The pomegranate (Punica granatum L.) draft genome dissects genetic divergence between soft- and hard-seeded cultivars.

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.

Complementary iTRAQ-based proteomic and RNA sequencing-based transcriptomic analyses reveal a complex network regulating pomegranate (Punica granatum L.) fruit peel colour.

Peel colour is an important factor affecting the marketability of pomegranate fruits. Therefore, elucidating the genetic mechanism of fruit peel colour development may be useful for breeding pomegranate cultivars with enhanced fruit peel colours. In this study, we combined an iTRAQ-based proteome-level analysis with an RNA sequencing-based transcriptome-level analysis to detect the proteins and genes related to fruit peel colour development in pomegranate. We analysed the 'Tunisia' (red fruit) and 'White' (white fruit) pomegranate cultivars at two stages of fruit development. A total of 27 differentially abundant proteins (increased abundance) and 54 differentially expressed genes (16 up-regulated and 38 down-regulated) were identified from our proteomics and transcriptomics data. The identified proteins and genes contribute to pomegranate fruit peel colour by participating in the biosynthesis of anthocyanins, stilbenoids, diarylheptanoids, gingerols, flavonoids, and phenylpropanoids. Several candidate proteins and genes corresponded to enzymes related to general reactions (PAL, 4CL, DFR, LDOX/ANS, CHS, and F3'5'H) and glycosylation (GT1 and UGAT) of compounds and pigments related to the colour of pomegranate fruit peel. Complementary proteome- and transcriptome-level analyses revealed a complex molecular network controlling fruit peel colour. The candidate genes identified in this study may be useful for the marker-based breeding of new pomegranate cultivars.

Integrated microRNA and mRNA expression profiling reveals a complex network regulating pomegranate (Punica granatum L.) seed hardness.

The breeding of new soft-seeded pomegranate cultivars provides new products for the market and increases farmers' incomes, yet the genetic architecture mediating seed hardness is largely unknown. Here, the seed hardness and hundred-seed weights of 26 cultivars were determined in 2 successive years. We conducted miRNA and mRNA sequencing to analyse the seeds of two varieties of Punica granatum: soft-seeded Tunisia and hard-seeded Sanbai, at 60 and 120 d after flowering. Seed hardness was strongly positively correlated with hundred-seed weight. We detected 25 and 12 differentially expressed miRNA-mRNA pairs with negative regulatory relationships between the two genotypes at 60 and 120 d after flowering, respectively. These miRNA-mRNA pairs mainly regulated seed hardness by altering cell wall structure. Transcription factors including NAC1, WRKY and MYC, which are involved in seed hardness, were targeted by differentially expressed mdm-miR164e and mdm-miR172b. Thus, seed hardness is the result of a complex biological process regulated by a miRNA-mRNA network in pomegranate. These results will help us understand the complexity of seed hardness and help to elucidate the miRNA-mediated molecular mechanisms that contribute to seed hardness in pomegranate.

Transcriptomic Analysis Reveals Candidate Genes for Female Sterility in Pomegranate Flowers.

Pomegranate has two types of flowers on the same plant: functional male flowers (FMF) and bisexual flowers (BF). BF are female-fertile flowers that can set fruits. FMF are female-sterile flowers that fail to set fruit and that eventually drop. The putative cause of pomegranate FMF female sterility is abnormal ovule development. However, the key stage at which the FMF pomegranate ovules become abnormal and the mechanism of regulation of pomegranate female sterility remain unknown. Here, we studied ovule development in FMF and BF, using scanning electron microscopy to explore the key stage at which ovule development was terminated and then analyzed genes differentially expressed (differentially expressed genes - DEGs) between FMF and BF to investigate the mechanism responsible for pomegranate female sterility. Ovule development in FMF ceased following the formation of the inner integument primordium. The key stage for the termination of FMF ovule development was when the bud vertical diameter was 5.0-13.0 mm. Candidate genes influencing ovule development may be crucial factors in pomegranate female sterility. INNER OUTER (INO/YABBY4) (Gglean016270) and AINTEGUMENTA (ANT) homolog genes (Gglean003340 and Gglean011480), which regulate the development of the integument, showed down-regulation in FMF at the key stage of ovule development cessation (ATNSII). Their upstream regulator genes, such as AGAMOUS-like (AG-like) (Gglean028014, Gglean026618, and Gglean028632) and SPOROCYTELESS (SPL) homolog genes (Gglean005812), also showed differential expression pattern between BF and FMF at this key stage. The differential expression of the ethylene response signal genes, ETR (ethylene-resistant) (Gglean022853) and ERF1/2 (ethylene-responsive factor) (Gglean022880), between FMF and BF indicated that ethylene signaling may also be an important factor in the development of pomegranate female sterility. The increase in BF observed after spraying with ethephon supported this interpretation. Results from qRT-PCR confirmed the findings of the transcriptomic analysis.

De novo transcriptome assembly and quantification reveal differentially expressed genes between soft-seed and hard-seed pomegranate (Punica granatum L.).

Pomegranate (Punica granatum L.) belongs to Punicaceae, and is valued for its social, ecological, economic, and aesthetic values, as well as more recently for its health benefits. The 'Tunisia' variety has softer seeds and big arils that are easily swallowed. It is a widely popular fruit; however, the molecular mechanisms of the formation of hard and soft seeds is not yet clear. We conducted a de novo assembly of the seed transcriptome in P. granatum L. and revealed differential gene expression between the soft-seed and hard-seed pomegranate varieties. A total of 35.1 Gb of data were acquired in this study, including 280,881,106 raw reads. Additionally, de novo transcriptome assembly generated 132,287 transcripts and 105,743 representative unigenes; approximately 13,805 unigenes (37.7%) were longer than 1,000 bp. Using bioinformatics annotation libraries, a total of 76,806 unigenes were annotated and, among the high-quality reads, 72.63% had at least one significant match to an existing gene model. Gene expression and differentially expressed genes were analyzed. The seed formation of the two pomegranate cultivars involves lignin biosynthesis and metabolism, including some genes encoding laccase and peroxidase, WRKY, MYB, and NAC transcription factors. In the hard-seed pomegranate, lignin-related genes and cellulose synthesis-related genes were highly expressed; in soft-seed pomegranates, expression of genes related to flavonoids and programmed cell death was slightly higher. We validated selection of the identified genes using qRT-PCR. This is the first transcriptome analysis of P. granatum L. This transcription sequencing greatly enriched the pomegranate molecular database, and the high-quality SSRs generated in this study will aid the gene cloning from pomegranate in the future. It provides important insights into the molecular mechanisms underlying the formation of soft seeds in pomegranate.