Wall-associated kinase BrWAK1 confers resistance to downy mildew in Brassica rapa.

The plant cell wall is the first line of defence against physical damage and pathogen attack. Wall-associated kinase (WAK) has the ability to perceive the changes in the cell wall matrix and transform signals into the cytoplasm, being involved in plant development and the defence response. Downy mildew, caused by Hyaloperonospora brassicae, can result in a massive loss in Chinese cabbage (Brassica rapa L. ssp. pekinensis) production. Herein, we identified a candidate resistant WAK gene, BrWAK1, in a major resistant quantitative trait locus, using a double haploid population derived from resistant inbred line T12-19 and the susceptible line 91-112. The expression of BrWAK1 could be induced by salicylic acid and pathogen inoculation. Expression of BrWAK1 in 91-112 could significantly enhance resistance to the pathogen, while truncating BrWAK1 in T12-19 increased disease susceptibility. Variation in the extracellular galacturonan binding (GUB) domain of BrWAK1 was found to mainly confer resistance to downy mildew in T12-19. Moreover, BrWAK1 was proved to interact with BrBAK1 (brassinosteroid insensitive 1 associated kinase), resulting in the activation of the downstream mitogen-activated protein kinase (MAPK) cascade to trigger the defence response. BrWAK1 is the first identified and thoroughly characterized WAK gene conferring disease resistance in Chinese cabbage, and the plant biomass is not significantly influenced by BrWAK1, which will greatly accelerate Chinese cabbage breeding for downy mildew resistance.

Promoter variations in a homeobox gene, BrLMI1, contribute to leaf lobe formation in Brassica rapa ssp. chinensis Makino.

Key message BrLMI1 is a positive regulatory factor of leaf lobe formation in non-heading Chinese cabbage, and cis-regulatory variations lead to the phenotype of lobed or entire leaf margins.Abstract Leaves are the main consumed organ in leafy non-heading Chinese cabbage (Brassica rapa L. ssp. chinensis Makino), and the shape of the leaves is an important economic trait. However, the molecular regulatory mechanism underlying the lobed-leaf trait in non-heading Chinese cabbage remains unclear. Here, we identified a stable incompletely dominant major locus, qLLA10, for lobed leaf formation in non-heading Chinese cabbage. Based on map-based cloning strategies, BrLMI1, a LATE MERISTEM IDENTITY1 (LMI1)-like gene, was predicted as the candidate gene for qLLA10. Genotyping analysis showed that promoter variations of BrLMI1 in the two parents are responsible for elevating the expression in the lobed-leaf parent and ultimately causing the difference in leaf shape between the two parents, and the promoter activity of BrLMI1 was significantly affected by the promoter variations. BrLMI1 was exclusively localized in the nucleus and expressed mainly at the tip of each lobe. Leaf lobe development was perturbed in BrLMI1-silenced plants produced by virus-induced gene silencing assays, and ectopic overexpression of BrLMI1 in Arabidopsis led to deeply lobed leaves never seen in the wild type, which indicates that BrLMI1 is required for leaf lobe formation in non-heading Chinese cabbage. These findings suggested that BrLMI1 is a positive regulatory factor of leaf lobe formation in non-heading Chinese cabbage and that cis-regulatory variations lead to the phenotype of lobed or entire leaf margins, thus providing a theoretical basis for unraveling the molecular mechanism underlying the lobed leaf phenotype in Brassica crops.

The chromosome-scale assembly of endive (Cichorium endivia) genome provides insights into the sesquiterpenoid biosynthesis.

Endive (Cichorium endivia L.) is a leafy vegetable in the Asteraceae family. Sesquiterpene lactones (STLs) in endive leaves bring a bitter taste that varies between varieties. Despite their importance in breeding varieties with unique flavours, sesquiterpenoid biosynthesis pathways in endive are poorly understood. We assembled a chromosome-scale endive genome of 641 Mb with a contig N50 of 5.16 Mb and annotated 46,711 protein-coding genes. Several gene families, especially terpene synthases (TPS) genes, expanded significantly in the C. endivia genome. STLs biosynthesis-related genes and TPS genes in more bitter varieties have shown a higher level of expression, which could be attributed to genomic variations. Our results penetrate the origin and diversity of bitter taste and facilitate the molecular breeding of endive varieties with unique bitter tastes. The high-quality endive assembly would provide a reference genome for studying the evolution and diversity of Asteraceae.

BrrA02.LMI1 Encodes a Homeobox Protein That Affects Leaf Margin Development in Brassica rapa.

Leaf margin morphology is an important quality trait affecting the commodity and environmental adaptability of crops. Brassica rapa is an ideal research material for exploring the molecular mechanisms underlying leaf lobe development. Here, we identified BrrA02.LMI1 to be a promising gene underlying the QTL qBrrLLA02 controlling leaf lobe formation in B. rapa, which was detected in our previous study. Sequence comparison analysis showed that the promoter divergences were the most obvious variations of BrrA02.LMI1 between parental lines. The higher expression level and promoter activity of BrrA02.LMI1 in the lobe-leafed parent indicated that promoter variations of BrrA02.LMI1 were responsible for elevating expression and ultimately causing different allele effects. Histochemical GUS staining indicated that BrrA02.LMI1 is mainly expressed at the leaf margin, with the highest expression at the tip of each lobe. Subcellular localization results showed that BrrA02.LMI1 was in the nucleus. The ectopic expression of BrrA02.LMI1 in A. thaliana resulted in a deep leaf lobe in the wild-type plants, and lobed leaf formation was disturbed in BrrA02.LMI11-downregulated plants. Our findings revealed that BrrA02.LMI1 plays a vital role in regulating the formation of lobed leaves, providing a theoretical basis for the selection and breeding of leaf-shape-diverse varieties of B. rapa.

Assembly of the non-heading pak choi genome and comparison with the genomes of heading Chinese cabbage and the oilseed yellow sarson.

Brassica rapa displays a wide range of morphological diversity which is exploited for a variety of food crops. Here we present a high-quality genome assembly for pak choi (Brassica rapa L. subsp. chinensis), an important non-heading leafy vegetable, and comparison with the genomes of heading type Chinese cabbage and the oilseed form, yellow sarson. Gene presence-absence variation (PAV) and genomic structural variations (SV) were identified, together with single nucleotide polymorphisms (SNPs). The structure and expression of genes for leaf morphology and flowering were compared between the three morphotypes revealing candidate genes for these traits in B. rapa. The pak choi genome assembly and its comparison with other B. rapa genome assemblies provides a valuable resource for the genetic improvement of this important vegetable crop and as a model to understand the diversity of morphological variation across Brassica species.

Natural variations of BrHISN2 provide a genetic basis for growth-flavour trade-off in different Brassica rapa subspecies.

Selection for yield during B. rapa breeding may have unintended consequences for other traits, such as flavour. LYH-type (light yellow head) Chinese cabbage (Brassica rapa ssp. pekinensis) and wucai (Brassica rapa L. ssp. chinensis var. rosularis) varieties are becoming popular because of their unique flavour and yellow leaves. However, the molecular mechanism underlying the interplay for these traits remains unknown. We conducted a fine mapping and genome-wide exploration analysis of the leaf yellowing of LYH and wucai, including transgenic plants, to identify causal genes. We identified that BrHISN2, a rate-limiting enzyme in histidine biosynthesis, causes leaf yellowing by destroying LYH chloroplasts. Normal growing Brhisn2 mutant plants became etiolated and senesced at the cotyledon-seedling stage. Sequence variations in the promoter confers cold-dependent expression on BrHISN2, probably resulting in leaf yellowing in LYH and wucai. Insertions of two DRE cis elements and the subsequent recruitment of two CBF2 proteins by the DREs to the promoter provided the cold-induced expression plasticity of BrHISN2 in plants. Both LYH and wucai are farmed in the fall, in which the temperature gradually decreases, therefore the CBF2-BrHISN2 module probably maximises the benefits of gene-environment interaction for breeding. We determined the mechanistic connections of chlorophyll synthesis and the growth-flavour trade-off in these B. rapa varieties.

A histone H4 gene prevents drought-induced bolting in Chinese cabbage by attenuating the expression of flowering genes.

Flowering is an important trait in Chinese cabbage, because premature flowering reduces yield and quality of the harvested products. Water deficit, caused by drought or other environmental conditions, induces early flowering. Drought resistance involves global reprogramming of transcription, hormone signaling, and chromatin modification. We show that a histone H4 protein, BrHIS4.A04, physically interacts with a homeodomain protein BrVIN3.1, which was selected during the domestication of late-bolting Chinese cabbage. Over-expression of BrHIS4.A04 resulted in premature flowering under normal growth conditions, but prevented further premature bolting in response to drought. We show that the expression of key abscisic acid (ABA) signaling genes, and also photoperiodic flowering genes was attenuated in BrHIS4.A04-overexpressing (BrHIS4.A04OE) plants under drought conditions. Furthermore, the relative change in H4-acetylation at these gene loci was reduced in BrHIS4.A04OE plants. We suggest that BrHIS4.A04 prevents premature bolting by attenuating the expression of photoperiodic flowering genes under drought conditions, through the ABA signaling pathway. Since BrHIS4.A04OE plants displayed no phenotype related to vegetative or reproductive development under laboratory-induced drought conditions, our findings contribute to the potential fine-tuning of flowering time in crops through genetic engineering without any growth penalty, although more data are necessary under field drought conditions.

Genome-wide analysis of mRNA and lncRNA expression and mitochondrial genome sequencing provide insights into the mechanisms underlying a novel cytoplasmic male sterility system, BVRC-CMS96, in Brassicarapa.

KEY MESSAGE: Characterization of a novel and valuable CMS system in Brassicarapa. Cytoplasmic male sterility (CMS) is extensively used to produce F1 hybrid seeds in a variety of crops. However, it has not been successfully used in Chinese cabbage (Brassicarapa L. ssp. pekinensis) because of degeneration or temperature sensitivity. Here, we characterize a novel CMS system, BVRC-CMS96, which originated in B.napus cybrid obtained from INRAE, France and transferred by us to B.rapa. Floral morphology and agronomic characteristics indicate that BVRC-CMS96 plants are 100% male sterile and show no degeneration in the BC7 generation, confirming its suitability for commercial use. We also sequenced the BVRC-CMS96 and maintainer line 18BCM mitochondrial genomes. Genomic analyses showed the presence of syntenic blocks and distinct structures between BVRC-CMS96 and 18BCM and the other known CMS systems. We found that BVRC-CMS96 has one orf222 from 'Nap'-type CMS and two copies of orf138 from 'Ogu'-type CMS. We analyzed expression of orf222, orf138, orf261b, and the mitochondrial energy genes (atp6, atp9, and cox1) in flower bud developmental stages S1-S5 and in four floral organs. orf138 and orf222 were both highly expressed in S4, S5-stage buds, calyx, and the stamen. RNA-seq identified differentially expressed mRNAs and lncRNAs (long non-coding RNAs) that were significantly enriched in pollen wall assembly, pollen development, and pollen coat. Our findings suggest that an energy supply disorder caused by orf222/orf138/orf261b may inhibit a series of nuclear pollen development-related genes. Our study shows that BVRC-CMS96 is a valuable CMS system, and our detailed molecular analysis will facilitate its application in Chinese cabbage breeding.

Identification and fine mapping of qSB.A09, a major QTL that controls shoot branching in Brassica rapa ssp. chinensis Makino.

KEY MESSAGE: QTL mapping plus bulked segregant analysis revealed a major QTL for shoot branching in non-heading Chinese cabbage. The candidate gene was then identified using sequence alignment and expression analysis. Shoot branching is a complex quantitative trait that contributes to plant architecture and ultimately yield. Although many studies have examined branching in grain crops, the genetic control of shoot branching in vegetable crops such as Brassica rapa L. ssp. chinensis remains poorly understood. In this study, we used bulked segregant analysis (BSA) of an F2 population to detect a major quantitative trait locus (QTL) for shoot branching, designated shoot branching 9 (qSB.A09) on the long arm of chromosome A09 in Brassica rapa L. ssp. chinensis. In addition, traditional QTL mapping of the F2 population revealed six QTLs in different regions. Of these, the mapping region on chromosome A09 was consistent with the results of BSA-seq analysis, as well as being stable over the 2-year study period, explaining 19.37% and 22.18% of the phenotypic variation across multiple genetic backgrounds. Using extreme recombinants, qSB.A09 was further delimited to a 127-kb genomic region harboring 28 annotated genes. We subsequently identified the GRAS transcription factor gene Bra007056 as a potential candidate gene; Bra007056 is an ortholog of MONOCULM 1 (MOC1), the key gene that controls tillering in rice. Quantitative RT-PCR further revealed that expression of Bra007056 was positively correlated with the shoot branching phenotype. Furthermore, an insertion/deletion marker specific to Bra007056 co-segregated with the shoot branching trait in the F2 populations. Overall, these results provide the basis for elucidating the molecular mechanism of shoot branching in Brassica rapa ssp. chinensis Makino.

Brittle Culm 1 Encodes a COBRA-Like Protein Involved in Secondary Cell Wall Cellulose Biosynthesis in Sorghum.

Plant mechanical strength contributes to lodging resistance and grain yield, making it an agronomically important trait in sorghum (Sorghum bicolor). In this study, we isolated the brittle culm 1 (bc1) mutant and identified SbBC1 through map-based cloning. SbBC1, a homolog of rice OsBC1 and Arabidopsis thaliana AtCOBL4, encodes a COBRA-like protein that exhibits typical structural features of a glycosylphosphatidylinositol-anchored protein. A single-nucleotide mutation in SbBC1 led to reduced mechanical strength, decreased cellulose content, and increased lignin content without obviously altering plant morphology. Transmission electron microscopy revealed reduced cell wall thickness in sclerenchyma cells of the bc1 mutant. SbBC1 is primarily expressed in developing sclerenchyma cells and vascular bundles in sorghum. RNA-seq analysis further suggested a possible mechanism by which SbBC1 mediates cellulose biosynthesis and cell wall remodeling. Our results demonstrate that SbBC1 participates in the biosynthesis of cellulose in the secondary cell wall and affects the mechanical strength of sorghum plants, providing additional genetic evidence for the roles of COBRA-like genes in cellulose biosynthesis in grasses.

Natural variation in a calreticulin gene causes reduced resistance to Ca2+ deficiency-induced tipburn in Chinese cabbage (Brassica rapa ssp. pekinensis).

Tipburn is an irreversible physiological disorder of Chinese cabbage that decreases crop value. Because of a strong environmental component, tipburn-resistant cultivars are the only solution, although tipburn resistance genes are unknown in Chinese cabbage. We studied three populations of Chinese cabbage over four growing seasons under field conditions: (a) 194 diverse inbred lines, (b) a doubled haploid (DH100) population, and (c) an F2 population. The 194 lines were genotyped using single nucleotide polymorphism markers, and genome-wide-association mapping showed that 24 gQTLs were significantly associated with tipburn disease index. Analysis of the DH100 and F2 populations identified a shared tipburn-associated locus, gqbTRA06, that was found to cover the region defined by one of the 24 gQTLs. Of 35 genes predicted in the 0.14-Mb quantitative trait locus region, Bra018575 (calreticulin family protein, BrCRT2) showed higher expression levels during disease development. We cloned the two BrCRT2 alleles from tipburn-resistant (BrCRT2R ) and tipburn-susceptible (BrCRT2S ) lines and identified a 51-bp deletion in BrCRT2S . Overexpression of BrCRT2R increased Ca2+ storage in the Arabidopsis crt2 mutant and also reduced cell death in leaf tips and margins under Ca2+ -depleted conditions. Our results suggest that BrCRT2 is a possible candidate gene for controlling tipburn in Chinese cabbage.

Population Genomics Reveals Low Genetic Diversity and Adaptation to Hypoxia in Snub-Nosed Monkeys.

Snub-nosed monkeys (genus Rhinopithecus) are a group of endangered colobines endemic to South Asia. Here, we re-sequenced the whole genomes of 38 snub-nosed monkeys representing four species within this genus. By conducting population genomic analyses, we observed a similar load of deleterious variation in snub-nosed monkeys living in both smaller and larger populations and found that genomic diversity was lower than that reported in other primates. Reconstruction of Rhinopithecus evolutionary history suggested that episodes of climatic variation over the past 2 million years, associated with glacial advances and retreats and population isolation, have shaped snub-nosed monkey demography and evolution. We further identified several hypoxia-related genes under selection in R. bieti (black snub-nosed monkey), a species that exploits habitats higher than any other nonhuman primate. These results provide the first detailed and comprehensive genomic insights into genetic diversity, demography, genetic burden, and adaptation in this radiation of endangered primates.

Effects of Endogenous Salicylic Acid During Calcium Deficiency-Induced Tipburn in Chinese Cabbage (Brassica rapa L. ssp. pekinensis).

By cultivating tipburn-susceptible plants in modified Hoagland's medium containing of gradient exogenous calcium (Ca2+), we have shown that Ca2+ deficiency is one of the main causes of tipburn in Chinese cabbage (Brassica rapa L. ssp. pekinensis). The effect of endogenous plant Ca2+ concentrations on tipburn was also studied in a doubled haploid (DH) population consisting of 100 individuals, but no correlation was found. We then examined the expression of 12 Ca2+ transporter genes that function in cytosolic Ca2+ homeostasis in both tipburn-susceptible and tipburn-resistant plants under normal and tipburn-inducing conditions. Expression patterns for most of these genes differed between the two types of plants. Salicylic acid (SA) accumulated in response to conditions of calcium deficiency in our study, and both total SA and SA ß-glucoside (SAG) in tipburn-susceptible plants was ∼3-fold higher than it was in resistant plants following Ca2+ deficiency treatment. Also, the changes observed in SA levels correlated well with cell death patterns revealed by trypan blue staining. Therefore, we speculate that the cytoplasmic Ca2+ fluctuation-induced downstream signaling events, as well as SA signaling or other biological events, are involved in the plant defense response to tipburn in Chinese cabbage.

Fine genetic mapping of target leaf spot resistance gene cca-3 in cucumber, Cucumis sativus L.

KEY MESSAGE: The cucumber target leaf spot resistance gene cca - 3 was fine mapped in a 79-kb region harboring a CC-NB-ARC type R gene that may be responsible for the hypersensitive responses to infection of the target leaf spot pathogen in cucumber. The target leaf spot (TLS) is one of the most important foliar diseases in cucumber (Cucumis sativus L.). In this study, we conducted fine genetic mapping of a simply inherited recessive resistance gene, cca-3 against TLS with 193 F2:3 families and 890 F2 plants derived from the resistant cucumber inbred line D31 and the susceptible line D5. Initial mapping with microsatellite markers and bulked segregant analysis placed cca-3 in a 2.5-Mbp region of cucumber chromosome 6. The D5 and D31 lines were re-sequenced at 10× genome coverage to explore new markers in the target region. Genetic mapping in the large F2 population delimited the cca-3 locus in a 79-kb region with flanking markers Indel16874230 and Indel16953846. Additional fine mapping and gene annotation in this region revealed that a CC-NB-ARC type resistance gene analog, Csa6M375730, seems to be the candidate gene for cca-3. One single nucleotide polymorphism (SNP) was found in the NB-ARC domain of this candidate gene sequence between D31 and D5 that may lead to amino acid change, thus altering the function of the conserved NB-ARC motif. This SNP was validated in the segregating population as well as 24 independent cucumber lines. There was significantly higher level of cca-3 expression in the leaves of D5 (susceptible) than in D31 (resistant), and the expression level was positively correlated with the areas of necrotic spots on leaves after inoculation. It seems the cca-3 resistance gene was able to induce hypersensitive responses to the infection by TLS pathogen.

A warm temperature-released negative feedback loop fine-tunes PIF4-mediated thermomorphogenesis in Arabidopsis.

Plants can sense temperature changes and adjust their growth accordingly. In Arabidopsis, high ambient temperatures stimulate stem elongation by activating a key thermoresponsive regulator, PHYTOCHROME INTERACTING FACTOR 4 (PIF4). Here, we show that warmth promotes the nighttime transcription of GI, which is necessary for the high temperature-induced transcription of TOC1. Genetic analyses suggest that GI prevents excessive thermoresponsive growth by inhibiting PIF4, with this regulatory mechanism being partially reliant on TOC1. GI transcription is repressed by ELF3 and HY5, which concurrently inhibit PIF4 expression and activity. Temperature elevation causes the deactivation or degradation of ELF3 and HY5, leading to PIF4 activation and relief of GI transcriptional repression at high temperatures. This allows PIF4 to further activate GI transcription in response to elevated temperatures. GI, in turn, inhibits PIF4, establishing a negative feedback loop that fine-tunes PIF4 activity. In addition, we demonstrate that ELF3, HY5, and PIF4 regulate GI transcription by modulating the enrichment of histone variant H2A.Z at the GI locus. Together, our findings suggest that thermal release of a negative feedback loop finely adjusts plant thermomorphogenesis.

Temperature-dependent jumonji demethylase modulates flowering time by targeting H3K36me2/3 in Brassica rapa.

Global warming has a severe impact on the flowering time and yield of crops. Histone modifications have been well-documented for their roles in enabling plant plasticity in ambient temperature. However, the factor modulating histone modifications and their involvement in habitat adaptation have remained elusive. In this study, through genome-wide pattern analysis and quantitative-trait-locus (QTL) mapping, we reveal that BrJMJ18 is a candidate gene for a QTL regulating thermotolerance in thermotolerant B. rapa subsp. chinensis var. parachinensis (or Caixin, abbreviated to Par). BrJMJ18 encodes an H3K36me2/3 Jumonji demethylase that remodels H3K36 methylation across the genome. We demonstrate that the BrJMJ18 allele from Par (BrJMJ18Par) influences flowering time and plant growth in a temperature-dependent manner via characterizing overexpression and CRISPR/Cas9 mutant plants. We further show that overexpression of BrJMJ18Par can modulate the expression of BrFLC3, one of the five BrFLC orthologs. Furthermore, ChIP-seq and transcriptome data reveal that BrJMJ18Par can regulate chlorophyll biosynthesis under high temperatures. We also demonstrate that three amino acid mutations may account for function differences in BrJMJ18 between subspecies. Based on these findings, we propose a working model in which an H3K36me2/3 demethylase, while not affecting agronomic traits under normal conditions, can enhance resilience under heat stress in Brassica rapa.

BrMYB108 confers resistance to Verticillium wilt by activating ROS generation in Brassica rapa.

Increasing plant resistance to Verticillium wilt (VW), which causes massive losses of Brassica rapa crops, is a challenge worldwide. However, few causal genes for VW resistance have been identified by forward genetic approaches, resulting in limited application in breeding. We combine a genome-wide association study in a natural population and quantitative trait locus mapping in an F2 population and identify that the MYB transcription factor BrMYB108 regulates plant resistance to VW. A 179 bp insertion in the BrMYB108 promoter alters its expression pattern during Verticillium longisporum (VL) infection. High BrMYB108 expression leads to high VL resistance, which is confirmed by disease resistance tests using BrMYB108 overexpression and loss-of-function mutants. Furthermore, we verify that BrMYB108 confers VL resistance by regulating reactive oxygen species (ROS) generation through binding to the promoters of respiratory burst oxidase genes (Rboh). A loss-of-function mutant of AtRbohF in Arabidopsis shows significant susceptibility to VL. Thus, BrMYB108 and its target ROS genes could be used as targets for genetic engineering for VL resistance of B. rapa.

Recent Advancements and Biotechnological Implications of Carotenoid Metabolism of Brassica.

Carotenoids were synthesized in the plant cells involved in photosynthesis and photo-protection. In humans, carotenoids are essential as dietary antioxidants and vitamin A precursors. Brassica crops are the major sources of nutritionally important dietary carotenoids. Recent studies have unraveled the major genetic components in the carotenoid metabolic pathway in Brassica, including the identification of key factors that directly participate or regulate carotenoid biosynthesis. However, recent genetic advances and the complexity of the mechanism and regulation of Brassica carotenoid accumulation have not been reviewed. Herein, we reviewed the recent progress regarding Brassica carotenoids from the perspective of forward genetics, discussed biotechnological implications and provided new perspectives on how to transfer the knowledge of carotenoid research in Brassica to the crop breeding process.

The Carotenoid Esterification Gene BrPYP Controls Pale-Yellow Petal Color in Flowering Chinese Cabbage (Brassica rapa L. subsp. parachinensis).

Carotenoid esterification plays indispensable roles in preventing degradation and maintaining the stability of carotenoids. Although the carotenoid biosynthetic pathway has been well characterized, the molecular mechanisms underlying carotenoid esterification, especially in floral organs, remain poorly understood. In this study, we identified a natural mutant flowering Chinese cabbage (Caixin, Brassica rapa L. subsp. chinensis var. parachinensis) with visually distinguishable pale-yellow petals controlled by a single recessive gene. Transmission electron microscopy (TEM) demonstrated that the chromoplasts in the yellow petals were surrounded by more fully developed plastoglobules compared to the pale-yellow mutant. Carotenoid analyses further revealed that, compared to the pale-yellow petals, the yellow petals contained high levels of esterified carotenoids, including lutein caprate, violaxanthin dilaurate, violaxanthin-myristate-laurate, 5,6epoxy-luttein dilaurate, lutein dilaurate, and lutein laurate. Based on bulked segregation analysis and fine mapping, we subsequently identified the critical role of a phytyl ester synthase 2 protein (PALE YELLOW PETAL, BrPYP) in regulating carotenoid pigmentation in flowering Chinese cabbage petals. Compared to the yellow wild-type, a 1,148 bp deletion was identified in the promoter region of BrPYP in the pale-yellow mutant, resulting in down-regulated expression. Transgenic Arabidopsis plants harboring beta-glucuronidase (GUS) driven by yellow (BrPYP Y ::GUS) and pale-yellow type (BrPYP PY ::GUS) promoters were subsequently constructed, revealing stronger expression of BrPYP Y ::GUS both in the leaves and petals. Furthermore, virus-induced gene silencing of BrPYP significantly altered petal color from yellow to pale yellow. These findings demonstrate the molecular mechanism of carotenoid esterification, suggesting a role of phytyl ester synthase in carotenoid biosynthesis of flowering Chinese cabbage.

Subgenome dominance and its evolutionary implications in crop domestication and breeding.

Polyploidization or whole-genome duplication (WGD) is a well-known speciation and adaptation mechanism in angiosperms, while subgenome dominance is a crucial phenomenon in allopolyploids, established following polyploidization. The dominant subgenomes contribute more to genome evolution and homoeolog expression bias, both of which confer advantages for short-term phenotypic adaptation and long-term domestication. In this review, we firstly summarize the probable mechanistic basis for subgenome dominance, including the effects of genetic [transposon, genetic incompatibility, and homoeologous exchange (HE)], epigenetic (DNA methylation and histone modification), and developmental and environmental factors on this evolutionary process. We then move to Brassica rapa, a typical allopolyploid with subgenome dominance. Polyploidization provides the B. rapa genome not only with the genomic plasticity for adapting to changeable environments, but also an abundant genetic basis for morphological variation, making it a representative species for subgenome dominance studies. According to the 'two-step theory', B. rapa experienced genome fractionation twice during WGD, in which most of the genes responding to the environmental cues and phytohormones were over-retained, enhancing subgenome dominance and consequent adaption. More than this, the pangenome of 18 B. rapa accessions with different morphotypes recently constructed provides further evidence to reveal the impacts of polyploidization and subgenome dominance on intraspecific diversification in B. rapa. Above and beyond the fundamental understanding of WGD and subgenome dominance in B. rapa and other plants, however, it remains elusive why subgenome dominance has tissue- and spatiotemporal-specific features and could shuffle between homoeologous regions of different subgenomes by environments in allopolyploids. We lastly propose acceleration of the combined application of resynthesized allopolyploids, omics technology, and genome editing tools to deepen mechanistic investigations of subgenome dominance, both genetic and epigenetic, in a variety of species and environments. We believe that the implications of genomic and genetic basis of a variety of ecologically, evolutionarily, and agriculturally interesting traits coupled with subgenome dominance will be uncovered and aid in making new discoveries and crop breeding.