Background/Objectives: Pepper (Capsicum annuum L.) is a widely grown vegetable and spice crop worldwide. This study aims to reveal the differences of metabolites among pepper accessions from different regions and explore candidate genes related to metabolites of pepper. Methods: The metabolome and transcriptome of 36 pepper accessions were determined by widely targeted metabolomics analysis and RNA sequencing technology, and the differential metabolites and differential genes among C. annuum from four important pepper production and consumption provinces of China, Hunan, Guizhou, Yunnan and Sichuan, were analyzed. Results: Flavonoids are the main characteristic metabolites that distinguish pepper accessions from Yun_Gui_Chuan Group and Hunan Group. The aglycones of characteristic flavonoids in each group are different; in Yun_Gui_Chuan Group mainly are luteolin, quercetin, chrysoeriol and isorhamnetin; in Hunan Group mainly are apigenin. Transcriptome data showed that two genes related to flavonoid 3′-monooxygenase differed significantly between the two groups of chili peppers, and we speculated that they may be the core enzymes regulating their flavonoid profile. And an SNP mutation located in gene Cgla06g001871 showed a strong correlation with pepper accessions from Yun_Gui_Chuan Group, which can be used as a DNA marker to identify pepper accessions from Yun_Gui_Chuan Group, and provide strong support for regional specialty variety conservation. In addition, we also analyzed the metabolites related to the taste and nutrition of pepper accessions in the four provinces, and the results showed that the sugar content of pepper accessions from Guizhou was low and the capsaicinoids content of pepper accessions from Sichuan was low, while no significant difference was found in acid and vitamin contents among pepper accessions from the four provinces. Conclusions: The metabolome and transcriptome of 36 pepper accessions from four important pepper production and consumption provinces of China were determined, and the characteristic metabolites and expressed genes of pepper accessions from each province were analyzed.

Pepper (Capsicum spp.) is a widely consumed vegetable with exceptionally large genomes in Solanaceae, yet its genomic evolutionary history remains largely unknown. Here we present 11 high-quality Capsicum genome assemblies, including two gap-free genomes, covering four wild and all five domesticated pepper species. We reconstructed the ancestral karyotype and inferred the evolutionary trajectory of peppers. The expanded and variable genome sizes were attributed to differential transposable element accumulations, which shaped 3D chromatin architecture and introduced mutations associated with traits such as fruit orientation and colour. Using a chromatin accessibility atlas of Capsicum, we highlight the influence of transposable elements on regulatory element evolution. Furthermore, by constructing a haploblock map of 124 pepper core germplasms, we uncover frequent introgressions that facilitate the formation of sweet blocky pepper and the acquisition of important traits such as resistance to pepper mild mottle virus. These findings on the genomic and functional evolution of Capsicum will benefit pepper breeding. This study presents 11 telomere-to-telomere genomes of wild and domesticated pepper, highlights how transposable elements have shaped the evolution of genome structure and regulatory elements and identifies structural variations and introgressions associated with key traits in cultivated pepper.

Petota includes more than 100 species (wild and cultivated), presenting a rich variety of corolla colors and associated traits. This variability provides important opportunities for investigating the differentiation of orthologous genes’ functions and their evolutionary pathways. However, the genetic underpinnings of this diversity in corolla colors are still to be further explored. In our previous study, a locus responsible for corolla color in potato was mapped to a 740 kb region on chromosome 10, which contains the AN2 gene previously identified as a regulation gene for corolla color. In the present study, this locus was further refined to a 380 kb interval through recombinant analysis. Targeted analysis of anthocyanidins and carotenoids revealed that purple corollas exhibit significantly higher levels of petunidin and delphinidin, while showing significantly lower levels of lutein and β-carotene compared to yellow corollas. Transcriptome and qRT-PCR analysis indicated that StMYB180, rather than AN2, is the candidate gene responsible for regulating coloration, specifically on the abaxial side of the corolla in potato. Expression analysis revealed that StMYB180 is exclusively highly expressed in corolla and leaf tissues, with purple coloration on the abaxial side of both corollas and leaves. Phylogenetic analysis further suggests that corolla color-regulatory genes may be closely tied to the origin and evolutionary trajectory of potato species. This study provides valuable insights into the regulation of tissue-specific expression of anthocyanin biosynthesis in potato and lays the groundwork for understanding the evolution of orthologous genes in the Petota section.

Background: Pepper (Capsicum annuum L.) is a widely cultivated vegetable crop worldwide, with its rich fruit colors providing unique visual traits and economic value. This study investigated the genetic basis of the immature green fruit color by constructing a F2 segregating population derived from a cross between yellow fruit C20 and green fruit C62 parent lines. Methods: Bulked segregant analysis sequencing (BSA-seq) was performed to identify genomic regions associated with fruit color. Candidate genes were pinpointed through functional annotation and genetic variation analysis, supported by SNP markers, genotype analysis, and transcriptome profiling. Results: Two genomic regions associated with fruit color were identified on chromosomes 1 (14.55-20.85 Mb) and 10 (10.15-22.85 Mb), corresponding to previously reported loci pc1 and pc10.1. Two chlorophyll synthesis-related genes, CaAPRR2 and CaGLK2, were identified as candidate regulators of fruit color. Mutations in these genes include a premature stop codon in both CaGLK2 and CaAPRR2. The mutation of CaAPRR2 and CaGLK2 jointly regulate the yellow fruit trait in pepper, with CaGLK2 being the major gene and CaAPRR2 being the minor gene. Transcriptome analysis showed that the expression levels of the two genes increased during the green ripening stage of the parent fruits, with higher expression levels of CaGLK2. Conclusions: This study identifies CaGLK2 and CaAPRR2 as key regulators of immature green fruit color in pepper, with CaGLK2 playing a predominant role. These findings provide a theoretical foundation and data support for elucidating the molecular regulatory mechanisms of fruit color and advancing marker-assisted breeding in pepper.

Polyploidization plays a crucial role in plant evolution and is becoming increasingly important in breeding. Structural variations and epigenomic repatterning have been observed in synthetic polyploidizations. However, the mechanisms underlying the occurrence and their effects on gene expression and phenotype remain unknown. Here, we investigated genome-wide large deletion/duplication regions (DelDups) and genomic methylation dynamics in leaf organs of progeny from the first eight generations of synthetic tetraploids derived from Chinese cabbage (Brassica rapa L. ssp. pekinensis) and cabbage (Brassica oleracea L. var. capitata). One- or two-copy DelDups, with a mean size of 5.70 Mb (400 kb to 65.85 Mb), occurred from the first generation of selfing and thereafter. The duplication of a fragment in one subgenome consistently coincided with the deletion of its syntenic fragment in the other subgenome, and vice versa, indicating that these DelDups were generated by homoeologous exchanges (HEs). Interestingly, the larger the genomic syntenic region, the higher the frequency of DelDups, further suggesting that the pairing of large homoeologous fragments is crucial for HEs. Moreover, we found that the active transcription of continuously distributed genes in local regions is positively associated with the occurrence of HE breakpoints. In addition, the expression of genes within DelDups exhibited a dosage effect, and plants with extra parental genomic fragments generally displayed phenotypes biased toward the corresponding parent. Genome-wide methylation fluctuated remarkably, which did not clearly affect gene expression on a large scale. Our findings provide insights into the early evolution of polyploid genomes, offering valuable knowledge for polyploidization-based breeding. A comprehensive multi-omics landscape across synthetic Brassica napus tetraploids reveals active transcription of homoeologous exchange-related genes.

SUMMARYDeep learning offers new approaches to investigate the mechanisms underlying complex biological phenomena, such as subgenome dominance. Subgenome dominance refers to the dominant expression and/or biased fractionation of genes in one subgenome of allopolyploids, which has shaped the evolution of a large group of plants. However, the underlying cause of subgenome dominance remains elusive. Here, we adopt deep learning to construct two convolutional neural network (CNN) models, binary expression model (BEM) and homoeolog contrast model (HCM), to investigate the mechanism underlying subgenome dominance using DNA sequence and methylation sites. We apply these CNN models to analyze three representative polyploidization systems, Brassica, Gossypium, and Cucurbitaceae, each with available ancient and neo/synthetic polyploidized genomes. The BEM shows that DNA sequence of the promoter region can accurately predict whether a gene is expressed or not. More importantly, the HCM shows that the DNA sequence of the promoter region predicts dominant expression status between homoeologous gene pairs retained from ancient polyploidizations, thus predicting subgenome dominance associated with these events. However, HCM fails to predict gene expression dominance between new homoeologous gene pairs arising from the neo/synthetic polyploidizations. These results are consistent across the three plant polyploidization systems, indicating broad applicability of our models. Furthermore, the two models based on methylation sites produce similar results. These results show that subgenome dominance is associated with long‐term sequence differentiation between the promoters of homoeologs, suggesting that subgenome expression dominance precedes and is the driving force or even the determining factor for sequence divergence between subgenomes following polyploidization.

Constructing inbred lines for self-incompatible species and species with long generation times is challenging, making the use of F1 outcross/segregating populations the main strategy for genetic studies of such species. However, there is a lack of dedicated algorithms/tools for rapid quantitative trait locus (QTL) mapping using the F1 populations. To this end, we have designed and developed an algorithm/tool called OcBSA specifically for QTL mapping of F1 populations. OcBSA transforms the four-haplotype inheritance problem from the two heterozygous diploid parents of the F1 population into the two-haplotype inheritance problem common in current genetic studies by removing the two haplotypes from the heterozygous parent that do not contribute to phenotype segregation in the F1 population. Testing of OcBSA on 1800 simulated F1 populations demonstrated its advantages over other currently available tools in terms of sensitivity and accuracy. In addition, the broad applicability of OcBSA was validated by QTL mapping using seven reported F1 populations of apple, pear, peach, citrus, grape, tea, and rice. We also used OcBSA to map the QTL for flower color in a newly constructed F1 population of potato generated in this study. The OcBSA mapping result was verified by the insertion or deletion markers to be consistent with a previously reported locus harboring the ANTHOCYANIN 2 gene, which regulates potato flower color. Taken together, these results highlight the power and broad utility of OcBSA for QTL mapping using F1 populations and thus a great potential for functional gene mining in outcrossing species. For ease of use, we have developed both Windows and Linux versions of OcBSA, which are freely available at: https://gitee.com/Bioinformaticslab/OcBSA.

Brassica oleracea , globally cultivated for its vegetable crops, consists of very diverse morphotypes, characterized by specialized enlarged organs as harvested products. This makes B. oleracea an ideal model for studying rapid evolution and domestication. We constructed a B. oleracea pan-genome from 27 high-quality genomes representing all morphotypes and their wild relatives. We identified structural variations (SVs) among these genomes and characterized these in 704 B. oleracea accessions using graph-based genome tools. We show that SVs exert bidirectional effects on the expression of numerous genes, either suppressing through DNA methylation or promoting probably by harboring transcription factor-binding elements. The following examples illustrate the role of SVs modulating gene expression: SVs promoting BoPNY and suppressing BoCKX3 in cauliflower/broccoli, suppressing BoKAN1 and BoACS4 in cabbage and promoting BoMYBtf in ornamental kale. These results provide solid evidence for the role of SVs as dosage regulators of gene expression, driving B. oleracea domestication and diversification.

The Solanaceae family contains many agriculturally important crops, including tomato, potato, pepper, and tobacco, as well as other species with potential for agricultural development, such as the orphan crops groundcherry, wolfberry, and pepino. Research progress varies greatly among these species, with model crops like tomato being far ahead. This disparity limits the broader agricultural application of other Solanaceae species. In this study, we constructed an interspecies pan-genome for the Solanaceae family and identified various gene retention patterns. Our findings reveal that the activity of specific transposable elements is closely associated with gene fractionation and transposition. The pan-genome was further resolved at the level of T subgenomes, which were generated by Solanaceae-specific paleo-hexaploidization (T event). We demonstrate substantial gene fractionation (loss) and divergence events following ancient duplications. For example, all class A and E flower model genes in Solanaceae originated from two tandemly duplicated genes, which expanded through the γ and T events before fractionating into 10 genes in tomato, each acquiring distinct functions critical for fruit development. Based on these results, we developed the Solanaceae Pan-Genome Database (SolPGD, http://www.bioinformaticslab.cn/SolPGD), which integrates datasets from both inter- and intra-species pan-genomes of Solanaceae. These findings and resources will facilitate future studies of solanaceous species, including orphan crops.

Phosphorus is critical for plant growth but often becomes less accessible due to its precipitation with cations in soil. Fabaceae, a diverse plant family, exhibits robust adaptability and includes species like Lupinus albus, known for its efficient phosphorus utilization via cluster roots. Here, we systematically identified phosphorus-utilization-efficiency (PUE) gene families across 35 Fabaceae species, highlighting significant gene amplification in PUE pathways in Fabaceae. Different PUE pathways exhibited variable amplification, evolution, and retention patterns among various Fabaceae crops. Additionally, the number of homologous genes of the root hair development gene RSL2 in L. albus was far more than that in other Fabaceae species. Multiple copies of the RSL2 gene were amplified and retained in L. albus after whole genome triplication. The gene structure and motifs specifically retained in L. albus were different from homologous genes in other plants. Combining transcriptome analysis under low-phosphorus treatment, it was found that most of the homologous genes of RSL2 in L. albus showed high expression in the cluster roots, suggesting that the RSL2 gene family plays an important role in the adaptation process of L. albus to low-phosphorus environments and the formation of cluster roots.

Brassica oleracea has been developed into many important crops, including cabbage, kale, cauliflower, broccoli and so on. The genome and gene annotation of cabbage (cultivar JZS), a representative morphotype of B. oleracea, has been widely used as a common reference in biological research. Although its genome assembly has been updated twice, the current gene annotation still lacks information on untranslated regions (UTRs) and alternative splicing (AS). Here, we constructed a high-quality gene annotation (JZSv3) using a full-length transcriptome acquired by nanopore sequencing, yielding a total of 59 452 genes and 75 684 transcripts. Additionally, we re-analyzed the previously reported transcriptome data related to the development of different tissues and cold response using JZSv3 as a reference, and found that 3843 out of 11 908 differentially expressed genes (DEGs) underwent AS during the development of different tissues and 309 out of 903 cold-related genes underwent AS in response to cold stress. Meanwhile, we also identified many AS genes, including BolLHCB5 and BolHSP70, that displayed distinct expression patterns within variant transcripts of the same gene, highlighting the importance of JZSv3 as a pivotal reference for AS analysis. Overall, JZSv3 provides a valuable resource for exploring gene function, especially for obtaining a deeper understanding of AS regulation mechanisms.

Polyploidization is important to the evolution of plants. Subgenome dominance is a distinct phenomenon associated with most allopolyploids. A gene on the dominant subgenome tends to express to higher RNA levels in all organs as compared to the expression of its syntenic paralogue (homoeolog). The mechanism that underlies the formation of subgenome dominance remains unknown, but there is evidence for the involvement of transposon/DNA methylation density differences nearby the genes of parents as being causal. The subgenome with lower density of transposon and methylation near genes is positively associated with subgenome dominance. Here, we generated eight generations of allotetraploid progenies from the merging of parental genomes Brassica rapa and Brassica oleracea. We found that transposon/methylation density differ near genes between the parental (rapa:oleracea) existed in the wide hybrid, persisted in the neotetraploids (the synthetic Brassica napus), but these neotetraploids expressed no expected subgenome dominance. This absence of B. rapa vs. B. oleracea subgenome dominance is particularly significant because, while there is no negative relationship between transposon/methylation level and subgenome dominance in the neotetraploids, the more ancient parental subgenomes for all Brassica did show differences in transposon/methylation densities near genes and did express, in the same samples of cells, biased gene expression diagnostic of subgenome dominance. We conclude that subgenome differences in methylated transposon near genes are not sufficient to initiate the biased gene expressions defining subgenome dominance. Our result was unexpected, and we suggest a "nuclear chimera" model to explain our data.

Dear Editor, Potato is a vital food security crop and is ranked as the world's third most important food crop after rice and wheat.In 2011,the first genome assembly of a doubled monoploid potato DM1-3 516 R44(DM)was released(Potato Genome Sequencing Consortium,2011),which has been widely used as one of the most popular reference genomes in the last decade and served as a valuable resource in plant genomics and potato genetics community(Leisner et al.,2018;Yang et al.,2020;Zheng et al.,2020).

Polyploidization is important in plant evolution and is becoming increasingly important in crop breeding and material creation. Studies have provided evidence for structural variations and epigenomic repatterning in synthetic polyploidizations, but the relationships between structural and epigenomic variations, as well as their effects on gene expression and phenotypic variations are unknown. Here, we investigated the genome-wide copy number variations of fragments (CVFs) and genomic methylation dynamics, in the leaf organ as a representative, of progenies from eight generations that derived from the synthetic tetraploidization between Brassica rapa and Brassica oleracea. We found that half or complete deletion/duplication of fragments ranging in size from 400 kb to 65.85 Mb, with a mean size of 5.70 Mb, occurred frequently from the first generation of selfing and thereafter. The genes located in these CVFs expressed at levels expected for a positive dosage effect, as indicated by the positive association between expression and the copy number of these genes. Plants containing these CVFs also showed distinct phenotypic variations. The whole genome methylation level experienced significant fluctuations in different generations and eventually decreased in the latter generations. Moreover, the CVFs did not show methylation changes from other individuals of the same generation, and the local regions with methylation alterations did not affect gene expression. Together, the dosage effect of CVFs on gene expression was likely the causal factor underlying phenotypic variations in synthetic tetraploids. Our findings provide new insights into the early evolution of polyploid genomes and guide the use of synthetic polyploidizations in breeding.### Competing Interest StatementThe authors have declared no competing interest.

ABSTRACTPolyploidization is important in plant evolution and is becoming increasingly important in crop breeding and material creation. Studies have provided evidence for structural variations and epigenomic repatterning in synthetic polyploidizations, but the relationships between structural and epigenomic variations, as well as their effects on gene expression and phenotypic variations are unknown. Here, we investigated the genome-wide large deletion/duplication regions (DelDups) and genomic methylation dynamics, in the leaf organ as a representative, of progenies from eight generations that derived from the synthetic tetraploidization betweenBrassica rapaandBrassica oleracea. We found that half or complete deletion/duplication of fragments ranging in size from 400 kb to 65.85 Mb, with a mean size of 5.70 Mb, occurred frequently from the first generation of selfing and thereafter. The genes located in these DelDups expressed at levels expected for a positive dosage effect, as indicated by the positive association between expression and the copy number of these genes. Plants containing these DelDups also showed distinct phenotypic variations. The whole genome methylation level experienced significant fluctuations in different generations and eventually decreased in the latter generations. Moreover, the DelDups did not show methylation changes from other individuals of the same generation, and the local regions with methylation alterations did not affect gene expression. Our findings provide new insights into the early evolution of polyploid genomes and guide the use of synthetic polyploidizations in breeding.

In order to trace the evolution of chromosomes of Solanaceae species，a systematic comparative genomic analysis was carried out on seven Solanaceae species. Using the genome of model plant tomato as a reference template，we comprehensively identified the collinear relationship among seven species of Solanaceae，and established the genome fragment unit system of Solanaceae. Based on this system，we further reconstructed the ancestral genome of Solanaceae with 12 chromosomes. The results showed that the ancestral genome gradually formed the current genome structure of Solanaceae mainly through translocation and inversion of genome fragments，and these rearrangement events mostly occurred in the regions of centromere and other repeated sequences were enriched. This study provided an important theoretical reference for further understanding the genome evolution and species differentiation of Solanaceae and other plants.

Whole genome duplication (WGD) and tandem duplication (TD) are important modes of gene amplification and functional innovation, and they are common in plant genome evolution. We analyzed the genomes of three Solanaceae species (Solanum lycopersicum, Capsicum annuum, and Petunia inflata), which share a common distant ancestor with Vitis vinifera, Theobroma cacao, and Coffea canephora but have undergone an extra whole genome triplication (WGT) event. The analysis was used to investigate the phenomenon of tandem gene evolution with (S. lycopersicum) or without WGT (V. vinifera). Among the tandem gene arrays in these genomes, we found that V. vinifera, which has not experienced the WGT event, retained relatively more and larger tandem duplicated gene (TDG) clusters than the Solanaceae species that experienced the WGT event. Larger TDG clusters tend to be derived from older TD events, so this indicates that continuous TDGs (absolute dosage) accumulated during long-term evolution. In addition, WGD and TD show a significant bias in the functional categories of the genes retained. WGD tends to retain dose-sensitive genes related to biological processes, including DNA-binding and transcription factor activity, while TD tends to retain genes involved in stress resistance. WGD and TD also provide more possibilities for gene functional innovation through gene fusion and fission. The TDG cluster containing the tomato fusarium wilt resistance gene I3 contains 15 genes, and one of these genes, Solyc07g055560, has undergone a fusion event after the duplication events. These data provide evidence that helps explain the new functionalization of TDGs in adapting to environmental changes.

Orychophragmus violaceus, referred to as "eryuelan" (February orchid) in China, is an early-flowering ornamental plant. The high oil content and abundance of unsaturated fatty acids in O. violaceus seeds make it a potential high-quality oilseed crop. Here, we generated a whole-genome assembly for O. violaceus using Nanopore and Hi-C sequencing technologies. The assembled genome of O. violaceus was ∼1.3 Gb in size, with 12 pairs of chromosomes. Through investigation of ancestral genome evolution, we determined that the genome of O. violaceus experienced a tetraploidization event from a diploid progenitor with the translocated proto-Calepineae karyotype. Comparisons between the reconstructed subgenomes of O. violaceus identified indicators of subgenome dominance, indicating that subgenomes likely originated via allotetraploidy. O. violaceus was phylogenetically close to the Brassica genus, and tetraploidy in O. violaceus occurred approximately 8.57 million years ago, close in time to the whole-genome triplication of Brassica that likely arose via an intermediate tetraploid lineage. However, the tetraploidization in Orychophragmus was independent of the hexaploidization in Brassica, as evidenced by the results from detailed phylogenetic analyses and comparisons of the break and fusion points of ancestral genomic blocks. Moreover, identification of multi-copy genes regulating the production of high-quality oil highlighted the contributions of both tetraploidization and tandem duplication to functional innovation in O. violaceus. These findings provide novel insights into the polyploidization evolution of plant species and will promote both functional genomic studies and domestication/breeding efforts in O. violaceus.

SUMMARYLeaf heading is an important and economically valuable horticultural trait in many vegetables. The formation of a leafy head is a specialized leaf morphogenesis characterized by the emergence of the enlarged incurving leaves. However, the transcriptional regulation mechanisms underlying the transition to leaf heading remain unclear. We carried out large‐scale time‐series transcriptome assays covering the major vegetative growth phases of two headingBrassica crops, Chinese cabbage and cabbage, with the non‐heading morphotype Taicai as the control. A regulatory transition stage that initiated the heading process is identified, accompanied by a developmental switch from rosette leaf to heading leaf in Chinese cabbages. This transition did not exist in the non‐heading control. Moreover, we reveal that the heading transition stage is also conserved in the cabbage clade. Chinese cabbage acquired through domestication a leafy head independently from the origins of heading in other cabbages; phylogenetics supports that the ancestor of all cabbages is non‐heading. The launch of the transition stage is closely associated with the ambient temperature. In addition, examination of the biological activities in the transition stage identified the ethylene pathway as particularly active, and we hypothesize that this pathway was targeted for selection for domestication to form the heading trait specifically in Chinese cabbage. In conclusion, our findings on the transcriptome transition that initiated the leaf heading in Chinese cabbage and cabbage provide a new perspective for future studies of leafy head crops.