIntegrating metabolomic and transcriptomic data identified differentially anthocyanins, expressed structural genes and R2R3-MYB genes from SG4, SG5, and SG6, revealing the color variation in red and purple-red pomegranate.

Summary Complete and highly accurate reference genomes and gene annotations are indispensable for basic biological research and trait improvement of woody tree species. In this study, we integrated single‐molecule sequencing and high‐throughput chromosome conformation capture techniques to produce a high‐quality and long‐range contiguity chromosome‐scale genome assembly of the soft‐seeded pomegranate cultivar ‘Tunisia’. The genome covers 320.31 Mb (scaffold N50 = 39.96 Mb; contig N50 = 4.49 Mb) and includes 33 594 protein‐coding genes. We also resequenced 26 pomegranate varieties that varied regarding seed hardness. Comparative genomic analyses revealed many genetic differences between soft‐ and hard‐seeded pomegranate varieties. A set of selective loci containing SUC8‐like, SUC6, FoxO and MAPK were identified by the selective sweep analysis between hard‐ and soft‐seeded populations. An exceptionally large selective region (26.2 Mb) was identified on chromosome 1. Our assembled pomegranate genome is more complete than other currently available genome assemblies. Our results indicate that genomic variations and selective genes may have contributed to the genetic divergence between soft‐ and hard‐seeded pomegranate varieties.

Summary Pomegranate is an important perennial fruit tree distributed worldwide. Reference genomes with gaps and limit gene identification controlling important agronomic traits hinder its functional genomics and genetic improvements. Here, we reported a telomere‐to‐telomere (T2T) gap‐free genome assembly of the distinctive cultivar ‘Moshiliu’. The Moshiliu reference genome was assembled into eight chromosomes without gaps, totalling ~366.71 Mb, with 32 158 predicted protein‐coding genes. All 16 telomeres and eight centromeres were characterized; combined with FISH analysis, we revealed the atypical telomere units in pomegranate as TTTTAGGG. Furthermore, a total of 16 loci associated with 15 important agronomic traits were identified based on GWAS of 146 accessions. Gene editing and biochemical experiments demonstrated that a 37.2‐Kb unique chromosome translocation disrupting the coding domain sequence of PgANS was responsible for anthocyanin‐less, knockout of PgANS in pomegranate exhibited a defect in anthocyanin production; a unique repeat expansion in the promoter of PgANR may affected its expression, resulting in black peel; notably, the G → A transversion located at the 166‐bp coding domain of PgNST3, which caused a E56K mutation in the PgNST3 protein, closely linked with soft‐seed trait. Overexpression of PgNST3A in tomato presented smaller and softer seed coats. The E56K mutation in PgNST3 protein, eliminated the binding ability of PgNST3 to the PgMYB46 promoter, which subsequently affected the thickness of the inner seed coat of soft‐seeded pomegranates. Collectively, the validated gap‐free genome, the identified genes controlling important traits and the CRISPR‐Cas9‐mediated gene knockout system all provided invaluable resources for pomegranate precise breeding.

Background The basic leucine zipper (bZIP) transcription factor is one of the most abundant and conserved gene families in eukaryotes. In addition to participating in plant development and growth, bZIP transcription factors play crucial roles in various abiotic stress responses and anthocyanin accumulation. Up to now, analysis of bZIP gene family members in pomegranate ( Punica granatum ) has not been reported. Three published pomegranate genome sequences provide valuable resources for further gene function analysis. Results Using bioinformatics analysis, 65 PgbZIPs were identified and analyzed from the ‘Taishanhong’ pomegranate genome. We divided them into 13 groups (A, B, C, D, E, F, G, H, I, J, K, M, and S) according to the phylogenetic relationship with those of Arabidopsis , each containing a different number of genes. The regularity of exon/intron number and distribution was consistent with the classification of groups in the evolutionary tree. Transcriptome analysis of different tissues showed that members of the PgbZIP gene family were differentially expressed in different developmental stages and tissues of pomegranate. Among them, we selected PgbZIP16 and PgbZIP34 as candidate genes which affect anthocyanin accumulation. The full-length CDS region of PgbZIP16 and PgbZIP34 were cloned from pomegranate petals by homologous cloning technique, encoding 170 and 174 amino acids, which were 510 bp and 522 bp, respectively. Subcellular localization assays suggested that both PgbZIP16 and PgbZIP34 were nucleus-localized. Real-time quantitative PCR (qPCR) was used to explore the expression of PgbZIP16 and PgbZIP34 in the petals of three kinds of ornamental pomegranates at the full flowering stage. The results demonstrated that the expression of PgbZIP16 in red petals was 5.83 times of that in white petals, while PgbZIP34 was 3.9 times. The results of transient expression in tobacco showed that consistent trends were observed in anthocyanin concentration and expression levels of related genes, which both increased and then decreased. Both PgbZIP16 and PgbZIP34 could promote anthocyanin accumulation in tobacco leaves. We obtained transgenic strains overexpressing PgbZIP16 , and the histochemical staining for GUS activity showed that overexpressed PgbZIP16 seedlings were expressed in the stem. Transgenic experiments indicated that overexpression of PgbZIP16 significantly upregulated UF3GT , ANS and DFR genes in Arabidopsis and enhanced anthocyanin accumulation. Conclusions The whole genome identification, gene structure, phylogeny, gene cloning, subcellular location and functional verification of the pomegranate bZIP gene family provide a theoretical foundation for the functional study of the PgbZIP gene family and candidate genes for anthocyanin biosynthesis.

4-Coumarate:CoA ligase (4CL, EC6.2.1.12), located at the end of the phenylpropanoid metabolic pathway, regulates the metabolic direction of phenylpropanoid derivatives and plays a pivotal role in the biosynthesis of flavonoids, lignin, and other secondary metabolites. In order to understand the molecular characteristics and potential biological functions of the 4CL gene family in the pomegranate, a bioinformatics analysis was carried out on the identified 4CLs. In this study, 12 Pg4CLs were identified in the pomegranate genome, which contained two conserved amino acid domains: AMP-binding domain Box I (SSGTTGLPKGV) and Box II (GEICIRG). During the identification, it was found that Pg4CL2 was missing Box II. The gene cloning and sequencing verified that this partial amino acid deletion was caused by genome sequencing and splicing errors, and the gene cloning results corrected the Pg4CL2 sequence information in the ‘Taishanhong’ genome. According to the phylogenetic tree, Pg4CLs were divided into three subfamilies, and each subfamily had 1, 1, and 10 members, respectively. Analysis of cis-acting elements found that all the upstream sequences of Pg4CLs contained at least one phytohormone response element. An RNA-seq and protein interaction network analysis suggested that Pg4CL5 was highly expressed in different tissues and may participate in lignin synthesis of pomegranate. The expression of Pg4CL in developing pomegranate fruits was analyzed by quantitative real-time PCR (qRT-PCR), and the expression level of Pg4CL2 demonstrated a decreasing trend, similar to the trend of flavonoid content, indicating Pg4CL2 may involve in flavonoid synthesis and pigment accumulation. Pg4CL3, Pg4CL7, Pg4CL8, and Pg4CL10 were almost not expressed or lowly expressed, the expression level of Pg4CL4 was higher in the later stage of fruit development, suggesting that Pg4CL4 played a crucial role in fruit ripening. The expression levels of 4CL genes were significantly different in various fruit development stages. The results laid the foundation for an in-depth analysis of pomegranate 4CL gene functions.

Members of the sugars will eventually be exported transporter (SWEET) family regulate the transport of different sugars through the cell membrane and control the distribution of sugars inside and outside the cell. The SWEET gene family also plays important roles in plant growth and development and physiological processes. So far, there are no reports on the SWEET family in pomegranate. Meanwhile, pomegranate is rich in sugar, and three published pomegranate genome sequences provide resources for the study of the SWEET gene family. 20 PgSWEETs from pomegranate and the known Arabidopsis and grape SWEETs were divided into four clades (Ⅰ, Ⅱ, Ⅲ and Ⅳ) according to the phylogenetic relationships. PgSWEETs of the same clade share similar gene structures, predicting their similar biological functions. RNA-Seq data suggested that PgSWEET genes have a tissue-specific expression pattern. Foliar application of tripotassium phosphate significantly increased the total soluble sugar content of pomegranate fruits and leaves and significantly affected the expression levels of PgSWEETs. The plant growth hormone regulator assay also significantly affected the PgSWEETs expression both in buds of bisexual and functional male flowers. Among them, we selected PgSWEET17a as a candidate gene that plays a role in fructose transport in leaves. The 798 bp CDS sequence of PgSWEET17a was cloned, which encodes 265 amino acids. The subcellular localization of PgSWEET17a showed that it was localized to the cell membrane, indicating its involvement in sugar transport. Transient expression results showed that tobacco fructose content was significantly increased with the up-regulation of PgSWEET17a, while both sucrose and glucose contents were significantly down-regulated. The integration of the PgSWEET phylogenetic tree, gene structure and RNA-Seq data provide a genome-wide trait and expression pattern. Our findings suggest that tripotassium phosphate and plant exogenous hormone treatments could alter PgSWEET expression patterns. These provide a reference for further functional verification and sugar metabolism pathway regulation of PgSWEETs.

This study describes the first genome sequence and analysis of Coniella granati, a fungal pathogen with a broad host range, which is responsible for postharvest crown rot, shoot blight, and canker diseases in pomegranates. C. granati is a geographically widespread pathogen which has been reported across Europe, Asia, the Americas, and Africa. Our analysis revealed a 46.8 Mb genome with features characteristic of hemibiotrophic fungi. Approximately one third of its genome was compartmentalised within ‘AT-rich’ regions exhibiting a low GC content (30 to 45%). These regions primarily comprised transposable elements that are repeated at a high frequency and interspersed throughout the genome. Transcriptome-supported gene annotation of the C. granati genome revealed a streamlined proteome, mirroring similar observations in other pathogens with a latent phase. The genome encoded a relatively compact set of 9568 protein-coding genes with a remarkable 95% having assigned functional annotations. Despite this streamlined nature, a set of 40 cysteine-rich candidate secreted effector-like proteins (CSEPs) was predicted as well as a gene cluster involved in the synthesis of a pomegranate-associated toxin. These potential virulence factors were predominantly located near repeat-rich and AT-rich regions, suggesting that the pathogen evades host defences through Repeat-Induced Point mutation (RIP)-mediated pseudogenisation. Furthermore, 23 of these CSEPs exhibited homology to known effector and pathogenicity genes found in other hemibiotrophic pathogens. The study establishes a foundational resource for the study of the genetic makeup of C. granati, paving the way for future research on its pathogenicity mechanisms and the development of targeted control strategies to safeguard pomegranate production.

Backgrounds Pomegranate is an excellent tree species with nutritional, medicinal, ornamental and ecological values. Studies have confirmed that SPL factors play an important role in floral transition and flower development. Results Used bioinformatics methods, 15 SPL (SQUAMOSA promoter-binding protein-like) genes were identified and analyzed from the ‘Taishanhong’ pomegranate ( P. granatum L.) genome. Phylogenetic analysis showed that PgSPLs were divided into six subfamilies (G1 ~ G6). PgSPL promoter sequences contained multiple cis -acting elements associated with abiotic stress or hormonal response. Based on the transcriptome data, expression profiles of different tissues and different developmental stages showed that PgSPL genes had distinct temporal and spatial expression characteristics. The expression analysis of miR156 in small RNA sequencing results showed that miR156 negatively regulated the expression of target genes. qRT-PCR analysis showed that the expression levels of PgSPL2 , PgSPL3 , PgSPL6 , PgSPL11 and PgSPL14 in leaves were significantly higher than those in buds and stems ( p  < 0.05). The expression levels of PgSPL5 , PgSPL12 and PgSPL13 in flower buds were significantly higher than that in leaves and stems ( p  < 0.05). The full-length of coding sequence of PgSPL5 and PgSPL13 were obtained by homologous cloning technology. The full length of PgSPL5 is 1020 bp, and PgSPL13 is 489 bp, which encodes 339 and 162 amino acids, respectively. Further investigation revealed that PgSPL5 and PgSPL13 proteins were located in the nucleus. Exogenous plant growth regulator induction experiments showed that PgSPL5 was up-regulated in leaves and stems. PgSPL13 was up-regulated in leaves and down-regulated in stems. When sprayed with 6-BA, IBA and PP333 respectively, PgSPL5 and PgSPL13 were up-regulated most significantly at P2 (bud vertical diameter was 5.1 ~ 12.0 mm) stage of bisexual and functional male flowers. Conclusions Our findings suggested that PgSPL2 , PgSPL3 , PgSPL6 , PgSPL11 and PgSPL14 played roles in leaves development of pomegranate. PgSPL5 , PgSPL12 and PgSPL13 played roles in pomegranate flower development. PgSPL5 and PgSPL13 were involved in the response process of different plant hormone signal transduction in pomegranate development. This study provided a robust basis for further functional analyses of SPL genes in pomegranate.

The peel color of pomegranates is an important exterior quality that determines market value. Anthocyanins are biosynthesized in the cytosol and then transported to the vacuole for storage. However, the molecular mechanism that determines the color variation between red and white pomegranates remains unclear. In this study, we identified an R2R3-MYB protein (PgMYB1) that interacts with the PgGSTF6 promoter and regulates its transcriptional expression, thus promoting the accumulation of anthocyanins in pomegranate. The expression of PgMYB1 and PgGSTF6 was positively correlated with the anthocyanin content in red and white pomegranates. Further investigation showed that the knockdown of PgMYB1 in red pomegranate ‘Taishanhong’ (TSH), by the virus-induced gene-silencing system, inhibited anthocyanin accumulation. Together, our results indicate that PgMYB1 controls the transport of anthocyanin via PgGSTF6 and thus promotes anthocyanin accumulation in red pomegranates. Our results have a certain reference value for further clarifying the regulation of anthocyanin synthesis and transport in pomegranate fruits.