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.     

Improved genome assembly provides new insights into genome evolution in a desert poplar (Populus euphratica)

Populus euphratica is well adapted to extreme desert environments and is an important model species for elucidating the mechanisms of abiotic stress resistance in trees. The current assembly of P. euphratica genome is highly fragmented with many gaps and errors, thereby impeding downstream applications. Here, we report an improved chromosome‐level reference genome of P. euphratica (v2.0) using single‐molecule sequencing and chromosome conformation capture (Hi‐C) technologies. Relative to the previous reference genome, our assembly represents a nearly 60‐fold improvement in contiguity, with a scaffold N50 size of 28.59 Mb. Using this genome, we have found that extensive expansion of Gypsy elements in P. euphratica led to its rapid increase in genome size compared to any other Salicaceae species studied to date, and potentially contributed to adaptive divergence driven by insertions near genes involved in stress tolerance. We also detected a wide range of unique structural rearrangements in P. euphratica, including 2,549 translocations, 454 inversions, 121 tandem and 14 segmental duplications. Several key genes likely to be involved in tolerance to abiotic stress were identified within these regions. This high‐quality genome represents a valuable resource for poplar breeding and genetic improvement in the future, as well as comparative genomic analysis with other Salicaceae species.     

Silver nitrate and aminoethoxyvinylglycine promote in vitro adventitious shoot regeneration of pomegranate (Punica granatum L.)

A protocol is presented for direct adventitous shoot organogenesis and complete plant regeneration from seedling-derived explants of pomegranate (Punica granatum L.), a tropical fruit tree. Murashige and Skoog (1962) (MS) medium enriched with 8.9 mumol/L benzyladenine (BA), 5.4 mumol/L naphthaleneacetic acid (NAA) and 10% coconut water (CW) induced adventitious shoot bud differentiation in axenic seedling-derived cotyledons as well as hypocotyl segments. The cotyledons were more responsive than the hypocotyls. Addition of ethylene inhibitors such as AgNO3 (10-40 mumol/L) and aminoethoxyvinylglycine (AVG) (5-15 mumol/L) to the medium markedly enhanced regeneration frequency as well as number of shoots obtained per explant. The promotive effect of AVG and AgNO3 on shoot organogenesis was observed only in cotyledon explants. The regeneration medium containing AgNO3 (20 mumol/L) or AVG (10 mumol/L) induced adventitious shoot buds from 57% or 53% of the cotyledon explants respectively. These shoot buds developed into shoots upon transfer to a regeneration medium without AgNO3 and AVG. The promotive effect of AVG on shoot regeneration was reversed by exogenous application of 20 mumol/L 2-chloroethylphosphonic acid (CEPA), an ethylene releasing compound. On the other hand, shoot regeneration stimulated by AgNO3 was relatively less affected by CEPA. Regenerated shoots were rooted in half-strength MS medium (1/2 MS) containing 0.54 mumol/L NAA. The well rooted plantlets were acclimatized and eventually established in soil.     

The anti-biofilm potential of pomegranate (Punica granatum L.) extract against human bacterial and fungal pathogens

Infectious diseases caused by bacteria and fungi are the major cause of morbidity and mortality across the globe. Multi-drug resistance in these pathogens augments the complexity and severity of the diseases. Various studies have shown the role of biofilms in multi-drug resistance, where the pathogen resides inside a protective coat made of extracellular polymeric substances. Since biofilms directly influence the virulence and pathogenicity of a pathogen, it is optimal to employ a strategy that effectively inhibits the formation of biofilm. Pomegranate is a common food and is also used traditionally to treat various ailments. This study assessed the anti-biofilm activity of a methanolic extract of pomegranate against bacterial and fungal pathogens. Methanolic extract of pomegranate was shown to inhibit the formation of biofilms by Staphylococcus aureus, methicillin resistant S. aureus, Escherichia coli, and Candida albicans. Apart from inhibiting the formation of biofilm, pomegranate extract disrupted pre-formed biofilms and inhibited germ tube formation, a virulence trait, in C. albicans. Characterization of the methanolic extract of pomegranate revealed the presence of ellagic acid (2,3,7,8-tetrahydroxy-chromeno[5,4,3-cde]chromene-5,10-dione) as the major component. Ellagic acid is a bioactive tannin known for its antioxidant, anticancer, and anti-inflammatory properties. Further studies revealed the ability of ellagic acid to inhibit the growth of all species in suspension at higher concentrations (>75?μg?ml^?1) and biofilm formation at lower concentrations (<40?μg?ml^?1) which warrants further investigation of the potential of ellagic acid or peel powders of pomegranate for the treatment of human ailments.     

Apolygus lucorum genome provides insights into omnivorousness and mesophyll feeding

Apolygus lucorum (Miridae) is an omnivorous pest that occurs worldwide and is notorious for the serious damage it causes to various crops and substantial economic losses. Although some studies have examined the biological characteristics of the mirid bug, no reference genome is available in Miridae, limiting in‐depth studies of this pest. Here, we present a chromosome‐scale reference genome of A. lucorum, the first sequenced Miridae species. The assembled genome size was 1.02 Gb with a contig N50 of 785 kb. With Hi‐C scaffolding, 1,016 Mb contig sequences were clustered, ordered and assembled into 17 large scaffolds with scaffold N50 length 68 Mb, each corresponding to a natural chromosome. Numerous transposable elements occur in this genome and contribute to the large genome size. Expansions of genes associated with omnivorousness and mesophyll feeding such as those related to digestion, chemosensory perception, and detoxification were observed in A. lucorum, suggesting that gene expansion contributed to its strong environmental adaptability and severe harm to crops. We clarified that a salivary enzyme polygalacturonase is unique in mirid bugs and has significantly expanded in A. lucorum, which may contribute to leaf damage from this pest. The reference genome of A. lucorum not only facilitates biological studies of Hemiptera as well as an understanding of the damage mechanism of mesophyll feeding, but also provides a basis on which to develop efficient control technologies for mirid bugs.     

The Chimonanthus salicifolius genome provides insight into magnoliid evolution and flavonoid biosynthesis

Chimonanthus salicifolius, a member of the Calycanthaceae of magnoliids, is one of the most famous medicinal plants in Eastern China. Here, we report a chromosome‐level genome assembly of C. salicifolius, comprising 820.1 Mb of genomic sequence with a contig N50 of 2.3 Mb and containing 36 651 annotated protein‐coding genes. Phylogenetic analyses revealed that magnoliids were sister to the eudicots. Two rounds of ancient whole‐genome duplication were inferred in the C. salicifolious genome. One is shared by Calycanthaceae after its divergence with Lauraceae, and the other is in the ancestry of Magnoliales and Laurales. Notably, long genes with > 20 kb in length were much more prevalent in the magnoliid genomes compared with other angiosperms, which could be caused by the length expansion of introns inserted by transposon elements. Homologous genes within the flavonoid pathway for C. salicifolius were identified, and correlation of the gene expression and the contents of flavonoid metabolites revealed potential critical genes involved in flavonoids biosynthesis. This study not only provides an additional whole‐genome sequence from the magnoliids, but also opens the door to functional genomic research and molecular breeding of C. salicifolius.     

The bottle gourd genome provides insights into Cucurbitaceae evolution and facilitates mapping of a Papaya ring‐spot virus resistance locus

Bottle gourd (Lagenaria siceraria) is an important vegetable crop as well as a rootstock for other cucurbit crops. In this study, we report a high‐quality 313.4‐Mb genome sequence of a bottle gourd inbred line, USVL1VR‐Ls, with a scaffold N50 of 8.7 Mb and the longest of 19.0 Mb. About 98.3% of the assembled scaffolds are anchored to the 11 pseudomolecules. Our comparative genomic analysis identifies chromosome‐level syntenic relationships between bottle gourd and other cucurbits, as well as lineage‐specific gene family expansions in bottle gourd. We reconstructed the genome of the most recent common ancestor of Cucurbitaceae, which revealed that the ancestral Cucurbitaceae karyotypes consisted of 12 protochromosomes with 18 534 protogenes. The 12 protochromosomes are largely retained in the modern melon genome, while have undergone different degrees of shuffling events in other investigated cucurbit genomes. The 11 bottle gourd chromosomes derive from the ancestral Cucurbitaceae karyotypes followed by 19 chromosomal fissions and 20 fusions. The bottle gourd genome sequence has facilitated the mapping of a dominant monogenic locus, Prs, conferring Papaya ring‐spot virus (PRSV) resistance in bottle gourd, to a 317.8‐kb region on chromosome 1. We have developed a cleaved amplified polymorphic sequence (CAPS) marker tightly linked to the Prs locus and demonstrated its potential application in marker‐assisted selection of PRSV resistance in bottle gourd. This study provides insights into the paleohistory of Cucurbitaceae genome evolution, and the high‐quality genome sequence of bottle gourd provides a useful resource for plant comparative genomics studies and cucurbit improvement.     

Chromosome‐level de novo genome assembly of Sarcophaga peregrina provides insights into the evolutionary adaptation of flesh flies

Sarcophaga peregrina is considered to be of great ecological, medical and forensic significance, and has unusual biological characteristics such as an ovoviviparous reproductive pattern and adaptation to feed on carrion. The availability of a high‐quality genome will help to further reveal the mechanisms underlying these charcateristics. Here we present a de novo‐assembled genome at chromosome scale for S. peregrina. The final assembled genome was 560.31 Mb with contig N50 of 3.84 Mb. Hi‐C scaffolding reliably anchored six pseudochromosomes, accounting for 97.76% of the assembled genome. Moreover, 45.70% of repeat elements were identified in the genome. A total of 14,476 protein‐coding genes were functionally annotated, accounting for 92.14% of all predicted genes. Phylogenetic analysis indicated that S. peregrina and S. bullata diverged ~ 7.14 million years ago. Comparative genomic analysis revealed expanded and positively selected genes related to biological features that aid in clarifying its ovoviviparous reproduction and carrion‐feeding adaptations, such as lipid metabolism, olfactory receptor activity, antioxidant enzymes, proteolysis and serine‐type endopeptidase activity. Protein‐coding genes associated with ovoviparity, such as yolk proteins, transferrin and acid sphingomyelinase, were identified. This study provides a valuable genomic resource for S. peregrina, and sheds insight into further revealing the underlying molecular mechanisms of adaptive evolution.     

The genome of cowpea (Vigna unguiculata [L.] Walp.)

Cowpea (Vigna unguiculata [L.] Walp.) is a major crop for worldwide food and nutritional security, especially in sub‐Saharan Africa, that is resilient to hot and drought‐prone environments. An assembly of the single‐haplotype inbred genome of cowpea IT97K‐499‐35 was developed by exploiting the synergies between single‐molecule real‐time sequencing, optical and genetic mapping, and an assembly reconciliation algorithm. A total of 519 Mb is included in the assembled sequences. Nearly half of the assembled sequence is composed of repetitive elements, which are enriched within recombination‐poor pericentromeric regions. A comparative analysis of these elements suggests that genome size differences between Vigna species are mainly attributable to changes in the amount of Gypsy retrotransposons. Conversely, genes are more abundant in more distal, high‐recombination regions of the chromosomes; there appears to be more duplication of genes within the NBS‐LRR and the SAUR‐like auxin superfamilies compared with other warm‐season legumes that have been sequenced. A surprising outcome is the identification of an inversion of 4.2 Mb among landraces and cultivars, which includes a gene that has been associated in other plants with interactions with the parasitic weed Striga gesnerioides. The genome sequence facilitated the identification of a putative syntelog for multiple organ gigantism in legumes. A revised numbering system has been adopted for cowpea chromosomes based on synteny with common bean (Phaseolus vulgaris). An estimate of nuclear genome size of 640.6 Mbp based on cytometry is presented.     

A draft genome of sweet cherry (Prunus avium L.) reveals genome‐wide and local effects of domestication

Sweet cherry (Prunus avium L.) trees are both economically important fruit crops but also important components of natural forest ecosystems in Europe, Asia and Africa. Wild and domesticated trees currently coexist in the same geographic areas with important questions arising on their historical relationships. Little is known about the effects of the domestication process on the evolution of the sweet cherry genome. We assembled and annotated the genome of the cultivated variety “Big Star*” and assessed the genetic diversity among 97 sweet cherry accessions representing three different stages in the domestication and breeding process (wild trees, landraces and modern varieties). The genetic diversity analysis revealed significant genome‐wide losses of variation among the three stages and supports a clear distinction between wild and domesticated trees, with only limited gene flow being detected between wild trees and domesticated landraces. We identified 11 domestication sweeps and five breeding sweeps covering, respectively, 11.0 and 2.4 Mb of the P. avium genome. A considerable fraction of the domestication sweeps overlaps with those detected in the related species, Prunus persica (peach), indicating that artificial selection during domestication may have acted independently on the same regions and genes in the two species. We detected 104 candidate genes in sweep regions involved in different processes, such as the determination of fruit texture, the regulation of flowering and fruit ripening and the resistance to pathogens. The signatures of selection identified will enable future evolutionary studies and provide a valuable resource for genetic improvement and conservation programs in sweet cherry.     

Construction and comparison of three reference‐quality genome assemblies for soybean

We report reference‐quality genome assemblies and annotations for two accessions of soybean (Glycine max) and for one accession of Glycine soja, the closest wild relative of G. max. The G. max assemblies provided are for widely used US cultivars: the northern line Williams 82 (Wm82) and the southern line Lee. The Wm82 assembly improves the prior published assembly, and the Lee and G. soja assemblies are new for these accessions. Comparisons among the three accessions show generally high structural conservation, but nucleotide difference of 1.7 single‐nucleotide polymorphisms (snps) per kb between Wm82 and Lee, and 4.7 snps per kb between these lines and G. soja. snp distributions and comparisons with genotypes of the Lee and Wm82 parents highlight patterns of introgression and haplotype structure. Comparisons against the US germplasm collection show placement of the sequenced accessions relative to global soybean diversity. Analysis of a pan‐gene collection shows generally high conservation, with variation occurring primarily in genomically clustered gene families. We found approximately 40–42 inversions per chromosome between either Lee or Wm82v4 and G. soja, and approximately 32 inversions per chromosome between Wm82 and Lee. We also investigated five domestication loci. For each locus, we found two different alleles with functional differences between G. soja and the two domesticated accessions. The genome assemblies for multiple cultivated accessions and for the closest wild ancestor of soybean provides a valuable set of resources for identifying causal variants that underlie traits for the domestication and improvement of soybean, serving as a basis for future research and crop improvement efforts for this important crop species.