Infernape uncovers cell type-specific and spatially resolved alternative polyadenylation in the brain.

Differential polyadenylation sites (PAs) critically regulate gene expression, but their cell type-specific usage and spatial distribution in the brain have not been systematically characterized. Here, we present Infernape, which infers and quantifies PA usage from single-cell and spatial transcriptomic data and show its application in the mouse brain. Infernape uncovers alternative intronic PAs and 3'-UTR lengthening during cortical neurogenesis. Progenitor-neuron comparisons in the excitatory and inhibitory neuron lineages show overlapping PA changes in embryonic brains, suggesting that the neural proliferation-differentiation axis plays a prominent role. In the adult mouse brain, we uncover cell type-specific PAs and visualize such events using spatial transcriptomic data. Over two dozen neurodevelopmental disorder-associated genes such as Csnk2a1 and Mecp2 show differential PAs during brain development. This study presents Infernape to identify PAs from scRNA-seq and spatial data, and highlights the role of alternative PAs in neuronal gene regulation.

PIE-seq: identifying RNA-binding protein targets by dual RNA-deaminase editing and sequencing.

RNA-binding proteins (RBPs) are essential for gene regulation, but it remains a challenge to identify their RNA targets across cell types. Here we present PIE-Seq to investigate Protein-RNA Interaction with dual-deaminase Editing and Sequencing by conjugating C-to-U and A-to-I base editors to RBPs. We benchmark PIE-Seq and demonstrate its sensitivity in single cells, its application in the developing brain, and its scalability with 25 human RBPs. Bulk PIE-Seq identifies canonical binding features for RBPs such as PUM2 and NOVA1, and nominates additional target genes for most tested RBPs such as SRSF1 and TDP-43/TARDBP. Homologous RBPs frequently edit similar sequences and gene sets in PIE-Seq while different RBP families show distinct targets. Single-cell PIE-PUM2 uncovers comparable targets to bulk samples and applying PIE-PUM2 to the developing mouse neocortex identifies neural-progenitor- and neuron-specific target genes such as App. In summary, PIE-Seq provides an orthogonal approach and resource to uncover RBP targets in mice and human cells.

Fine mapping of BnDM1-the gene regulating indeterminate inflorescence in Brassica napus.

KEY MESSAGE: A candidate gene Bndm1 related to determinate inflorescence was mapped to a 128-kb interval on C02 in Brassica napus. Brassica napus plants with determinate inflorescence exhibit improved traits in field production, such as lower plant height, improved lodging resistance, and consistent maturity. Compared to plants with indeterminate inflorescence, such features are favorable for mechanized harvesting techniques. Here, using a natural mutant 6138 with determinate inflorescence, it is demonstrated that determinate inflorescence reduces plant height significantly without affecting thousand-grain weight and yield per plant. Determinacy was regulated by a single recessive gene, Bndm1. Using a combination of SNP arrays and map-based cloning, we mapped the locus of determinacy to a 128-kb region on C02. Based on sequence comparisons and the reported functions of candidate genes in this region, we predicted BnaC02.knu (a homolog of KNU in Arabidopsis) as a possible candidate gene of Bndm1 for controlling determinate inflorescence. We found a 623-bp deletion in a region upstream of the KNU promoter in the mutant. This deletion led to the significant overexpression of BnaC02.knu in the mutant compared to that in the ZS11 line. The correlation between this deletion and determinate inflorescence was examined in natural populations. The results indicated that the deletion affected the normal transcription of BnaC02.knu in the plants with determinate inflorescence and played an important role in maintaining flower development. This study presents as a new material for optimizing plant architecture and breeding novel canola varieties suitable for mechanized production. Moreover, our findings provide a theoretical basis for analyzing the molecular mechanisms underlying the formation of determinate inflorescence in B. napus.

The Loss of YTHDC1 in Gut Macrophages Exacerbates Inflammatory Bowel Disease.

The nuclear N6 -methyladenosine (m6 A) reader YT521-B homology-domain-containing protein 1 (YTHDC1) is required to maintain embryonic stem cell identity. However, little is known about its biological functions in intestinal-resident macrophages and inflammatory bowel disease (IBD). Herein, it is demonstrated that macrophage-specific depletion or insufficiency of YTHDC1 accelerates IBD development in animal models. On the molecular basis, YTHDC1 reduction in IBD-derived macrophages is attributed to Zinc finger protein 36 (ZFP36)-induced mRNA degradation. Importantly, transcriptome profiling and mechanistic assays unveil that YTHDC1 in macrophages regulates Ras homolog family member H (RHOH) to suppress inflammatory responses and fine-tunes NME nucleoside diphosphate kinase 1 (NME1) to enhance the integrity of colonic epithelial barrier, respectively. Collectively, this study identifies YTHDC1 as an important factor for the resolution of inflammatory responses and restoration of colonic epithelial barrier in the setting of IBD.

Upregulation of SYNGAP1 expression in mice and human neurons by redirecting alternative splicing.

The Ras GTPase-activating protein SYNGAP1 plays a central role in synaptic plasticity, and de novo SYNGAP1 mutations are among the most frequent causes of autism and intellectual disability. How SYNGAP1 is regulated during development and how to treat SYNGAP1-associated haploinsufficiency remain challenging questions. Here, we characterize an alternative 3' splice site (A3SS) of SYNGAP1 that induces nonsense-mediated mRNA decay (A3SS-NMD) in mouse and human neural development. We demonstrate that PTBP1/2 directly bind to and promote SYNGAP1 A3SS inclusion. Genetic deletion of the Syngap1 A3SS in mice upregulates Syngap1 protein and alleviates the long-term potentiation and membrane excitability deficits caused by a Syngap1 knockout allele. We further report a splice-switching oligonucleotide (SSO) that converts SYNGAP1 unproductive isoform to the functional form in human iPSC-derived neurons. This study describes the regulation and function of SYNGAP1 A3SS-NMD, the genetic rescue of heterozygous Syngap1 knockout mice, and the development of an SSO to potentially alleviate SYNGAP1-associated haploinsufficiency.

Transcription factor WRKY28 curbs WRKY33-mediated resistance to Sclerotinia sclerotiorum in Brassica napus.

Sclerotinia sclerotiorum causes substantial damage and loss of yield in oilseed rape (Brassica napus). The molecular mechanisms of oilseed rape defense against Sclerotinia remain elusive. In this study, we found that in the early stages of B. napus infection a conserved mitogen-activated protein kinase (MAPK) cascade mediated by BnaA03.MKK5-BnaA06.MPK3/BnaC03.MPK3 module phosphorylates the substrate BnWRKY33, enhancing its transcriptional activity. The activated BnWRKY33 binds to its own promoter and triggers a transcriptional burst of BnWRKY33, thus helping plants effectively resist the pathogenic fungi by enhancing the expression of phytoalexin synthesis-related genes. The expression of BnWRKY33 is fine-tuned during defense. Ongoing Sclerotinia infection induces BnaA03.WRKY28 and BnaA09.VQ12 expression. BnaA09.VQ12 interacts physically with BnaA03.WRKY28 to form a protein complex, causing BnaA03.WRKY28 to outcompete BnWRKY33 and bind to the BnWRKY33 promoter. BnaA03.WRKY28 induction suppresses BnWRKY33 expression in the later stages of infection but promotes branch formation in the leaf axils by regulating the expression of branching-related genes such as BnBRC1. BnaA03.WRKY28 participates in the trade-off between defense and growth. These findings suggest that oilseed rape plants may modulate defense-response strength and develop alternative reproduction and survival strategies in the face of lethal pathogens.

Comparative transcriptomic analysis reveals the molecular mechanism underlying seedling biomass heterosis in Brassica napus.

BACKGROUND: Heterosis is an important biological phenomenon in which the hybrids exceed the parents in many traits. However, the molecular mechanism underlying seedling heterosis remains unclear. RESULTS: In the present study, we analyzed the leaf transcriptomes of strong hybrids (AM, HM) and weak hybrids (CM, HW) and their parents (A, C, H, M, and W) at two periods. Phenotypically, hybrids had obvious biomass heterosis at the seedling stage, with statistically significant differences between the strong and weak hybrids. The transcriptomic analysis demonstrated that the number of differentially expressed genes (DEGs) between parents was the highest. Further analysis showed that most DEGs were biased toward parental expression. The biological processes of the two periods were significantly enriched in the plant hormone signal transduction and photosynthetic pathways. In the plant hormone signaling pathway, DEG expression was high in hybrids, with expression differences between strong and weak hybrids. In addition, DEGs related to cell size were identified. Similar changes were observed during photosynthesis. The enhanced leaf area of hybrids generated an increase in photosynthetic products, which was consistent with the phenotype of the biomass. Weighted gene co-expression network analysis of different hybrids and parents revealed that hub genes in vigorous hybrid were mainly enriched in the plant hormone signal transduction and regulation of plant hormones. CONCLUSION: Plant hormone signaling and photosynthesis pathways, as well as differential expression of plant cell size-related genes, jointly regulate the dynamic changes between strong and weak hybrids and the generation of seedling-stage heterosis. This study may elucidate the molecular mechanism underlying early biomass heterosis and help enhance canola yield.

Variants in ADD1 cause intellectual disability, corpus callosum dysgenesis, and ventriculomegaly in humans.

PURPOSE: Adducins interconnect spectrin and actin filaments to form polygonal scaffolds beneath the cell membranes and form ring-like structures in neuronal axons. Adducins regulate mouse neural development, but their function in the human brain is unknown. METHODS: We used exome sequencing to uncover ADD1 variants associated with intellectual disability (ID) and brain malformations. We studied ADD1 splice isoforms in mouse and human neocortex development with RNA sequencing, super resolution imaging, and immunoblotting. We investigated 4 variant ADD1 proteins and heterozygous ADD1 cells for protein expression and ADD1-ADD2 dimerization. We studied Add1 functions in vivo using Add1 knockout mice. RESULTS: We uncovered loss-of-function ADD1 variants in 4 unrelated individuals affected by ID and/or structural brain defects. Three additional de novo copy number variations covering the ADD1 locus were associated with ID and brain malformations. ADD1 is highly expressed in the neocortex and the corpus callosum, whereas ADD1 splice isoforms are dynamically expressed between cortical progenitors and postmitotic neurons. Human variants impair ADD1 protein expression and/or dimerization with ADD2. Add1 knockout mice recapitulate corpus callosum dysgenesis and ventriculomegaly phenotypes. CONCLUSION: Our human and mouse genetics results indicate that pathogenic ADD1 variants cause corpus callosum dysgenesis, ventriculomegaly, and/or ID.

Overdominance at the Gene Expression Level Plays a Critical Role in the Hybrid Root Growth of Brassica napus.

Despite heterosis contributing to genetic improvements in crops, root growth heterosis in rapeseed plants is poorly understood at the molecular level. The current study was performed to discover key differentially expressed genes (DEGs) related to heterosis in two hybrids with contrasting root growth performance (FO; high hybrid and FV; low hybrid) based on analysis of the root heterosis effect. Based on comparative transcriptomic analysis, we believe that the overdominance at the gene expression level plays a critical role in hybrid roots' early biomass heterosis. Our findings imply that a considerable increase in up-regulation of gene expression underpins heterosis. In the FO hybrid, high expression of DEGs overdominant in the starch/sucrose and galactose metabolic pathways revealed a link between hybrid vigor and root growth. DEGs linked to auxin, cytokinin, brassinosteroids, ethylene, and abscisic acid were also specified, showing that these hormones may enhance mechanisms of root growth and the development in the FO hybrid. Moreover, transcription factors such as MYB, ERF, bHLH, NAC, bZIP, and WRKY are thought to control downstream genes involved in root growth. Overall, this is the first study to provide a better understanding related to the regulation of the molecular mechanism of heterosis, which assists in rapeseed growth and yield improvement.

QTL Mapping and Diurnal Transcriptome Analysis Identify Candidate Genes Regulating Brassica napus Flowering Time.

Timely flowering is important for seed formation and maximization of rapeseed (Brassica napus) yield. Here, we performed flowering-time quantitative trait loci (QTL) mapping using a double haploid (DH) population grown in three environments to study the genetic architecture. Brassica 60 K Illumina Infinium™ single nucleotide polymorphism (SNP) array and simple sequence repeat (SSR) markers were used for genotyping of the DH population, and a high-density genetic linkage map was constructed. QTL analysis of flowering time from the three environments revealed five consensus QTLs, including two major QTLs. A major QTL located on chromosome A03 was detected specifically in the semi-winter rapeseed growing region, and the one on chromosome C08 was detected in all environments. Ribonucleic acid sequencing (RNA-seq) was performed on the parents' leaves at seven time-points in a day to determine differentially expressed genes (DEGs). The biological processes and pathways with significant enrichment of DEGs were obtained. The DEGs in the QTL intervals were analyzed, and four flowering time-related candidate genes were found. These results lay a foundation for the genetic regulation of rapeseed flowering time and create a rapeseed gene expression library for seven time-points in a day.

Fine Mapping and Identification of BnaC06.FtsH1, a Lethal Gene That Regulates the PSII Repair Cycle in Brassica napus.

Photosystem II (PSII) is an important component of the chloroplast. The PSII repair cycle is crucial for the relief of photoinhibition and may be advantageous when improving stress resistance and photosynthetic efficiency. Lethal genes are widely used in the efficiency detection and method improvement of gene editing. In the present study, we identified the naturally occurring lethal mutant 7-521Y with etiolated cotyledons in Brassica napus, controlled by double-recessive genes (named cyd1 and cyd2). By combining whole-genome resequencing and map-based cloning, CYD1 was fine-mapped to a 29 kb genomic region using 15,167 etiolated individuals. Through cosegregation analysis and functional verification of the transgene, BnaC06.FtsH1 was determined to be the target gene; it encodes an filamentation temperature sensitive protein H 1 (FtsH1) hydrolase that degrades damaged PSII D1 in Arabidopsis thaliana. The expression of BnaC06.FtsH1 was high in the cotyledons, leaves, and flowers of B. napus, and localized in the chloroplasts. In addition, the expression of EngA (upstream regulation gene of FtsH) increased and D1 decreased in 7-521Y. Double mutants of FtsH1 and FtsH5 were lethal in A. thaliana. Through phylogenetic analysis, the loss of FtsH5 was identified in Brassica, and the remaining FtsH1 was required for PSII repair cycle. CYD2 may be a homologous gene of FtsH1 on chromosome A07 of B. napus. Our study provides new insights into lethal mutants, the findings may help improve the efficiency of the PSII repair cycle and biomass accumulation in oilseed rape.

Disruption of carotene biosynthesis leads to abnormal plastids and variegated leaves in Brassica napus.

Leaf color is an important characteristic of normal chloroplast development. Variegated plants have green- and white-sectored leaves, which can be used to identify important pathways and molecular mechanisms of chloroplast development. We studied two Brassica napus variegation mutants from same one variegated ancestor, designated ZY-4 and ZY-8, which have different degrees of variegation. When grown in identical conditions, the ratio of white sectors in ZY-4 leaves is higher than in ZY-8. In both mutants, the cells in green sectors contain normal chloroplasts; while, the cells in white sectors contain abnormal plastids. Seedling chloroplasts ultrastructure of both mutants showed that the biogenesis of chloroplasts was blocked in early stages; delayed development and structual damage in ZY-4 were more serious than in ZY-8. Employing bulked segregant analysis(BSA), two bulks (BY142 and BY137) from BC2F1 lines derived from ZY-4 and ZS11, and one bulk (BY56) from BC2F1 lines derived from ZY-8 and ZS11, and screening by Brassica 60K SNP BeadChip Array, showed the candidate regions localized in chromosome A08 (BY142), C04 (BY137), and A08 (BY56), respectively. Transcriptome analysis of five seedling development stages of ZY-4, ZY-8, and ZS11 showed that photosynthesis, energy metabolism-related pathways and translation-related pathways were important for chloroplast biogenesis. The number of down- or up-regulated genes related to immune system process in ZY-4 was more than in ZY-8. The retrograde signaling pathway was mis-regulated in both mutants. DEG analysis indicated that both mutants showed photooxidative damages. By coupling transcriptome and BSA CHIP analyses, some candidate genes were identified. The gene expression pattern of carotene biosynthesis pathway was disrupted in both mutants. However, histochemical analysis of ROS revealed that there was no excessive accumulation of ROS in ZY-4 and ZY-8. Taken together, our data indicate that the disruption of carotene biosynthetic pathways leads to the variegation phenotypes of ZY-4 and ZY-8 and there are some functions that can compensate for the disruption of carotene biosynthesis in ZY-4 and ZY-8 to reduce ROS and prevent seedling mortality.

Two young genes reshape a novel interaction network in Brassica napus.

New genes often drive the evolution of gene interaction networks. In Brassica napus, the widely used genic male sterile breeding system 7365ABC is controlled by two young genes, Bnams4b and BnaMs3. However, the interaction mechanism of these two young genes remains unclear. Here, we confirmed that Bnams4b interacts with the nuclear localised E3 ligase BRUTUS (BTS). Ectopic expression of AtBRUTUS (AtBTS) and comparison between Bnams4b -transgenic Arabidopsis and bts mutants suggested that Bnams4b may drive translocation of BTS to cause various toxic defects. BnaMs3 gained an exclusive interaction with the plastid outer-membrane translocon Toc33 compared with Bnams3 and AtTic40, and specifically compensated for the toxic effects of Bnams4b . Heat shock treatment also rescued the sterile phenotype, and high temperature suppressed the interaction between Bnams4b and BTS in yeast. Furthermore, the ubiquitin system and TOC (translocon at the outer envelope membrane of chloroplasts) component accumulation were affected in Bnams4b -transgenic Arabidopsis plants. Taken together, these results indicate that new chimeric Bnams4b carries BTS from nucleus to chloroplast, which may disrupt the normal ubiquitin-proteasome system to cause toxic effects, and these defects can be compensated by BnaMs3-Toc33 interaction or environmental heat shock. It reveals a scenario in which two population-specific coevolved young genes reshape a novel interaction network in plants.

Helitron distribution in Brassicaceae and whole Genome Helitron density as a character for distinguishing plant species.

BACKGROUND: Helitron is a rolling-circle DNA transposon; it plays an important role in plant evolution. However, Helitron distribution and contribution to evolution at the family level have not been previously investigated. RESULTS: We developed the software easy-to-annotate Helitron (EAHelitron), a Unix-like command line, and used it to identify Helitrons in a wide range of 53 plant genomes (including 13 Brassicaceae species). We determined Helitron density (abundance/Mb) and visualized and examined Helitron distribution patterns. We identified more than 104,653 Helitrons, including many new Helitrons not predicted by other software. Whole genome Helitron density is independent from genome size and shows stability at the species level. Using linear discriminant analysis, de novo genomes (next-generation sequencing) were successfully classified into Arabidopsis thaliana groups. For most Brassicaceae species, Helitron density negatively correlated with gene density, and Helitron distribution patterns were similar to those of A. thaliana. They preferentially inserted into sequence around the centromere and intergenic region. We also associated 13 Helitron polymorphism loci with flowering-time phenotypes in 18 A. thaliana ecotypes. CONCLUSION: EAHelitron is a fast and efficient tool to identify new Helitrons. Whole genome Helitron density can be an informative character for plant classification. Helitron insertion polymorphism could be used in association analysis.

Mitochondrial genome and transcriptome analysis of five alloplasmic male-sterile lines in Brassica juncea.

BACKGROUND: Alloplasmic lines, in which the nuclear genome is combined with wild cytoplasm, are often characterized by cytoplasmic male sterility (CMS), regardless of whether it was derived from sexual or somatic hybridization with wild relatives. In this study, we sequenced and analyzed the mitochondrial genomes of five such alloplasmic lines in Brassica juncea. RESULTS: The assembled and annotated mitochondrial genomes of the five alloplasmic lines were found to have virtually identical gene contents. They preserved most of the ancestral mitochondrial segments, and the same candidate male sterility gene (orf108) was found harbored in mitotype-specific sequences. We also detected promiscuous sequences of chloroplast origin that were conserved among plants of the Brassicaceae, and found the RNA editing profiles to vary across the five mitochondrial genomes. CONCLUSIONS: On the basis of our characterization of the genetic nature of five alloplasmic mitochondrial genomes, we speculated that the putative candidate male sterility gene orf108 may not be responsible for the CMS observed in Brassica oxyrrhina and Diplotaxis catholica. Furthermore, we propose the potential coincidence of CMS in alloplasmic lines. Our findings lay the foundation for further elucidation of male sterility gene.

Transcriptomic Analysis of Seed Coats in Yellow-Seeded Brassica napus Reveals Novel Genes That Influence Proanthocyanidin Biosynthesis.

Yellow seeds are a favorable trait for Brassica crops breeding due to better quality than their black-seeded counterparts. Here, we compared the Brassica napus seed coat transcriptomes between yellow- and brown-seeded near-isogenic lines (Y-NIL and B-NIL) that were developed from the resynthesized yellow-seeded line No. 2127-17. A total of 4,974 differentially expressed genes (DEG) were identified during seed development, involving 3,128 up-regulated and 1,835 down-regulated genes in yellow seed coats. Phenylpropanoid and flavonoid biosynthesis pathways were enriched in down-regulated genes, whereas the top two pathways for up-regulated genes were plant-pathogen interaction and plant hormone signal transduction. Twelve biosynthetic genes and three regulatory genes involved in the flavonoid pathway exhibited similar expression patterns in seed coats during seed development, of which the down-regulation mainly contributed to the reduction of proanthocyanidins (PAs) in yellow seed coats, indicating that these genes associated with PA biosynthesis may be regulated by an unreported common regulator, possibly corresponding to the candidate for the dominant black-seeded gene D in the NILs. Three transcription factor (TF) genes, including one bHLH gene and two MYB-related genes that are located within the previous seed coat color quantitative trait locus (QTL) region on chromosome A09, also showed similar developmental expression patterns to the key PA biosynthetic genes and they might thus potentially involved participate in flavonoid biosynthesis regulation. Our study identified novel potential TFs involved in PAs accumulation and will provide pivotal information for identifying the candidate genes for seed coat color in B. napus.

Trilocular phenotype in Brassica juncea L. resulted from interruption of CLAVATA1 gene homologue (BjMc1) transcription.

As a desirable agricultural trait, multilocular trait of rapeseed (Brassica rapa; Brassica napus; Brassica juncea), always represents higher yield per plant compared with bilocular plants. We previously isolated a trilocular gene locus, Bjmc1, and identified a set of molecular markers linked to the trilocular gene. With a map-based cloning, we identified that the BjMc1 was located in B genome of Brassica juncea, and it was a CLAVATA1 (CLV1) gene homologue. The insertion of a copia-LTR retrotransposable element 1 (RTE1) into the coding region of BjMc1 interrupted its transcription in rapeseed, leading to the trilocular phenotype. Phylogenetic analysis showed that Mc1 genes were conserved and widespread in land plants. Two amino acid sites had undergone positive selection in the ancestor of Mc1 genes, and then purifying selection was the dominant force after the divergence of dicots and monocots from their common ancestor in the evolutionary process, indicating that Mc1 genes are conserved in modern land plants. Our results provided new insights in molecular regulatory mechanism of multilocularity in rapeseed, and better understanding of molecular mechanism in crop yield improvement.

Identification of different cytoplasms based on newly developed mitotype-specific markers for marker-assisted selection breeding in Brassica napus L.

KEY MESSAGE: Different mitotype-specific markers were developed to distinguish different cytoplasms in Brassica napus L. Mitotype-specific markers have been developed to distinguish different mitotypes in plant. And use of molecular markers to identify different mitotypes in Brassica napus would enhance breeding efficiency. Here, we comparatively analyzed six sequenced mitochondrial genomes in Brassica napus and identified collinear block sequences and mitotype-specific sequences (MSSs) of these mitochondrial genomes. The collinear block sequences between mitochondrial genomes of nap, cam, and pol cytoplasmic male sterility (CMS) lines were higher than those of other lines. After comparative analysis of the six sequenced mitochondrial genomes (cam, nap, ole, pol CMS, ogu CMS, and hau CMS), 90 MSSs with sizes ranging from 101 to 9981 bp and a total length of 103,756 bp (accounting for 6.77% of the mitochondrial genome sequences) were identified. Additionally, 12 mitotype-specific markers were developed based on the mitochondrial genome-specific sequences in order to distinguish among these different mitotypes. Cytoplasms of 570 different inbred lines collected across scientific research institutes in China were identified using the MSS markers developed in our study. In addition to confirming the accuracy of the cytoplasmic identification, we also identified mitotypes that have not been reported in Brassica napus. Our study may provide guidance for the classification of different mitotypes in B. napus breeding.

Joint genome-wide association and transcriptome sequencing reveals a complex polygenic network underlying hypocotyl elongation in rapeseed (Brassica napus L.).

Hypocotyl elongation is considered an important typical seedling trait contributing directly to an increase in and stabilization of the yield in Brassica napus, but its molecular genetic mechanism is poorly understood. In the present study, hypocotyl lengths of 210 lines were measured in an illuminated culture room. A genome-wide association study (GWAS) was performed with 23,435 single nucleotide polymorphisms (SNPs) for hypocotyl length. Three lines with long hypocotyl length and three lines with short hypocotyl length from one doubled haploid line (DH) population were used for transcriptome sequencing. A GWAS followed by transcriptome analysis identified 29 differentially expressed genes associated with significant SNPs in B. napus. These genes regulate hypocotyl elongation by mediating flowering morphogenesis, circadian clock, hormone biosynthesis, or important metabolic signaling pathways. Among these genes, BnaC07g46770D negatively regulates hypocotyl elongation directly, as well as flowering time. Our results indicate that a joint GWAS and transcriptome analysis has significant potential for identifying the genes responsible for hypocotyl elongation; The extension of hypocotyl is a complex biological process regulated by a polygenic network.

Overexpression of the Novel Arabidopsis Gene At5g02890 Alters Inflorescence Stem Wax Composition and Affects Phytohormone Homeostasis.

The cuticle is composed of cutin and cuticular wax. It covers the surfaces of land plants and protects them against environmental damage. At5g02890 encodes a novel protein in Arabidopsis thaliana. In the current study, protein sequence analysis showed that At5g02890 is highly conserved in the Brassicaceae. Arabidopsis lines overexpressing At5g02890 (OE-At5g02890 lines) and an At5g02890 orthologous gene from Brassica napus (OE-Bn1 lines) exhibited glossy stems. Chemical analysis revealed that overexpression of At5g02890 caused significant reductions in the levels of wax components longer than 28 carbons (C28) in inflorescence stems, whereas the levels of wax molecules of chain length C28 or shorter were significantly increased. Transcriptome analysis indicated that nine of 11 cuticular wax synthesis-related genes with different expression levels in OE-At5g02890 plants are involved in very-long-chain fatty acid (VLCFA) elongation. At5g02890 is localized to the endoplasmic reticulum (ER), which is consistent with its function in cuticular wax biosynthesis. These results demonstrate that the overexpression of At5g02890 alters cuticular wax composition by partially blocking VLCFA elongation of C28 and higher. In addition, detailed analysis of differentially expressed genes associated with plant hormones and endogenous phytohormone levels in wild-type and OE-At5g02890 plants indicated that abscisic acid (ABA), jasmonic acid (JA), and jasmonoyl-isoleucine (JA-Ile) biosynthesis, as well as polar auxin transport, were also affected by overexpression of At5g02890. Taken together, these findings indicate that overexpression of At5g02890 affects both cuticular wax biosynthesis and phytohormone homeostasis in Arabidopsis.