The evolution and diversification of oakleaf butterflies.

Oakleaf butterflies in the genus Kallima have a polymorphic wing phenotype, enabling these insects to masquerade as dead leaves. This iconic example of protective resemblance provides an interesting evolutionary paradigm that can be employed to study biodiversity. We integrated multi-omic data analyses and functional validation to infer the evolutionary history of Kallima species and investigate the genetic basis of their variable leaf wing patterns. We find that Kallima butterflies diversified in the eastern Himalayas and dispersed to East and Southeast Asia. Moreover, we find that leaf wing polymorphism is controlled by the wing patterning gene cortex, which has been maintained in Kallima by long-term balancing selection. Our results provide macroevolutionary and microevolutionary insights into a model species originating from a mountain ecosystem.

Multiple Origins of Bioluminescence in Beetles and Evolution of Luciferase Function.

Bioluminescence in beetles has long fascinated biologists, with diverse applications in biotechnology. To date, however, our understanding of its evolutionary origin and functional variation mechanisms remains poor. To address these questions, we obtained high-quality reference genomes of luminous and nonluminous beetles in 6 Elateroidea families. We then reconstructed a robust phylogenetic relationship for all luminous families and related nonluminous families. Comparative genomic analyses and biochemical functional experiments suggested that gene evolution within Elateroidea played a crucial role in the origin of bioluminescence, with multiple parallel origins observed in the luminous beetle families. While most luciferase-like proteins exhibited a conserved nonluminous amino acid pattern (TLA346 to 348) in the luciferin-binding sites, luciferases in the different luminous beetle families showed divergent luminous patterns at these sites (TSA/CCA/CSA/LVA). Comparisons of the structural and enzymatic properties of ancestral, extant, and site-directed mutant luciferases further reinforced the important role of these sites in the trade-off between acyl-CoA synthetase and luciferase activities. Furthermore, the evolution of bioluminescent color demonstrated a tendency toward hypsochromic shifts and variations among the luminous families. Taken together, our results revealed multiple parallel origins of bioluminescence and functional divergence within the beetle bioluminescent system.

Comprehensive identification and systematical characterization of BRX gene family and the functional of GhBRXL5A in response to salt stress.

BACKGROUND: BRVIS RADIX (BRX) family is a small gene family with the highly conserved plant-specific BRX domains, which plays important roles in plant development and response to abiotic stress. Although BRX protein has been studied in other plants, the biological function of cotton BRX-like (BRXL) gene family is still elusive. RESULT: In this study, a total of 36 BRXL genes were identified in four cotton species. Whole genome or segmental duplications played the main role in the expansion of GhBRXL gene family during evolutionary process in cotton. These BRXL genes were clustered into 2 groups, α and ß, in which structural and functional conservation within same groups but divergence among different groups were found. Promoter analysis indicated that cis-elements were associated with the phytohormone regulatory networks and the response to abiotic stress. Transcriptomic analysis indicated that GhBRXL2A/2D and GhBRXL5A/5D were up/down-regulated in response to the different stress. Silencing of GhBRXL5A gene via virus-induced gene silencing (VIGS) improved salt tolerance in cotton plants. Furthermore, yeast two hybrid analysis suggested homotypic and heterotypic interactions between GhBRXL1A and GhBRXL5D. CONCLUSIONS: Overall, these results provide useful and valuable information for understanding the evolution of cotton GhBRXL genes and their functions in salt stress.

Genome-wide association analysis of kernel nutritional quality in two natural maize populations.

As one of the three staple crops, nutritional traits in maize are important for human and animal nutrition. Grain quality-related traits are closely related to grain commercial value. Understanding the genetic basis of quality-related traits in maize would be helpful for breeding high-quality maize varieties. In this study, two association panels (AM122 and AM180) were subjected to genome-wide association analysis of grain quality-related traits, including protein content, oil content, starch content, and fiber content. In total, 98 SNPs (P < 1 × 10-4) were identified to be significantly associated with these four grain quality-related traits. By integrating two sets of public transcriptome data, 31 genes located in 200 kb regions flanking the associated SNP showed high expression during kernel development and were differentially expressed in two maize inbred lines, KA225 and KB035, with significantly different quality. These genes might regulate maize grain quality by participating in plant hormone processes, autophagy processes, and others. All these results could provide important reference information for breeding high­quality maize varieties. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-023-01360-w.

Genome-wide survey of open chromatin regions in two swallowtail butterflies Papilio machaon and P. bianor.

Papilio machaon was assigned as the type species for all butterflies by Linnaeus and P. bianor is a congener but exhibits a great difference in morphology (especially larva and adult color pattern) and larval host plants from P. machaon. Thus, they are the ideal models to investigate genetic mechanisms underlying morphology and plasticity between congeners. The reference genomes of both species were dissected in our previous studies, but little is known about their regulatory genome and the epigenetic regulation of gene expression throughout developmental stages. Here, we profiled the chromatin accessibility and gene expression of three developmental stages (the 4th instar larva [L4], the 5th instar larva [L5], and pupa [P]) using transposase accessible chromatin sequencing (ATAC-seq) and RNA-seq. Results showed that many accessible chromatin peaks were identified at three developmental stages (peak number, P. machaon: 44,977 [L4], 36,919 [L5], 47,147 [P]; P. bianor: 20,341 [L4], 44,668 [L5], 62,249 [P]). Moreover, the number of differentially accessible peaks and differentially expressed genes between larval stages of each butterfly species are significantly fewer than that between larval and pupal stages, suggesting a higher similarity within larvae and a significant difference between larvae and pupae. This study added the annotated information of chromatin accessibility genome-wide of the two papilionid species and will promote the investigation of gene regulation in butterfly evolution.

Genome-wide identification and gene-editing of pigment transporter genes in the swallowtail butterfly Papilio xuthus.

BACKGROUND: Insect body coloration often functions as camouflage to survive from predators or mate selection. Transportation of pigment precursors or related metabolites from cytoplasm to subcellular pigment granules is one of the key steps in insect pigmentation and usually executed via such transporter proteins as the ATP-binding cassette (ABC) transmembrane transporters and small G-proteins (e.g. Rab protein). However, little is known about the copy numbers of pigment transporter genes in the butterfly genomes and about the roles of pigment transporters in the development of swallowtail butterflies. RESULTS: Here, we have identified 56 ABC transporters and 58 Rab members in the genome of swallowtail butterfly Papilio xuthus. This is the first case of genome-wide gene copy number identification of ABC transporters in swallowtail butterflies and Rab family in lepidopteran insects. Aiming to investigate the contribution of the five genes which are orthologous to well-studied pigment transporters (ABCG: white, scarlet, brown and ok; Rab: lightoid) of fruit fly or silkworm during the development of swallowtail butterflies, we performed CRISPR/Cas9 gene-editing of these genes using P. xuthus as a model and sequenced the transcriptomes of their morphological mutants. Our results indicate that the disruption of each gene produced mutated phenotypes in the colors of larvae (cuticle, testis) and/or adult eyes in G0 individuals but have no effect on wing color. The transcriptomic data demonstrated that mutations induced by CRISPR/Cas9 can lead to the accumulation of abnormal transcripts and the decrease or dosage compensation of normal transcripts at gene expression level. Comparative transcriptomes revealed 606 ~ 772 differentially expressed genes (DEGs) in the mutants of four ABCG transporters and 1443 DEGs in the mutants of lightoid. GO and KEGG enrichment analysis showed that DEGs in ABCG transporter mutants enriched to the oxidoreductase activity, heme binding, iron ion binding process possibly related to the color display, and DEGs in lightoid mutants are enriched in glycoprotein binding and protein kinases. CONCLUSIONS: Our data indicated these transporter proteins play an important role in body color of P. xuthus. Our study provides new insights into the function of ABC transporters and small G-proteins in the morphological development of butterflies.

A panel of four plasma amino acids is a promising biomarker for newly diagnosed bladder cancer.

BACKGROUND: Metastasis and recurrence are the main causes of death in post-operative bladder cancer (BC), emphasizing the importance of exploring early-stage diagnostic markers. Serum biomarkers constitute a promising diagnostic approach for asymptomatic stage cancer as they are non-invasive, have high accuracy and low cost. AIMS: To correlate concentrations of plasma amino acids with BC progression to assess their utility as an early-stage diagnostic. METHODS: Newly diagnosed BC patients (n = 95) and normal controls (n = 96) were recruited during the period from 1 December 2018 to 30 December 2020. General and food frequency questionnaires established their basic information and dietary intake data. Venous blood samples were collected from fasting subjects and used to detect levels of plasma amino acids by liquid chromatography-mass spectrometry. Verification was performed on the GSE13507 transcriptome gene expression matrix of BC from Gene Expression Omnibus (GEO) database. RESULTS: Eleven amino acids have been identified as altered in the plasma of newly diagnosed BC patients compared to controls (P < 0.05). Adjusted by gender, education, smoking and other factors, plasma ornithine level (OR = 0.256, 95% CI: 0.104-0.630) is a protective factor for BC, plasma levels of methionine (OR = 3.460, 95% CI: 1.384-8.651), arginine (OR = 3.851, 95% CI: 1.542-9.616), and glutamate (OR = 3.813, 95% CI: 1.543-9.419) are all risk factors for BC. ROC analysis demonstrated that the combination of plasma ornithine, methionine, arginine and glutamate could accurately diagnose BC (AUC = 0.84, 95% CI: 0.747-0.833). In addition, the mRNA level of arginase 1 was decreased (P < 0.05), while the inducible nitric oxide synthase was increased significantly, which may be linked with the disturbance of arginine metabolism in BC patients. Further analysis of GEO database confirmed the role of arginine metabolism. CONCLUSION: A biomarker panel containing four amino acids may provide a feasible strategy for the early diagnosis of BC. However, further validation is required through prospective studies.

Hagfish genome elucidates vertebrate whole-genome duplication events and their evolutionary consequences.

Polyploidy or whole-genome duplication (WGD) is a major event that drastically reshapes genome architecture and is often assumed to be causally associated with organismal innovations and radiations. The 2R hypothesis suggests that two WGD events (1R and 2R) occurred during early vertebrate evolution. However, the timing of the 2R event relative to the divergence of gnathostomes (jawed vertebrates) and cyclostomes (jawless hagfishes and lampreys) is unresolved and whether these WGD events underlie vertebrate phenotypic diversification remains elusive. Here we present the genome of the inshore hagfish, Eptatretus burgeri. Through comparative analysis with lamprey and gnathostome genomes, we reconstruct the early events in cyclostome genome evolution, leveraging insights into the ancestral vertebrate genome. Genome-wide synteny and phylogenetic analyses support a scenario in which 1R occurred in the vertebrate stem-lineage during the early Cambrian, and 2R occurred in the gnathostome stem-lineage, maximally in the late Cambrian-earliest Ordovician, after its divergence from cyclostomes. We find that the genome of stem-cyclostomes experienced an additional independent genome triplication. Functional genomic and morphospace analyses demonstrate that WGD events generally contribute to developmental evolution with similar changes in the regulatory genome of both vertebrate groups. However, appreciable morphological diversification occurred only in the gnathostome but not in the cyclostome lineage, calling into question the general expectation that WGDs lead to leaps of bodyplan complexity.

HMOX1 silencing prevents doxorubicin-induced cardiomyocyte injury, mitochondrial dysfunction, and ferroptosis by downregulating CTGF.

OBJECTIVES: Doxorubicin is a type of effective antitumor drug but can contribute to cardiomyocyte injuries. We aimed to dissect the mechanism of the HMOX1/CTGF axis in DOX-induced cardiomyocyte injury, mitochondrial dysfunction, and ferroptosis. METHODS: Bioinformatics analysis was conducted to retrieve differentially expressed genes in a DOX-induced mouse model. Mouse cardiomyocytes, HL-1 cells, were induced with l µM DOX, after which gain- or loss-of-function assays were applied. CCK-8, fluorescent probe assay, flow cytometry, and corresponding kits were employed to detect cell viability, ROS levels, mitochondrial membrane potential and cell apoptosis, and GSH and Fe2+ contents, respectively. qRT-PCR or Western blot assay was adopted to test HMOX1, CTGF, BCL-2, Caspase3, Cleaved-Caspase3, and GPX4 expression. RESULTS: Bioinformatics analysis showed that HMOX1 and CTGF were highly expressed in DOX-induced mice and correlated with each other. Also, HMOX1 and CTGF expression was high in HL-1 cells after DOX treatment, along with an obvious decrease in cell viability and GSH and GPX4 expression, an increase in ROS levels, apoptosis, and Fe2+ contents, and mitochondrial membrane potential dysfunction or loss. HMOX1 or CTGF silencing diminished cell apoptosis, Cleaved-Caspase3 expression, Fe2+ contents, and ROS levels, enhanced cell viability and the expression of GSH, GPX4, and BCL-2, and recovered mitochondrial membrane potential in DOX-induced HL-1 cells. Nevertheless, the effects of HMOX1 silencing on the viability, apoptosis, ferroptosis, and mitochondrial dysfunction of DOX-induced HL-1 cells were counteracted by CTGF overexpression. CONCLUSIONS: In conclusion, HMOX1 silencing decreased CTGF expression to alleviate DOX-induced injury, mitochondrial dysfunction, and ferroptosis of mouse cardiomyocytes.

Fatty foods and the risk of bladder cancer: A case-control study.

OBJECTIVE: The aim of this study was to explore the association between dietary fatty foods and the risk for bladder cancer. METHODS: Patients newly diagnosed with bladder cancer (n = 113) and 292 controls were recruited. A food frequency questionnaire (FFQ) was used to investigate the food intake within 1 y. Multivariate logistic regression model was used to estimated odds ratio (OR) between different types of fatty food consumption and bladder cancer. RESULTS: The consumption of soybean oil, the largest proportion of cooking oil, in both groups were much higher than the Chinese recommended dietary intake, especially in the control group. Higher intake of red meat was also observed in bladder cancer cases, although lower intakes of marine fish, egg, milk, and dairy products and nuts were observed in controls. After adjusting for potential confounders, the intakes of marine fish and milk and dairy products were negatively correlated with bladder cancer, with the adjusted OR of 0.28 (95% confidence interval [CI], 0.15-0.55) and 0.36 (95% CI, 0.19-0.69). Total nuts were related to a 76% reduction in bladder cancer risk (OR, 0.24; 95% CI, 0.12-0.48). There was clear and positive association between soybean oil and bladder cancer risk with OR of 3.47 (95 % CI, 1.69-7.14). In stratified analyses by sex and smoking status, the relationship was similar for most results, except for milk and dairy products. The negative correlation between milk and dairy products and bladder cancer risk was only found in men; and milk and dairy products and bladder cancer risk were irrelevant by smoking status. No significant association was found between the intakes of other foods and bladder cancer risk. CONCLUSIONS: Intake of nuts and marine fish may be beneficial for the prevention of bladder cancer. The protective effect of milk and dairy products was only found in men with bladder cancer. High soybean oil intake was a risk factor for bladder cancer.

Branched Chain Amino Acids, New Target of Germinated Brown Rice against Type 2 Diabetes Mellitus: A Randomized Controlled Trial.

SCOPE: Adequate intake of whole grain foods is beneficial to type 2 diabetes mellitus (T2DM). Whether the preventive effects are related with metabolism of branched-chain amino acids (BCAAs) is unclear. The study aims to evaluate the effects of germinated brown rice (GBR) intervention on BCAAs metabolism in T2DM patients. METHODS AND RESULTS: In this randomized controlled trial, subjects with T2DM are instructed to consume 100 g day-1 GBR (GBR group, n=42) or equal staple food (Control group, n=25) for 3 months. Food frequency questionnaires (FFQ) and serum samples are collected before and after the intervention. In the GBR group, fasting blood glucose (FBG), fasting insulin (FINS), and serum BCAAs are decreased, and islet function is improved (p<0.05). Logistic regression analysis showed that FBG (odds ratios [OR]: 1.55, 95% confidence interval [CI]: 1.01-1.84) and energy (OR: 1.21, 95% CI: 1.09-1.30) are positively associated with serum total BCAAs level, while FINS is negatively associated (OR: 0.20, 95% CI: 0.04-0.88). Simultaneously, the key enzymes of BCAAs decomposition, which promotes glycolysis by activating pyruvate dehydrogenase (PDH), are significantly increased. CONCLUSION: GBR improves the indicators of T2DM patients, and the underlying mechanisms include improving insulin resistance and accelerating catabolism of BCAAs.

Germinated Brown rice enhanced n-3 PUFA metabolism in type 2 diabetes patients: A randomized controlled trial.

BACKGROUND: Brown rice (BR) has been considered as a potential strategy in improving T2DM. However, there are a lack of population-based trials on the association of Germinated brown rice (GBR) and diabetes. AIMS: We aimed to explore the influence of GBR diet in T2DM patients for 3 months and whether this effect relates to serum fatty acids. METHODS: Two hundred and twenty T2DM patients have been enrolled and eligible subjects (n = 112, 61 female, 51 male) were randomly divided into GBR intervention group (n = 56) and control group (n = 56). Except those who lost follow-up and withdrew, final GBR group and control group consisted of 42 and 43 patients, respectively. Participants in GBR group were asked to consume 100 g/d GBR instead of equal refined grain (RG) for 3 months, while control group maintain their usual eating habits. A structured questionnaire was used for demographic information at baseline, and basic indicators were measured both at the beginning and end of the trail to evaluate plasma glucose and lipids levels. RESULTS: In GBR group, mean dietary inflammation index (DII) decreased, indicating GBR intervention retarded patient inflammation. Besides, glycolipid related parameters, including FBG, HbA1c, TC and HDL, were all significantly lower than those in control group. Excitingly, fatty acid composition was changed by intake of GBR, especially n-3 PUFA and n-3/n-6 PUFA rate were significantly increased. Moreover, subjects in GBR group had higher levels of n-3 metabolites, such as RVE, MaR1 and PD1, reducing inflammatory effect. In contrast, n-6 metabolites, like LTB4 and PGE2 which could promote inflammatory effect, were lower in GBR group. CONCLUSION: We confirmed that diet with 100 g/d GBR for 3 months could really improve T2DM to some extent. This beneficial effect may be related to n-3 metabolites, namely inflammation changes. TRIAL REGISTRATION: ChiCRT-IOR-17013999, www.chictr.org.cn.

High-quality reference genomes of swallowtail butterflies provide insights into their coloration evolution.

Swallowtail butterflies (Papilionidae) are a historically significant butterfly group due to their colorful wing patterns, extensive morphological diversity, and phylogenetically important position as a sister group to all other butterflies and have been widely studied regarding ecological adaption, phylogeny, genetics, and evolution. Notably, they contain a unique class of pigments, i.e., papiliochromes, which contribute to their color diversity and various biological functions such as predator avoidance and mate preference. To date, however, the genomic and genetic basis of their color diversity and papiliochrome origin in a phylogenetic and evolutionary context remain largely unknown. Here, we obtained high-quality reference genomes of 11 swallowtail butterfly species covering all tribes of Papilioninae and Parnassiinae using long-read sequencing technology. Combined with previously published butterfly genomes, we obtained robust phylogenetic relationships among tribes, overcoming the challenges of incomplete lineage sorting (ILS) and gene flow. Comprehensive genomic analyses indicated that the evolution of Papilionidae-specific conserved non-exonic elements (PSCNEs) and transcription factor binding sites (TFBSs) of patterning and transporter/cofactor genes, together with the rapid evolution of transporters/cofactors, likely promoted the origin and evolution of papiliochromes. These findings not only provide novel insights into the genomic basis of color diversity, especially papiliochrome origin in swallowtail butterflies, but also provide important data resources for exploring the evolution, ecology, and conservation of butterflies.

Integrated Analysis of Transcriptome and Proteome to Reveal Pupal Color Switch in Papilio xuthus Butterflies.

Pupal color polyphenism in Papilio butterflies, including green, intermediate, or brown, is an excellent study system for understanding phenotypic plasticity. Previous studies suggested that development of brown pupae may be controlled by a hormone called pupal-cuticle-melanizing-hormone (PCMH) which is synthesized and secreted from brain-suboesophageal ganglion and prothoracic ganglion complexes (Br-SG-TG1) during the pre-pupa stage. However, detailed molecular mechanisms of neuroendocrine regulation in pupal color development remain unknown. In this study, we integrated the expression profiles of transcriptome and proteome at pre-pupa stages [2 h after gut purge (T1) and 3 h after forming the garter around the body (T2)] and pigmentation stages [10 h after ecdysis (T3) and 24 h after ecdysis (T4)] to identify important genes and pathways underlying the development of green and brown pupa in the swallowtail butterfly Papilio xuthus. Combined comparisons of each developmental stage and each tissue under green and brown conditions, a total of 1042 differentially expressed genes (DEGs) and 430 different abundance proteins (DAPs) were identified. Weighted gene co-expression network analysis (WGCNA) and enrichment analysis indicate that these DEGs were mainly related to oxidation-reduction, structural constituent of cuticle, and pigment binding. Soft clustering by Mfuzz and enrichment analysis indicate that these DAPs are mainly involved in tyrosine metabolism, insect hormone biosynthesis, and melanogenesis. By homologous alignment, we further identified those genes encoding neuropeptides (51), GPCRs (116), G-proteins (8), cuticular proteins (226), chitinases (16), and chitin deacetylases (8) in the whole genome of P. xuthus and analyzed their expression profiles. Although we identified no gene satisfying with hypothesized expression profile of PCMH, we found some genes in the neuropeptide cascade showed differentially expressed under two pupal color conditions. We also found that Toll signaling pathway genes, juvenile hormone (JH) related genes, and multiple cuticular proteins play important roles in the formation of selective pupal colors during the prepupal-pupal transition. Our data also suggest that both green and brown pupa include complex pigment system that is regulated by genes involved in black, blue, and yellow pigments. Our results provide important insights into the evolution of pupal protective colors among swallowtail butterflies.

Derivedness Index for Estimating Degree of Phenotypic Evolution of Embryos: A Study of Comparative Transcriptomic Analyses of Chordates and Echinoderms.

Species retaining ancestral features, such as species called living fossils, are often regarded as less derived than their sister groups, but such discussions are usually based on qualitative enumeration of conserved traits. This approach creates a major barrier, especially when quantifying the degree of phenotypic evolution or degree of derivedness, since it focuses only on commonly shared traits, and newly acquired or lost traits are often overlooked. To provide a potential solution to this problem, especially for inter-species comparison of gene expression profiles, we propose a new method named "derivedness index" to quantify the degree of derivedness. In contrast to the conservation-based approach, which deals with expressions of commonly shared genes among species being compared, the derivedness index also considers those that were potentially lost or duplicated during evolution. By applying our method, we found that the gene expression profiles of penta-radial phases in echinoderm tended to be more highly derived than those of the bilateral phase. However, our results suggest that echinoderms may not have experienced much larger modifications to their developmental systems than chordates, at least at the transcriptomic level. In vertebrates, we found that the mid-embryonic and organogenesis stages were generally less derived than the earlier or later stages, indicating that the conserved phylotypic period is also less derived. We also found genes that potentially explain less derivedness, such as Hox genes. Finally, we highlight technical concerns that may influence the measured transcriptomic derivedness, such as read depth and library preparation protocols, for further improvement of our method through future studies. We anticipate that this index will serve as a quantitative guide in the search for constrained developmental phases or processes.

Chromatin accessibility profiling provides insights into larval cuticle color and adult longevity in butterflies.

Butterflies are diverse in virtually all aspects of their ontogeny, including morphology, life history, and behavior. However, the developmental regulatory mechanisms underlying the important phenotypic traits of butterflies at different developmental stages remain unknown. Here, we investigated the developmental regulatory profiles of butterflies based on transposase accessible chromatin sequencing (ATAC-seq) at three developmental stages in two representative species ( Papilio xuthus and Kallima inachus). Results indicated that 15%-47% of open chromatin peaks appeared in associated genes located 3 kb upstream (i.e., promoter region) of their transcription start site (TSS). Comparative analysis of the different developmental stages indicated that chromatin accessibility is a dynamic process and associated genes with differentially accessible (DA) peaks show functions corresponding to their phenotypic traits. Interestingly, the black color pattern in P. xuthus 4th instar larvae may be attributed to promoter peak-related genes involved in the melanogenesis pathway. Furthermore, many longevity genes in 5th instar larvae and pupae showed open peaks 3 kb upstream of their TSS, which may contribute to the overwintering diapause observed in K. inachus adults. Combined with RNA-seq analysis, our data demonstrated that several genes enriched in the melanogenesis and longevity pathways also exhibit higher expression, confirming that the expression of genes may be closely related to their phenotypic traits. This study offers new insights into larval cuticle color and adult longevity in butterflies and provides a resource for investigating the developmental regulatory mechanisms underlying butterfly ontogeny.

Large-scale sequencing of flatfish genomes provides insights into the polyphyletic origin of their specialized body plan.

The evolutionary and genetic origins of the specialized body plan of flatfish are largely unclear. We analyzed the genomes of 11 flatfish species representing 9 of the 14 Pleuronectiforme families and conclude that Pleuronectoidei and Psettodoidei do not form a monophyletic group, suggesting independent origins from different percoid ancestors. Genomic and transcriptomic data indicate that genes related to WNT and retinoic acid pathways, hampered musculature and reduced lipids might have functioned in the evolution of the specialized body plan of Pleuronectoidei. Evolution of Psettodoidei involved similar but not identical genes. Our work provides valuable resources and insights for understanding the genetic origins of the unusual body plan of flatfishes.

Identification of Key Genes for the Ultrahigh Yield of Rice Using Dynamic Cross-tissue Network Analysis.

Significantly increasing crop yield is a major and worldwide challenge for food supply and security. It is well-known that rice cultivated at Taoyuan in Yunnan of China can produce the highest yield worldwide. Yet, the gene regulatory mechanism underpinning this ultrahigh yield has been a mystery. Here, we systematically collected the transcriptome data for seven key tissues at different developmental stages using rice cultivated both at Taoyuan as the case group and at another regular rice planting place Jinghong as the control group. We identified the top 24 candidate high-yield genes with their network modules from these well-designed datasets by developing a novel computational systems biology method, i.e., dynamic cross-tissue (DCT) network analysis. We used one of the candidate genes, OsSPL4, whose function was previously unknown, for gene editing experimental validation of the high yield, and confirmed that OsSPL4 significantly affects panicle branching and increases the rice yield. This study, which included extensive field phenotyping, cross-tissue systems biology analyses, and functional validation, uncovered the key genes and gene regulatory networks underpinning the ultrahigh yield of rice. The DCT method could be applied to other plant or animal systems if different phenotypes under various environments with the common genome sequences of the examined sample. DCT can be downloaded from https://github.com/ztpub/DCT.

Draft Genome of the European Mouflon (Ovis orientalis musimon).

Mouflon (Ovis orientalis) with its huge and beautiful horns is considered as one of the ancestors of domesticated sheep. The European mouflon (Ovis orientalis musimon) is in the Asiatic mouflon (O. orientalis) clade. In order to provide novel genome information for mouflon, moreover promote genetic analysis of genus Ovis both domestic and wild, we propose to sequence the mouflon genome. We assembled the highly heterozygous mouflon genome based on Illumina HiSeq platform using the next-generation sequencing technology. Finally, the draft genome we accessed approximately 2.69 Gb (42.15% GC), while N50 sizes of contig and scaffold are 110.1 kb and 10.4 Mb, respectively. The contiguity of this assembly is obviously better than earlier versions. Further analyses predicted 20,814 protein-coding genes in the mouflon genome and 12,390 shared gene families among bovine species. It is estimated that the divergence time between O. orientalis musimon and Ovis aries was 7.6 million years ago. The draft mouflon genome assembly will provide data support and theoretical basis for various investigations of the genus Ovis species in future.

Chromosome-level reference genome assembly and gene editing of the dead-leaf butterfly Kallima inachus.

The leaf resemblance of Kallima (Nymphalidae) butterflies is an important ecological adaptive mechanism that increases their survival. However, the genetic mechanism underlying ecological adaptation remains unclear owing to a dearth of genomic information. Here, we determined the karyotype (n = 31) of the dead-leaf butterfly Kallima inachus, and generated a high-quality, chromosome-level assembly (568.92 Mb; contig N50: 19.20 Mb). We also identified candidate Z and W chromosomes. To our knowledge, this is the first study to report on these aspects of this species. In the assembled genome, 15,309 protein-coding genes and 49.86% repeat elements were annotated. Phylogenetic analysis showed that K. inachus diverged from Melitaea cinxia (no leaf resemblance), both of which are in Nymphalinae, around 40 million years ago. Demographic analysis indicated that the effective population size of K. inachus decreased during the last interglacial period in the Pleistocene. The wings of adults with the pigmentary gene ebony knocked out using CRISPR/Cas9 showed phenotypes in which the orange dorsal region and entire ventral surface darkened, suggesting its vital role in the ecological adaption of dead-leaf butterflies. Our results provide important genome resources for investigating the genetic mechanism underlying protective resemblance in dead-leaf butterflies and insights into the molecular basis of protective coloration.