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Abstract Background Long non-coding RNAs (lncRNAs) play a crucial role in regulating gene expression vital for the growth and development of plants. Despite this, the role of lncRNAs in Chinese cabbage (Brassica rapa L. ssp. pekinensis) pollen development and male fertility remains poorly understood. Results In this study, we characterized a recessive genic male sterile mutant (366–2 S), where the delayed degradation of tapetum and the failure of tetrad separation primarily led to the inability to form single microspores, resulting in male sterility. To analyze the role of lncRNAs in pollen development, we conducted a comparative lncRNA sequencing using anthers from the male sterile mutant line (366–2 S) and the wild-type male fertile line (366–2 F). We identified 385 differentially expressed lncRNAs between the 366–2 F and 366–2 S lines, with 172 of them potentially associated with target genes. To further understand the alterations in mRNA expression and explore potential lncRNA-target genes (mRNAs), we performed comparative mRNA transcriptome analysis in the anthers of 366–2 S and 366–2 F at two stages. We identified 1,176 differentially expressed mRNAs. Remarkably, GO analysis revealed significant enrichment in five GO terms, most notably involving mRNAs annotated as pectinesterase and polygalacturonase, which play roles in cell wall degradation. The considerable downregulation of these genes might contribute to the delayed degradation of tapetum in 366–2 S. Furthermore, we identified 15 lncRNA-mRNA modules through Venn diagram analysis. Among them, MSTRG.9997-BraA04g004630.3 C (β-1,3-glucanase) is associated with callose degradation and tetrad separation. Additionally, MSTRG.5212-BraA02g040020.3 C (pectinesterase) and MSTRG.13,532-BraA05g030320.3 C (pectinesterase) are associated with cell wall degradation of the tapetum, indicating that these three candidate lncRNA-mRNA modules potentially regulate pollen development. Conclusion This study lays the foundation for understanding the roles of lncRNAs in pollen development and for elucidating their molecular mechanisms in regulating male sterility in Chinese cabbage.

Clubroot disease poses a significant threat to Brassica crops, necessitating ongoing updates on resistance gene sources. In F2 segregants of the clubroot-resistant inbred line BrT18-6-4-3 and susceptible DH line Y510, the genetic analysis identified a single dominant gene responsible for clubroot resistance. Through bulk segregant sequencing analysis and kompetitive allele-specific polymerase chain reaction assays, CRA8.1.6 was mapped within 110 kb (12,255–12,365 Mb) between markers L-CR11 and L-CR12 on chromosome A08. We identified B raA08g015220.3.5C as the candidate gene of CRA8.1.6. Upon comparison with the sequence of disease-resistant material BrT18-6-4-3, we found 249 single-nucleotide polymorphisms, seven insertions, six deletions, and a long terminal repeat (LTR) retrotransposon (5,310 bp) at 909 bp of the first intron. However, the LTR retrotransposon was absent in the coding sequence of the susceptible DH line Y510. Given the presence of a non-functional LTR insertion in other materials, it showed that the LTR insertion might not be associated with susceptibility. Sequence alignment analysis revealed that the fourth exon of the susceptible line harbored two deletions and an insertion, resulting in a frameshift mutation at 8,551 bp, leading to translation termination at the leucine-rich repeat domain’s C-terminal in susceptible material. Sequence alignment of the CDS revealed a 99.4% similarity to Crr1a, which indicate that CRA8.1.6 is likely an allele of the Crr1a gene. Two functional markers, CRA08-InDel and CRA08-KASP1, have been developed for marker-assisted selection in CR turnip cultivars. Our findings could facilitate the development of clubroot-resistance turnip cultivars through marker-assisted selection.

Flower color is an important trait in Brassica species. However, genes responsible for the dark yellow flower trait in Chinese cabbage have not been reported. In this study, we identified a dark-yellow-flowered Chinese cabbage line SD369. Genetic analysis indicated that the dark yellow flower trait in SD369 was controlled by a single recessive locus, Br-dyp1 (dark yellow petal color 1 in Brassica rapa). Using bulked segregant RNA sequencing and kompetitive allele-specific PCR assays, Br-dyp1 was fine-mapped to an interval of 53.6 kb on chromosome A09. Functional annotation analysis, expression analysis, and sequence variation analysis revealed that Bra037130 (BraA09.ZEP), which encodes a zeaxanthin epoxidase, was the most likely candidate gene for Br-dyp1. Carotenoid profile analysis suggested that Bra037130 (BraA09.ZEP) might participate in the epoxidation from zeaxanthin to violaxanthin. The 679 bp insertion in dark yellow petal caused premature stop codon, thus caused the loss-of-function of the enzyme zeaxanthin epoxidase (ZEP), which disturbed the carotenoid metabolism, and caused the increased accumulation of total carotenoid, and finally converted the flower color from yellow to dark yellow. Comparative transcriptome analysis also showed that the “carotenoid biosynthesis” pathway was significantly enriched, and genes involved in carotenoid degradation and abscisic acid biosynthesis and metabolism were significantly downregulated. Furthermore, we developed and validated the functional marker Br-dyp1-InDel for Br-dyp1. Overall, these results provide insight into the molecular basis of carotenoid-based flower coloration in B. rapa and reveal valuable information for marker-assisted selection breeding in Chinese cabbage.

Plasmodiophora brassicae, an obligate biotrophic pathogen-causing clubroot disease, can seriously affect Brassica crops worldwide, especially Chinese cabbage. Understanding the transcriptome and metabolome profiling changes during the infection of P. brassicae will provide key insights in understanding the defense mechanism in Brassica crops. In this study, we estimated the phytohormones using targeted metabolome assays and transcriptomic changes using RNA sequencing (RNA-seq) in the roots of resistant (BrT24) and susceptible (Y510-9) plants at 0, 3, 9, and 20 days after inoculation (DAI) with P. brassicae. Differentially expressed genes (DEGs) in resistant vs. susceptible lines across different time points were identified. The weighted gene co-expression network analysis of the DEGs revealed six pathways including “Plant–pathogen interaction” and “Plant hormone signal transduction” and 15 hub genes including pathogenic type III effector avirulence factor gene (RIN4) and auxin-responsive protein (IAA16) to be involved in plants immune response. Inhibition of Indoleacetic acid, cytokinin, jasmonate acid, and salicylic acid contents and changes in related gene expression in R-line may play important roles in regulation of clubroot resistance (CR). Based on the combined metabolome profiling and hormone-related transcriptomic responses, we propose a general model of hormone-mediated defense mechanism. This study definitely enhances our current understanding and paves the way for improving CR in Brassica rapa.

Clubroot, caused by the soil-borne protist Plasmodiophora brassicae, is one of the most destructive diseases of Chinese cabbage worldwide. However, the clubroot resistance mechanisms remain unclear. In this study, in both clubroot-resistant (DH40R) and clubroot-susceptible (DH199S) Chinese cabbage lines, the primary (root hair infection) and secondary (cortical infection) infection stages started 2 and 5 days after inoculation (dai), respectively. With the extension of the infection time, cortical infection was blocked and complete P. brassica resistance was observed in DH40R, while disease scales of 1, 2, and 3 were observed at 8, 13, and 22 dai in DH199S. Transcriptome analysis at 0, 2, 5, 8, 13, and 22 dai identified 5,750 relative DEGs (rDEGs) between DH40R and DH199S. The results indicated that genes associated with auxin, PR, disease resistance proteins, oxidative stress, and WRKY and MYB transcription factors were involved in clubroot resistance regulation. In addition, weighted gene coexpression network analysis (WGCNA) identified three of the modules whose functions were highly associated with clubroot-resistant, including ten hub genes related to clubroot resistance (ARF2, EDR1, LOX4, NHL3, NHL13, NAC29, two AOP1, EARLI 1, and POD56). These results provide valuable information for better understanding the molecular regulatory mechanism of Chinese cabbage clubroot resistance.

Along with being important pigments that determining the flower color in many plants, anthocyanins also perform crucial functions that attract pollinators and reduce abiotic stresses. Purple and white are two different colors of radish petals. In this study, two cDNA libraries constructed with purple and white petal plants were sequenced for transcriptome profiling. Transcriptome results implied that the expression level of the genes participating in the anthocyanin biosynthetic pathway was commonly higher in the purple petals than that in the white petals. In particular, two genes, F3′H and DFR, had a significantly higher expression pattern in the purple petals, suggesting the important roles these genes playing in radish petal coloration. BSA-seq aided-Next Generation Sequencing of two DNA pools revealed that the radish purple petal gene (RsPP) was located on chromosome 7. With additional genotyping of 617 F2 population plants, the RsPP was further confined within a region of 93.23 kb. Transcriptome and Sanger sequencing analysis further helped identify the target gene, Rs392880. Rs392880 is a homologous gene to F3′H, a key gene in the anthocyanin biosynthetic pathway. These results will aid in elucidating the molecular mechanism of plant petal coloration and developing strategies to modify flower color through genetic transformation.

Flower color is an important trait in plants. However, genes responsible for the white flower trait in Chinese cabbage are rarely reported. In this study, we constructed an F2 population derived from the Y640-288 (white flower) and Y641-87 (yellow flower) lines for the fine mapping of the white flower gene BrWF3 in Chinese cabbage. Genetic analysis indicated that BrWF3 was controlled by a single recessive gene. Using BSA-seq and KASP assays, BrWF3 was fine-mapped to an interval of 105.6 kb. Functional annotation, expression profiling, and sequence variation analyses confirmed that the AtPES2 homolog, Bra032957, was the most likely candidate gene for BrWF3. Carotenoid profiles and transmission electron microscopy analysis suggested that BrWF3 might participate in the production of xanthophyll esters (particularly violaxanthin esters), which in turn disrupt chromoplast development and the formation of plastoglobules (PGs). A SNP deletion in the third exon of BrWF3 caused the loss of protein function, and interfered with the normal assembly of PGs, which was associated with reduced expression levels of genes involved in carotenoid metabolism. Furthermore, we developed and validated the functional marker TXBH83 for BrWF3. Our results provide insight into the molecular mechanism underlying flower color pigmentation and reveal valuable information for marker-assisted selection (MAS) breeding in Chinese cabbage.

Chinese cabbage is one of the most important and widely consumed vegetables in China. The developmental transition from the vegetative to reproductive phase is a crucial process in the life cycle of flowering plants. In spring-sown Chinese cabbage, late bolting is desirable over early bolting. In this study, we analyzed double haploid (DH) lines of late bolting (“Y410-1” and “SY2004”) heading Chinese cabbage (Brassica rapa var. pekinensis) and early-bolting Chinese cabbage (“CX14-1”) (B. rapa ssp. chinensis var. parachinensis) by comparative transcriptome profiling using the Illumina RNA-seq platform. We assembled 721.49 million clean high-quality paired-end reads into 47,363 transcripts and 47,363 genes, including 3,144 novel unigenes. There were 12,932, 4,732, and 4,732 differentially expressed genes (DEGs) in pairwise comparisons of Y410-1 vs. CX14-1, SY2004 vs. CX14-1, and Y410-1 vs. SY2004, respectively. The RNA-seq results were confirmed by reverse transcription quantitative real-time PCR (RT-qPCR). A Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of DEGs revealed significant enrichment for plant hormone and signal transduction as well as starch and sucrose metabolism pathways. Among DEGs related to plant hormone and signal transduction, six unigenes encoding the indole-3-acetic acid-induced protein ARG7 (BraA02g009130), auxin-responsive protein SAUR41 (BraA09g058230), serine/threonine-protein kinase BSK11 (BraA07g032960), auxin-induced protein 15A (BraA10g019860), and abscisic acid receptor PYR1 (BraA08g012630 and BraA01g009450), were upregulated in both late bolting Chinese cabbage lines (Y410-1 and SY2004) and were identified as putative candidates for the trait. These results improve our understanding of the molecular mechanisms underlying flowering in Chinese cabbage and provide a foundation for studies of this key trait in related species.

Clubroot disease caused by Plasmodiophora brassicae is one of the major threats to Brassica crops. New clubroot resistant varieties of Chinese cabbage (B. rapa ssp. pekinensis) have been developed through breeding, but the underlying genetic mechanism of clubroot resistance is still unclear. In this study, two Chinese cabbage DH lines, clubroot-resistant Y635-10 and susceptible Y177-47 were crossed to develop F2 population for fine mapping and cloning resistance gene CRq. After sequence analysis, the expression vector was constructed by gateway technology and transferred into Arabidopsis thaliana for functional characterization. Bulked segregant analysis sequencing (BSA-seq) confirmed that CRq is located in the 80 kb genomic region on chromosome A03 between markers GC30-FW/RV and BGA. In silico tools confirmed that the gene length was 3959 bp with 3675 bp coding sequences (CDs), and it has three exons and two introns. In addition, we found 72bp insertion in the third exon of CRq in the susceptible line. We developed and verified functional marker Br-insert1, by which genotyping results showed that 72bp insertion might lead to the destruction of the LRR region of Y177-47, resulting in a loss of resistance relative to clubroot. The results of genetic transformation showed that the roots for wild-type Arabidopsis thaliana were significantly enlarged compared with T2 generation transgenic Arabidopsis after treatment by P. brassicae spores, and transgenic Arabidopsis had certain resistance. Therefore, CRq is a candidate gene of clubroot disease resistance in Chinese cabbage, which could be used as a reference for elucidating disease resistance mechanisms and the marker-assisted breeding of clubroot resistant varieties.

Chinese cabbage (Brassica rapa L.) is one of the most important and highly nutritious vegetables in China belonging to the Brassicaceae family. Flowering or bolting is one of the most critical developmental stages in flowering plants. For the spring-sown Chinese cabbage, late-bolting is desirable over early-bolting according to consumer preferences. We determined the inheritance pattern of the late-bolting trait using F1 and F2 generated from a cross between ‘SY2004’ (late-bolting) and ‘CX14-1’ (early-bolting). The genetic analysis revealed that the late-bolting to early-bolting trait was controlled by an incomplete dominant gene that we named BrLb-1. Furthermore, we performed bulked segregant analysis (BSA) via whole genome re-sequencing and the results showed that this gene was harbored on the chromosome A07 at the intersections of 20,070,000 to 25,290,000 bp and 20,330,000 to 25,220,000, an interval distance of 4.89 Mb. In this candidate interval, totals of 2321 and 1526 SNPs with non-synonymous mutations, and 229 and 131 InDels with frameshift mutations, were found between the parents and the bulked pools, respectively. Furthermore, we identified three putative candidate genes for the late-bolting trait, including BraA07g029500, BraA07g029530 and BraA07g030360, which code for the AGAMOUS-like MADS-box protein AGL12, a pentatricopeptide repeat-containing protein and NAC transcription factor 29, respectively; however, further functional analysis is required. These genetic variants could be utilized for the further development of molecular markers for marker-assisted breeding in Chinese cabbage.

Kompetitive allele-specific PCR (KASP) is a cost-effective single-step SNP genotyping technology, With an objective to enhance the marker repertoire and develop high efficient KASP-SNP markers in Chinese cabbage, we re-sequenced four Chinese cabbage doubled haploid (DH) lines, Y177-47, Y635-10, Y510-1 and Y510-9, and generated a total of more than 38.5 billion clean base pairs. A total of 827,720 SNP loci were identified with an estimated density of 3,217 SNPs/Mb. Further, a total of 387,354 SNPs with at least 30 bp to the next most adjacent SNPs on either side were selected as resource for KASP markers. From this resource, 258 (96.27%) of 268 SNP loci were successfully transformed into KASP-SNP markers using a Roche LightCycler 480-II instrument. Among these markers, 221 (85.66%) were co-dominant markers, 220 (85.27%) were non-synonymous SNPs, and 257 (99.6%) were newly developed markers. In addition, 53 markers were applied for genotyping of 34 Brassica rapa accessions. Cluster analysis separated these 34 accessions into three clusters based on heading types. The millions of SNP loci, a large set of resource for KASP markers, as well as the newly developed KASP markers in this study may facilitate further genetic and molecular breeding studies in Brassica rapa.

Ogura cytoplasmic male sterility (Ogura CMS), originally identified in wild radish (Raphanus sativus), has enabled complete pollen sterility in Brassica plants, but the underlying mechanism in Ogura CMS Chinese cabbage (Brassica rapa ssp. pekinensis) remains unclear. In this study cytological analysis showed that during microsporogenesis the meiosis occurred normally, and the uninucleated pollens subsequently formed, but the development of both binucleated and trinucleated pollens was obviously disrupted due to defects of pollen mitosis in the Ogura CMS line (Tyms) compared with the corresponding maintainer line (231–330). In transcriptome profiling a total of 8052 differentially expressed genes (DEGs) were identified, among which 3890 were up-regulated and 4162 were down-regulated at the pollen abortion stages in an Ogura CMS line. KOG cluster analysis demonstrated that a large number of DEGs were related to the cytoskeleton’s dynamics, which may account for the failure of pollen mitosis during development in the Ogura CMS line. The pivotal genes related to the phenylpropane synthesis pathway (PAL, 4CL and CAD) were significantly down-regulated, which probably affected the formation and disposition of anther lignin and sporopollenin, and eventually led to abnormality in the pollen exine structure. In addition, several key up-regulated genes (GPX7, G6PD and PGD1) related to the glutathione oxidation-reduction (REDOX) reaction indicated that the accumulation of peroxides in Ogura CMS lines during this period affected the pollen development. Taken together, this cytological and molecular evidence is expected to advance our understanding of pollen abortion induced by Ogura cytoplasmic action in Chinese cabbage.

Background: The large and complex hexaploid genome has greatly hindered genomics studies of common wheat (Triticum aestivum, AABBDD). Here, we investigated transcripts in common wheat developing caryopses using the emerging single-molecule real-time (SMRT) sequencing technology PacBio RSII, and assessed the resultant data for improving common wheat genome annotation and grain transcriptome research. Results: We obtained 197,709 full-length non-chimeric (FLNC) reads, 74.6 % of which were estimated to carry complete open reading frame. A total of 91,881 high-quality FLNC reads were identified and mapped to 16,188 chromosomal loci, corresponding to 13,162 known genes and 3026 new genes not annotated previously. Although some FLNC reads could not be unambiguously mapped to the current draft genome sequence, many of them are likely useful for studying highly similar homoeologous or paralogous loci or for improving chromosomal contig assembly in further research. The 91,881 high-quality FLNC reads represented 22,768 unique transcripts, 9591 of which were newly discovered. We found 180 transcripts each spanning two or three previously annotated adjacent loci, suggesting that they should be merged toform correct gene models. Finally, our data facilitated the identification of 6030 genes differentially regulated during caryopsis development, and full-length transcripts for 72 transcribed gluten gene members that are important for the end-use quality control of common wheat. Conclusions: Our work demonstrated the value of PacBio transcript sequencing for improving common wheat genome annotation through uncovering the loci and full-length transcripts not discovered previously. The resource obtained may aid further structural genomics and grain transcriptome studies of common wheat. [ABSTRACT FROM AUTHOR]

Wnt/β-catenin signaling plays critical roles in embryonic development and disease. Here, we identify RNF220, a RING domain E3 ubiquitin ligase, as a new regulator of β-catenin. RNF220 physically interacts with β-catenin, but instead of promoting its ubiquitination and proteasomal degradation, it stabilizes β-catenin and promotes canonical Wnt signaling. Our analysis showed that RNF220 interacts with USP7, a ubiquitin-specific peptidase, which is required for RNF220 to stabilize β-catenin. The RNF220/USP7 complex deubiquitinates β-catenin and enhances canonical Wnt signaling. Interestingly, the stability of RNF220 itself is negatively regulated by Gsk3β, which is a key component of the β-catenin destruction complex and is inhibited upon Wnt stimulation. Accordingly, the RNF220/USP7 complex works as a positive feedback regulator of β-catenin signaling. In colon cancer cells with stimulated Wnt signaling, knockdown of RNF220 or USP7 impairs Wnt signaling and expression of Wnt target genes, suggesting a potentially novel role of RNF220 in Wnt-related tumorigenesis. [ABSTRACT FROM AUTHOR]