A high-quality chromosome-scale genome assembly of blood orange, an important pigmented sweet orange variety.

Blood orange (BO) is a rare red-fleshed sweet orange (SWO) with a high anthocyanin content and is associated with numerous health-related benefits. Here, we reported a high-quality chromosome-scale genome assembly for Neixiu (NX) BO, reaching 336.63 Mb in length with contig and scaffold N50 values of 30.6 Mb. Furthermore, 96% of the assembled sequences were successfully anchored to 9 pseudo-chromosomes. The genome assembly also revealed the presence of 37.87% transposon elements and 7.64% tandem repeats, and the annotation of 30,395 protein-coding genes. A high level of genome synteny was observed between BO and SWO, further supporting their genetic similarity. The speciation event that gave rise to the Citrus species predated the duplication event found within them. The genome-wide variation between NX and SWO was also compared. This first high-quality BO genome will serve as a fundamental basis for future studies on functional genomics and genome evolution.

GhiPLATZ17 and GhiPLATZ22, zinc-dependent DNA-binding transcription factors, promote salt tolerance in upland cotton.

KEY MESSAGE: The utilization of transcriptome analysis, functional validation, VIGS, and DAB techniques have provided evidence that GhiPLATZ17 and GhiPLATZ22 play a pivotal role in improving the salt tolerance of upland cotton. PLATZ (Plant AT-rich sequences and zinc-binding proteins) are known to be key regulators in plant growth, development, and response to salt stress. In this study, we comprehensively analyzed the PLATZ family in ten cotton species in response to salinity stress. Gossypium herbaceum boasts 25 distinct PLATZ genes, paralleled by 24 in G. raimondii, 25 in G. arboreum, 46 in G. hirsutum, 48 in G. barbadense, 43 in G. tomentosum, 67 in G. mustelinum, 60 in G. darwinii, 46 in G. ekmanianum, and a total of 53 PLATZ genes attributed to G. stephensii. The PLATZ gene family shed light on the hybridization and allopolyploidy events that occurred during the evolutionary history of allotetraploid cotton. Ka/Ks analysis suggested that the PLATZ gene family underwent intense purifying selection during cotton evolution. Analysis of synteny and gene collinearity revealed a complex pattern of segmental and dispersed duplication events to expand PLATZ genes in cotton. Cis-acting elements and gene expressions revealed that GhiPLATZ exhibited salt stress resistance. Transcriptome analysis, functional validation, virus-induced gene silencing (VIGS), and diaminobenzidine staining (DAB) demonstrated that GhiPLATZ17 and GhiPLATZ22 enhance salt tolerance in upland cotton. The study can potentially advance our understanding of identifying salt-resistant genes in cotton.

Genome-wide identification of GH28 family and insight into its contributions to pod shattering resistance in Brassica napus L.

Rapeseed (Brassica napus L.), accounts for nearly 16% of vegetable oil, is the world's second produced oilseed. However, pod shattering has caused significant yield loses in rapeseed production, particularly during mechanical harvesting. The GH28 genes can promote pod shattering by changing the structure of the pod cell wall in Arabidopsis. However, the role of the GH28 gene family in rapeseed was largely unknown. Therefore, a genome-wide comprehensive analysis was conducted to classify the role of GH28 gene family on rapeseed pod shattering. A total of 37 BnaGH28 genes in the rapeseed genome were identified. These BnaGH28s can be divided into five groups (Group A-E), based on phylogenetic and synteny analysis. Protein property, gene structure, conserved motif, cis-acting element, and gene expression profile of BnaGH28 genes in the same group were similar. Specially, the expression level of genes in group A-D was gradually decreased, but increased in group E with the development of silique. Among eleven higher expressed genes in group E, two BnaGH28 genes (BnaA07T0199500ZS and BnaC06T0206500ZS) were significantly regulated by IAA or GA treatment. And the significant effects of BnaA07T0199500ZS variation on pod shattering resistance were also demonstrated in present study. These results could open a new window for insight into the role of BnaGH28 genes on pod shattering resistance in rapeseed.

The promise and pitfalls of synteny in phylogenomics.

Reconstructing the tree of life remains a central goal in biology. Early methods, which relied on small numbers of morphological or genetic characters, often yielded conflicting evolutionary histories, undermining confidence in the results. Investigations based on phylogenomics, which use hundreds to thousands of loci for phylogenetic inquiry, have provided a clearer picture of life's history, but certain branches remain problematic. To resolve difficult nodes on the tree of life, 2 recent studies tested the utility of synteny, the conserved collinearity of orthologous genetic loci in 2 or more organisms, for phylogenetics. Synteny exhibits compelling phylogenomic potential while also raising new challenges. This Essay identifies and discusses specific opportunities and challenges that bear on the value of synteny data and other rare genomic changes for phylogenomic studies. Synteny-based analyses of highly contiguous genome assemblies mark a new chapter in the phylogenomic era and the quest to reconstruct the tree of life.

KmerAperture: Retaining k-mer synteny for alignment-free extraction of core and accessory differences between bacterial genomes.

By decomposing genome sequences into k-mers, it is possible to estimate genome differences without alignment. Techniques such as k-mer minimisers, for example MinHash, have been developed and are often accurate approximations of distances based on full k-mer sets. These and other alignment-free methods avoid the large temporal and computational expense of alignment. However, these k-mer set comparisons are not entirely accurate within-species and can be completely inaccurate within-lineage. This is due, in part, to their inability to distinguish core polymorphism from accessory differences. Here we present a new approach, KmerAperture, which uses information on the k-mer relative genomic positions to determine the type of polymorphism causing differences in k-mer presence and absence between pairs of genomes. Single SNPs are expected to result in k unique contiguous k-mers per genome. On the other hand, contiguous series > k may be caused by accessory differences of length S-k+1; when the start and end of the sequence are contiguous with homologous sequence. Alternatively, they may be caused by multiple SNPs within k bp from each other and KmerAperture can determine whether that is the case. To demonstrate use cases KmerAperture was benchmarked using datasets including a very low diversity simulated population with accessory content independent from the number of SNPs, a simulated population where SNPs are spatially dense, a moderately diverse real cluster of genomes (Escherichia coli ST1193) with a large accessory genome and a low diversity real genome cluster (Salmonella Typhimurium ST34). We show that KmerAperture can accurately distinguish both core and accessory sequence diversity without alignment, outperforming other k-mer based tools.

Shared single copy genes are generally reliable for inferring phylogenetic relationships among polyploid taxa.

Polyploidy, or whole-genome duplication, is expected to confound the inference of species trees with phylogenetic methods for two reasons. First, the presence of retained duplicated genes requires the reconciliation of the inferred gene trees to a proposed species tree. Second, even if the analyses are restricted to shared single copy genes, the occurrence of reciprocal gene loss, where the surviving genes in different species are paralogs from the polyploidy rather than orthologs, will mean that such genes will not have evolved under the corresponding species tree and may not produce gene trees that allow inference of that species tree. Here we analyze three different ancient polyploidy events, using synteny-based inferences of orthology and paralogy to infer gene trees from nearly 17,000 sets of homologous genes. We find that the simple use of single copy genes from polyploid organisms provides reasonably robust phylogenetic signals, despite the presence of reciprocal gene losses. Such gene trees are also most often in accord with the inferred species relationships inferred from maximum likelihood models of gene loss after polyploidy: a completely distinct phylogenetic signal present in these genomes. As seen in other studies, however, we find that methods for inferring phylogenetic confidence yield high support values even in cases where the underlying data suggest meaningful conflict in the phylogenetic signals.

Extensive Loss and Gain of Conserved Noncoding Elements During Early Teleost Evolution.

Conserved noncoding elements in vertebrates are enriched around transcription factor loci associated with development. However, loss and rapid divergence of conserved noncoding elements has been reported in teleost fish, albeit taking only few genomes into consideration. Taking advantage of the recent increase in high-quality teleost genomes, we focus on studying the evolution of teleost conserved noncoding elements, carrying out targeted genomic alignments and comparisons within the teleost phylogeny to detect conserved noncoding elements and reconstruct the ancestral teleost conserved noncoding elements repertoire. This teleost-centric approach confirms previous observations of extensive vertebrate conserved noncoding elements loss early in teleost evolution, but also reveals massive conserved noncoding elements gain in the teleost stem-group over 300 million years ago. Using synteny-based association to link conserved noncoding elements to their putatively regulated target genes, we show the most teleost gained conserved noncoding elements are found in the vicinity of orthologous loci involved in transcriptional regulation and embryonic development that are also associated with conserved noncoding elements in other vertebrates. Moreover, teleost and vertebrate conserved noncoding elements share a highly similar motif and transcription factor binding site vocabulary. We suggest that early teleost conserved noncoding element gains reflect a restructuring of the ancestral conserved noncoding element repertoire through both extreme divergence and de novo emergence. Finally, we support newly identified pan-teleost conserved noncoding elements have potential for accurate resolution of teleost phylogenetic placements in par with coding sequences, unlike ancestral only elements shared with spotted gar. This work provides new insight into conserved noncoding element evolution with great value for follow-up work on phylogenomics, comparative genomics, and the study of gene regulation evolution in teleosts.

Whole-genome assembly of a novel invertebrate herpesvirus from the gastropod Babylonia areolata.

Molluscan herpesviruses cause disease in species of major importance to aquaculture and are the only known herpesviruses to infect invertebrates, which lack an adaptive immune system. Understanding the evolution of malacoherpesviruses in relation to their hosts will likely require comparative genomic studies on multiple phylogenetic scales. Currently, only two malacoherpesvirus species have genomes that have been fully assembled, which limits the ability to perform comparative genomic studies on this family of viruses. In the present study, we fully assemble a herpesvirus from Illumina and Nanopore sequence data that were previously used to assemble the genome of the gastropod Babylonia areolata. We tentatively assign this novel herpesvirus to the genus Aurivirus within the family Malacoherpesviridae based on a phylogenetic analysis of DNA polymerase. While structurally similar to other malacoherpesvirus genomes, a synteny analysis of the novel herpesvirus with another Aurivirus species indicates that genomic rearrangements might be an important process in the evolution of this genus. We anticipate that future complete assemblies of malacoherpesviruses will be a valuable resource in comparative herpesvirus research.

Plant genome evolution in the genus Eucalyptus is driven by structural rearrangements that promote sequence divergence.

Genomes have a highly organized architecture (nonrandom organization of functional and nonfunctional genetic elements within chromosomes) that is essential for many biological functions, particularly gene expression and reproduction. Despite the need to conserve genome architecture, a high level of structural variation has been observed within species. As species separate and diverge, genome architecture also diverges, becoming increasingly poorly conserved as divergence time increases. However, within plant genomes, the processes of genome architecture divergence are not well described. Here we use long-read sequencing and de novo assembly of 33 phylogenetically diverse, wild and naturally evolving Eucalyptus species, covering 1-50 million years of diverging genome evolution to measure genome architectural conservation and describe architectural divergence. The investigation of these genomes revealed that following lineage divergence, genome architecture is highly fragmented by rearrangements. As genomes continue to diverge, the accumulation of mutations and the subsequent divergence beyond recognition of rearrangements become the primary driver of genome divergence. The loss of syntenic regions also contribute to genome divergence but at a slower pace than that of rearrangements. We hypothesize that duplications and translocations are potentially the greatest contributors to Eucalyptus genome divergence.

Genome-Wide Identification and Expression Analysis of Kiwifruit Leucine-Rich Repeat Receptor-Like Proteins Reveal Their Roles in Biotic and Abiotic Stress Responses.

Leucine-rich repeat receptor-like proteins (LRR-RLPs), a major group of receptor-like proteins in plants, have diverse functions in plant physiology, including growth, development, signal transduction, and stress responses. Despite their importance, the specific roles of kiwifruit LRR-RLPs in response to biotic and abiotic stresses remain poorly understood. In this study, we performed family identification, characterization, transcriptome data analysis, and differential gene expression analysis of kiwifruit LRR-RLPs. We identified totals of 101, 164, and 105 LRR-RLPs in Actinidia chinensis 'Hongyang', Actinidia eriantha 'Huate', and Actinidia chinensis 'Red5', respectively. Synteny analysis revealed that the expansion of kiwifruit LRR-RLPs was primarily attributed to segmental duplication events. Based on RNA-seq data from pathogen-infected kiwifruits, we identified specific LRR-RLP genes potentially involved in different stages of pathogen infection. Additionally, we observed the potential involvement of kiwifruit LRR-RLPs in abiotic stress responses, with upstream transcription factors possibly regulating their expression. Furthermore, protein interaction network analysis unveiled the participation of kiwifruit LRR-RLP in the regulatory network of abiotic stress responses. These findings highlight the crucial roles of LRR-RLPs in mediating both biotic and abiotic stress responses in kiwifruit, offering valuable insights for the breeding of stress-resistant kiwifruit varieties.

Extreme genome scrambling in marine planktonic Oikopleura dioica cryptic species.

Genome structural variations within species are rare. How selective constraints preserve gene order and chromosome structure is a central question in evolutionary biology that remains unsolved. Our sequencing of several genomes of the appendicularian tunicate Oikopleura dioica around the globe reveals extreme genome scrambling caused by thousands of chromosomal rearrangements, although showing no obvious morphological differences between these animals. The breakpoint accumulation rate is an order of magnitude higher than in ascidian tunicates, nematodes, Drosophila, or mammals. Chromosome arms and sex-specific regions appear to be the primary unit of macrosynteny conservation. At the microsyntenic level, scrambling did not preserve operon structures, suggesting an absence of selective pressure to maintain them. The uncoupling of the genome scrambling with morphological conservation in O. dioica suggests the presence of previously unnoticed cryptic species and provides a new biological system that challenges our previous vision of speciation in which similar animals always share similar genome structures.

OrthoRefine: automated enhancement of prior ortholog identification via synteny.

BACKGROUND: Identifying orthologs continues to be an early and imperative step in genome analysis but remains a challenging problem. While synteny (conservation of gene order) has previously been used independently and in combination with other methods to identify orthologs, applying synteny in ortholog identification has yet to be automated in a user-friendly manner. This desire for automation and ease-of-use led us to develop OrthoRefine, a standalone program that uses synteny to refine ortholog identification. RESULTS: We developed OrthoRefine to improve the detection of orthologous genes by implementing a look-around window approach to detect synteny. We tested OrthoRefine in tandem with OrthoFinder, one of the most used software for identification of orthologs in recent years. We evaluated improvements provided by OrthoRefine in several bacterial and a eukaryotic dataset. OrthoRefine efficiently eliminates paralogs from orthologous groups detected by OrthoFinder. Using synteny increased specificity and functional ortholog identification; additionally, analysis of BLAST e-value, phylogenetics, and operon occurrence further supported using synteny for ortholog identification. A comparison of several window sizes suggested that smaller window sizes (eight genes) were generally the most suitable for identifying orthologs via synteny. However, larger windows (30 genes) performed better in datasets containing less closely related genomes. A typical run of OrthoRefine with ~ 10 bacterial genomes can be completed in a few minutes on a regular desktop PC. CONCLUSION: OrthoRefine is a simple-to-use, standalone tool that automates the application of synteny to improve ortholog detection. OrthoRefine is particularly efficient in eliminating paralogs from orthologous groups delineated by standard methods.

Genome assembly of Melilotus officinalis provides a new reference genome for functional genomics.

BACKGROUND: Sweet yellow clover (Melilotus officinalis) is a diploid plant (2n = 16) that is native to Europe. It is an excellent legume forage. It can both fix nitrogen and serve as a medicine. A genome assembly of Melilotus officinalis that was collected from Best corporation in Beijing is available based on Nanopore sequencing. The genome of Melilotus officinalis was sequenced, assembled, and annotated. RESULTS: The latest PacBio third generation HiFi assembly and sequencing strategies were used to produce a Melilotus officinalis genome assembly size of 1,066 Mbp, contig N50 = 5 Mbp, scaffold N50 = 130 Mbp, and complete benchmarking universal single-copy orthologs (BUSCOs) = 96.4%. This annotation produced 47,873 high-confidence gene models, which will substantially aid in our research on molecular breeding. A collinear analysis showed that Melilotus officinalis and Medicago truncatula shared conserved synteny. The expansion and contraction of gene families showed that Melilotus officinalis expanded by 565 gene families and shrank by 56 gene families. The contacted gene families were associated with response to stimulus, nucleotide binding, and small molecule binding. Thus, it is related to a family of genes associated with peptidase activity, which could lead to better stress tolerance in plants. CONCLUSIONS: In this study, the latest PacBio technology was used to assemble and sequence the genome of the Melilotus officinalis and annotate its protein-coding genes. These results will expand the genomic resources available for Melilotus officinalis and should assist in subsequent research on sweet yellow clover plants.

Genome-Wide Identification of the WRKY Gene Family and Functional Characterization of CpWRKY5 in Cucurbita pepo.

The WRKY gene family is crucial for regulating plant growth and development. However, the WRKY gene is rarely studied in naked kernel formation in hull-less Cucurbita pepo L. (HLCP), a natural mutant that lacks the seed coat. In this research, 76 WRKY genes were identified through bioinformatics-based methods in C. pepo, and their phylogenetics, conserved motifs, synteny, collinearity, and temporal expression during seed coat development were analyzed. The results showed that 76 CpWRKYs were identified and categorized into three main groups (I-III), with Group II further divided into five subgroups (IIa-IIe). Moreover, 31 segmental duplication events were identified in 49 CpWRKY genes. A synteny analysis revealed that C. pepo shared more collinear regions with cucumber than with melon. Furthermore, quantitative RT-PCR (qRT-PCR) results indicated the differential expression of CpWRKYs across different varieties, with notable variations in seed coat development between HLCP and CP being attributed to differences in CpWRKY5 expression. To investigate this further, CpWRKY5-overexpression tobacco plants were generated, resulting in increased lignin content and an upregulation of related genes, as confirmed by qRT-PCR. This study offers valuable insights for future functional investigations of CpWRKY genes and presents novel information for understanding the regulation mechanism of lignin synthesis.

Evolutionary and synteny analysis of HIS1, BADH2, GBSS1, and GBSS2 in rice: insights for effective introgression breeding strategies.

The key genes BADH2, GBSS1, GBSS2, and HIS1 regulate the fragrance, starch synthesis, and herbicide resistance in rice. Although the molecular functions of four genes have been investigated in the Oryza sativa species, little is known regarding their evolutionary history in the Oryza genus. Here, we studied the evolution of four focal genes in 10 Oryza species using phylogenetic and syntenic approaches. The HIS1 family underwent several times of tandem duplication events in the Oryza species, resulting in copy number variation ranging from 2 to 7. At most one copy of BADH2, GBSS1, and GBSS2 orthologs were identified in each Oryza species, and gene loss events of BADH2 and GBSS2 were identified in three Oryza species. Gene transfer analysis proposed that the functional roles of GBSS1 and GBSS2 were developed in the Asian and African regions, respectively, and most allelic variations of BADH2 in japonica rice emerged after the divergence between the Asian and African rice groups. These results provide clues to determine the origin and evolution of the key genes in rice breeding as well as valuable information for molecular breeders and scientists to develop efficient strategies to simultaneously improve grain quality and yield potential in rice.

Genomic insights into the chromosomal elongation in a family of Collembola.

Collembola is a highly diverse and abundant group of soil arthropods with chromosome numbers ranging from 5 to 11. Previous karyotype studies indicated that the Tomoceridae family possesses an exceptionally long chromosome. To better understand chromosome size evolution in Collembola, we obtained a chromosome-level genome of Yoshiicerus persimilis with a size of 334.44 Mb and BUSCO completeness of 97.0% (n = 1013). Both genomes of Y. persimilis and Tomocerus qinae (recently published) have an exceptionally large chromosome (ElChr greater than 100 Mb), accounting for nearly one-third of the genome. Comparative genomic analyses suggest that chromosomal elongation occurred independently in the two species approximately 10 million years ago, rather than in the ancestor of the Tomoceridae family. The ElChr elongation was caused by large tandem and segmental duplications, as well as transposon proliferation, with genes in these regions experiencing weaker purifying selection (higher dN/dS) than conserved regions. Moreover, inter-genomic synteny analyses indicated that chromosomal fission/fusion events played a crucial role in the evolution of chromosome numbers (ranging from 5 to 7) within Entomobryomorpha. This study provides a valuable resource for investigating the chromosome evolution of Collembola.

Chromosome-Level Assembly and Annotation of the Pearly Heath Coenonympha arcania Butterfly Genome.

We present the first chromosome-level genome assembly and annotation of the pearly heath Coenonympha arcania, generated with a PacBio HiFi sequencing approach and complemented with Hi-C data. We additionally compare synteny, gene, and repeat content between C. arcania and other Lepidopteran genomes. This reference genome will enable future population genomics studies with Coenonympha butterflies, a species-rich genus that encompasses some of the most highly endangered butterfly taxa in Europe.

Chromosome-level genome assembly of the European green woodpecker Picus viridis.

The European green woodpecker, Picus viridis, is a widely distributed species found in the Western Palearctic region. Here, we assembled a highly contiguous genome assembly for this species using a combination of short- and long-read sequencing and scaffolded with chromatin conformation capture (Hi-C). The final genome assembly was 1.28 Gb and features a scaffold N50 of 37 Mb and a scaffold L50 of 39.165 Mb. The assembly incorporates 89.4% of the genes identified in birds in OrthoDB. Gene and repetitive content annotation on the assembly detected 15,805 genes and a ∼30.1% occurrence of repetitive elements, respectively. Analysis of synteny demonstrates the fragmented nature of the P. viridis genome when compared to the chicken (Gallus gallus). The assembly and annotations produced in this study will certainly help for further research into the genomics of P. viridis and the comparative evolution of woodpeckers. Five historical and seven contemporary samples have been resequenced and may give insights on the population history of this species.

The hagfish genome and the evolution of vertebrates.

As the only surviving lineages of jawless fishes, hagfishes and lampreys provide a crucial window into early vertebrate evolution1-3. Here we investigate the complex history, timing and functional role of genome-wide duplications4-7 and programmed DNA elimination8,9 in vertebrates in the light of a chromosome-scale genome sequence for the brown hagfish Eptatretus atami. Combining evidence from syntenic and phylogenetic analyses, we establish a comprehensive picture of vertebrate genome evolution, including an auto-tetraploidization (1RV) that predates the early Cambrian cyclostome-gnathostome split, followed by a mid-late Cambrian allo-tetraploidization (2RJV) in gnathostomes and a prolonged Cambrian-Ordovician hexaploidization (2RCY) in cyclostomes. Subsequently, hagfishes underwent extensive genomic changes, with chromosomal fusions accompanied by the loss of genes that are essential for organ systems (for example, genes involved in the development of eyes and in the proliferation of osteoclasts); these changes account, in part, for the simplification of the hagfish body plan1,2. Finally, we characterize programmed DNA elimination in hagfish, identifying protein-coding genes and repetitive elements that are deleted from somatic cell lineages during early development. The elimination of these germline-specific genes provides a mechanism for resolving genetic conflict between soma and germline by repressing germline and pluripotency functions, paralleling findings in lampreys10,11. Reconstruction of the early genomic history of vertebrates provides a framework for further investigations of the evolution of cyclostomes and jawed vertebrates.

Systematic analysis of Heat Shock Protein 70 (HSP70) gene family in radish and potential roles in stress tolerance.

The 70 kD heat shock proteins (HSP70s) represent a class of molecular chaperones that are widely distributed in all kingdoms of life, which play important biological roles in plant growth, development, and stress resistance. However, this family has not been systematically characterized in radish (Raphanus sativus L.). In this study, we identified 34 RsHSP70 genes unevenly distributed within nine chromosomes of R. sativus. Phylogenetic and multiple sequence alignment analyses classified the RsHSP70 proteins into six distinct groups (Group A-F). The characteristics of gene structures, motif distributions, and corresponding cellular compartments were more similar in closely linked groups. Duplication analysis revealed that segmental duplication was the major driving force for the expansion of RsHSP70s in radish, particularly in Group C. Synteny analysis identified eight paralogs (Rs-Rs) in the radish genome and 19 orthologs (Rs-At) between radish and Arabidopsis, and 23 orthologs (Rs-Br) between radish and Chinese cabbage. RNA-seq analysis showed that the expression change of some RsHSP70s were related to responses to heat, drought, cadmium, chilling, and salt stresses and Plasmodiophora brassicae infection, and the expression patterns of these RsHSP70s were significantly different among 14 tissues. Furthermore, we targeted a candidate gene, RsHSP70-23, the product of which is localized in the cytoplasm and involved in the responses to certain abiotic stresses and P. brassicae infection. These findings provide a reference for further molecular studies to improve yield and stress tolerance of radish.