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Abstract Background The low efficiency of genetic transformation in Chinese cabbage (Brassica rapa L. ssp. pekinensis) is the key problem affecting functional verification. Particle bombardment is a widely used method along with the Agrobacterium-mediated method. As a physical means, it has almost no restrictions on the type of host and a wide range of receptor types, which largely avoids the restriction of explants. The bombardment parameters, which include the number of bombardments, the bombardment pressure, and the bombardment distance, may affect the microspores' genetic transformation efficiency. Results The transformation efficiency was improved using the particle bombardment method under the combination of bombardment shot times (3, 4, 5) × bombardment pressure (900, 1100, 1350 psi) × bombardment distance (3, 6, 9 cm). The average viability of microspores in the treatment group ranged from 74.76 to 88.55%, while the control group was 88.09%. When the number of shot times was 4, the number of embryos incubated in the treatment group ranged from 16 to 236 per dish, and the control group had 117 embryos per dish. When the bombardment parameters of the biolistic method were 4 shot times—1350 psi—3 cm, 4 times—1100 psi—3 cm, and 4 times—900 psi—3 cm, they had high transient expression efficiency, and the average number of transformed microspores was 21.67, 11.67, and 11.67 per dish (3.5 mL), respectively. When the bombardment parameters were 4 times, 900 psi, and 6 cm, the highest genetically transformed embryos were obtained, and the transformation efficiency reached 10.82%. Conclusion A new genetic transformation system with proper parameters for Chinese cabbage microspores was established using particle bombardment. This proper transformation system could provide a useful tool for the improvement of cultivar quality and the investigation of functional genes in Chinese cabbage.

The purple-leaf phenotype in pak choi is due to the accumulation of anthocyanin. The main regulated genes are unclear. The gene controlling the purple-leaf phenotype was mapped on A03 using BSA-seq, but the candidate interval was not further narrowed with InDel markers. Based on our previous study, we hypothesized that the candidate gene that regulates purple leaves in pak choi may also be the Dark_Pur gene from B. juncea. Using the Dark_Pur-1 marker to identify P1, P2, F1, and F2, it was confirmed that the purple trait in purple-leaf pak choi was controlled by the Dark_Pur gene from B. juncea through distant hybridization. A DNA segment of approximately 514 Kb containing the Dark_Pur gene was reintroduced into pak choi from B. juncea. Meanwhile, a new purple pak choi germplasm line was created with green pak choi × purple B. juncea via distant hybridization, which proved that distant hybridization is an effective method for creating new germplasms. Furthermore, the purple-leaf phenotypes of 20 pak choi varieties were identified, and the purple-leaf traits of all lines were derived from B. juncea via distant hybridization. At present, few studies have focused on the background of the purple trait in pak choi; however, in this study, our results suggest that there is a high probability that the purple trait in pak choi may be completely derived from purple B. juncea via distant hybridization. This study also lays a good foundation for research on the creation of new germplasms through distant hybridization among the Brassica species.

The soil-borne pathogen Plasmodiophora brassicae is the causal agent of clubroot, a major disease in Chinese cabbage (Brassica rapa ssp. pekinensis). The host’s resistance genes often confer immunity to only specific pathotypes and may be rapidly overcome. Identification of novel clubroot resistance (CR) from germplasm sources is necessary. In this study, Bap246 was tested by being crossed with different highly susceptible B. rapa materials and showed recessive resistance to clubroot. An F2 population derived from Bap246 × Bac1344 was used to locate the resistance Quantitative Trait Loci (QTL) by Bulk Segregant Analysis Sequencing (BSA-Seq) and QTL mapping methods. Two QTL on chromosomes A01 (4.67–6.06 Mb) and A08 (10.42–11.43 Mb) were found and named Cr4Ba1.1 and Cr4Ba8.1, respectively. Fifteen and eleven SNP/InDel markers were used to narrow the target regions in the larger F2 population to 4.67–5.17 Mb (A01) and 10.70–10.84 Mb (A08), with 85 and 19 candidate genes, respectively. The phenotypic variation explained (PVE) of the two QTL were 30.97% and 8.65%, respectively. Combined with gene annotation, mutation site analysis, and real-time quantitative polymerase chain reaction (qRT-PCR) analysis, one candidate gene in A08 was identified, namely Bra020861. And an insertion and deletion (InDel) marker (co-segregated) named Crr1-196 was developed based on the gene sequence. Bra013275, Bra013299, Bra013336, Bra013339, Bra013341, and Bra013357 in A01 were the candidate genes that may confer clubroot resistance in Chinese cabbage. The resistance resource and the developed marker will be helpful in Brassica breeding programs.

Vernalization plays a crucial role in the flowering and yield of Chinese cabbage, a process intricately influenced by long non-coding RNAs (lncRNAs). Our research focused on lncFLC1, lncFLC2a, and lncFLC2b, which emerged as key players in this process. These lncRNAs exhibited an inverse expression pattern to the flowering repressor genes FLOWERING LOCUS C 1 (BrFLC1) and FLOWERING LOCUS C 2 (BrFLC2) during vernalization, suggesting a complex regulatory mechanism. Notably, their expression in the shoot apex and leaves was confirmed through in fluorescent in situ hybridization (FISH). Furthermore, when these lncRNAs were overexpressed in Arabidopsis, a noticeable acceleration in flowering was observed, unveiling functional similarities to Arabidopsis’s COLD ASSISTED INTRONIC NONCODING RNA (COOLAIR). This resemblance suggests a potentially conserved regulatory mechanism across species. This study not only enhances our understanding of lncRNAs in flowering regulation, but also opens up new possibilities for their application in agricultural practices.

In order to make the identification and utilization of nutritional quality components in Chinese cabbage more predictive, to obtain ideal raw materials, and to help screen functional Chinese cabbage varieties that have high nutritional value, we conducted a comprehensive evaluation of the nutritional quality of different Chinese cabbage varieties. In this study, 17 nutritional quality indexes of 35 Chinese cabbage varieties, including crude fiber (CF), crude protein (CP), vitamin C (VC), glucose (Glc), fructose (Fru), malic acid (MA), citric acid (CA), oxalic acid (OA), total amino acid (TAA) and CA, K, Mg, P, Cu, Fe, Mn and Zn, were analyzed using diversity analysis, correlation analysis, principal component analysis, membership function analysis and cluster analysis. The results showed that there were different degrees of variation in the 17 nutritional quality indexes, and the coefficients of variation ranged from 11.45% to 91.47%. The correlation analysis found that there were significant or extremely significant correlations between different nutrient elements of Chinese cabbage, which indicated that principal component analysis could be carried out, and the comprehensive score (D value) of different materials could be obtained using principal component analysis and the membership function method. The nutritional quality of Chinese cabbage was classified into five grades by cluster analysis. Finally, a mathematical model for evaluating the nutritional quality of Chinese cabbage was established using the D value and multiple stepwise regression methods, and 10 key indexes were selected from the 17 indexes, which could be used for the rapid identification of the nutritional quality of Chinese cabbage. This study provided a theoretical basis for the nutritional quality evaluation and variety breeding of Chinese cabbage.

Abstract Chinese cabbage that prefers cold conditions is also affected by low-temperature stress, such as the accumulation of leaf anthocyanins. Research on anthocyanin biosynthesis and regulation mechanisms has made great progress. However, research on anthocyanin accumulation for resistance to biological and non-biological stress is still lacking. To study the relationship between anthocyanin accumulation of Chinese cabbage and resistance under low-temperature conditions, RNA sequencing (RNA-seq) was performed on Chinese cabbage ‘Xiao Baojian’ grown at a low temperature for four time periods and at a control temperature for five time periods. In Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, 7954 differentially expressed genes (DEGs) were enriched, of which 587 DEGs belonged to "biosynthesis of other secondary metabolites." Gene temporal expression patterns were used to discover enriched genes related to phenylpropanoid biosynthesis; flavonoid biosynthesis and anthocyanin biosynthesis pathways were found in cluster 1. The interaction networks were constructed, and hub genes were selected, showing that flavonoid biosynthesis pathway genes (DFR, ANS, F3H, FLS1, CHS1, CHS3, and TT8) and defense mechanisms-related genes (DFR, SNL6, and TKPR1) interact with each other. Anthocyanin biosynthesis DEGs in Chinese cabbage were evaluated under low-temperature conditions to map the relevant pathways, and expression maps of transcription factors in the flavonoid pathway were created at various periods. Low temperature upregulated the expression of genes related to anthocyanin biosynthesis. Taken together, our results provide further analysis of the relationship between plant anthocyanin synthesis and stress resistance and may also provide further insights for the future development of high-quality color and cold-tolerant Chinese cabbage germplasm resources.

Vernalization is a phenomenon in which plants must undergo a period of continuous low temperatures to change from the vegetative growth stage to the reproductive growth stage. Chinese cabbage is a heading vegetable, and flowering time is an essential developmental trait. Premature vernalization leads to premature bolting, which causes a loss of product value and yield. While research into vernalization has provided a wealth of information, a complete understanding of the molecular mechanism for controlling vernalization requirements has not yet been elucidated. In this study, using high-throughput RNA sequencing, we analyzed the plumule-vernalization response of mRNA and long noncoding RNA in the bolting-resistant Chinese cabbage double haploid (DH) line ‘Ju Hongxin’ (JHX). A total of 3382 lncRNAs were identified, of which 1553 differentially expressed (DE) lncRNAs were characterized as plumule-vernalization responses. The ceRNA network revealed that 280 ceRNA pairs participated in the plumule-vernalization reaction of Chinese cabbage. Through identifying DE lncRNAs in Chinese cabbage and analyzing anti-, cis-, and trans-functional analysis, some candidate lncRNAs related to vernalization promoting flowering of Chinese cabbage and their regulated mRNA genes were found. Moreover, the expression of several critical lncRNAs and their targets was verified using qRT-PCR. Furthermore, we identified the candidate plumule-vernalization-related long noncoding RNAs that regulate BrFLCs in Chinese cabbage, which was interesting and different from previous studies and was a new discovery. Our findings expand the knowledge of lncRNAs in the vernalization of Chinese cabbage, and the identified lncRNAs provide rich resources for future comparative and functional studies.

Ogura cytoplasmic male sterility (CMS) is one of the important methods for hybrid seed production in cruciferous crops. The lack of a restorer of fertility gene (Rfo) in Brassica rapa L. restricts the development and utilization of its germplasm resources. In this research, Brassica napus with the Rfo gene was used to restore the fertility of Ogura CMS B. rapa with the golden heart trait. Through the distant cross of two B. rapa and four B. napus, six interspecific hybrid combinations received F1 seeds. The six combinations were different in seed receiving. By morphological observation and molecular marker-assisted selection (MAS), in F1, individuals containing the Rfo gene all appeared fertile, while those without it remained male-sterile. The pollen viability of the fertile individuals was measured, and the fertile lines of the six interspecific hybrid combinations were different (40.68–80.49%). Three individuals (containing both Rfo and GOLDEN genes) with the highest pollen vitality (≥60%) were backcrossed with fertile cytoplasmic B. rapa, resulting in a total of 800 plants. Based on the MAS, a total of 144 plants with GOLDEN but no Rfo were screened (18%). Moreover, through morphological investigation, one individual with normal cytoplasm, stable fertility but without the restoring gene Rfo, the GOLDEN gene, and morphological characteristics similar to those of B. rapa was obtained. These results increased the diversity of B. rapa germplasm and provided a new method for the utilization of CMS germplasm in Brassica crops.

Abstract Background Vernalization is a type of low temperature stress used to promote rapid bolting and flowering in plants. Although rapid bolting and flowering promote the reproduction of Chinese cabbages (Brassica rapa L. ssp. pekinensis), this process causes their commercial value to decline. Clarifying the mechanisms of vernalization is essential for its further application. We performed RNA sequencing of gradient-vernalization in order to explore the reasons for the different bolting process of two Chinese cabbage accessions during vernalization. Results There was considerable variation in gene expression between different-bolting Chinese cabbage accessions during vernalization. Comparative transcriptome analysis and weighted gene co-expression network analysis (WGCNA) were performed for different-bolting Chinese cabbage during different vernalization periods. The biological function analysis and hub gene annotation of highly relevant modules revealed that shoot system morphogenesis and polysaccharide and sugar metabolism caused early-bolting ‘XBJ’ to bolt and flower faster; chitin, ABA and ethylene-activated signaling pathways were enriched in late-bolting ‘JWW’; and leaf senescence and carbohydrate metabolism enrichment were found in the two Chinese cabbage-related modules, indicating that these pathways may be related to bolting and flowering. The high connectivity of hub genes regulated vernalization, including MTHFR2, CPRD49, AAP8, endoglucanase 10, BXLs, GATLs, and WRKYs. Additionally, five genes related to flower development, BBX32 (binds to the FT promoter), SUS1 (increases FT expression), TSF (the closest homologue of FT), PAO and NAC029 (plays a role in leaf senescence), were expressed in the two Chinese cabbage accessions. Conclusion The present work provides a comprehensive overview of vernalization-related gene networks in two different-bolting Chinese cabbages during vernalization. In addition, the candidate pathways and hub genes related to vernalization identified here will serve as a reference for breeders in the regulation of Chinese cabbage production.

Plants protect against viruses through passive and active resistance mechanisms, and in most cases characterized thus far, natural recessive resistance to potyviruses has been mapped to mutations in the eukaryotic initiation factor eIF4E or eIF(iso)4E genes. Five eIF4E copies and three eIF(iso)4E copies were detected in Brassica rapa. The eIF4E and eIF(iso)4E genes could interact with turnip mosaic virus (TuMV) viral protein linked to the genome (VPg) to initiate virus translation. From the yeast two-hybrid system (Y2H) and bimolecular fluorescence complementation (BiFC) assays, the TuMV-CHN2/CHN3 VPgs could not interact with BraA.eIF4E.a/c or BraA.eIF(iso)4E.c, but they could interact with BraA.eIF(iso)4E.a in B. rapa. Further analysis indicated that the amino acid substitution L186F (nt T556C) in TuMV-UK1 VPg was important for the interaction networks between the TuMV VPg and eIF(iso)4E proteins. An interaction model of the BraA. eIF(iso)4E protein with TuMV VPg was constructed to infer the effect of the significant amino acids on the interaction of TuMV VPgs-eIF(iso)4Es, particularly whether the L186F in TuMV-UK1 VPg could change the structure of the TuMV-UK1 VPg protein, which may terminate the interaction of the BraA.eIF(iso)4E and TuMV VPg protein. This study provides new insights into the interactions between plant viruses and translation initiation factors to reveal the working of key amino acids.

Clubroot is a serious soil-borne disease of crucifers caused by the obligate parasite Plasmodiophora brassicae. The genetic basis and histopathology of clubroot resistance in two Chinese cabbage (Brassica rapa ssp. pekinensis) inbred lines Bap055 and Bap246, challenged with pathotype 4 of P. brassicae, was evaluated. The Chinese cabbage cultivar “Juxin” served as a susceptible check. The resistance in Bap055 was found to be controlled by the CRa gene, while resistance in Bap246 fit a model of control by unknown recessive gene. Infection of the roots by P. brassicae was examined by inverted microscopy. Despite their resistance, primary and secondary infection were observed to occur in Bap055 and Bap246. Primary infection was detected at 2 days post-inoculation (DPI) in “Juxin,” at 4 DPI in Bap055, and at 6 DPI in Bap246. Infection occurred most quickly on “Juxin,” with 60% of the root hairs infected at 10 DPI, followed by Bap055 (31% of the root hairs infected at 12 DPI) and Bap246 (20% of the root hairs infected at 14 DPI). Secondary infection of “Juxin” was first observed at 8 DPI, while in Bap055 and Bap246, secondary infection was first observed at 10 DPI. At 14 DPI, the percentage of cortical infection in “Juxin,” Bap055 and Bap246 was 93.3, 20.0, and 11.1%, respectively. Although cortical infection was more widespread in Bap055 than in Bap246, secondary infection in both of these hosts was restricted relative to the susceptible check, and the vascular system remained intact. A large number of binucleate secondary plasmodia were observed in “Juxin” and the vascular system was disrupted at 16 DPI; in Bap055 and Bap246, only a few secondary plasmodia were visible, with no binucleate secondary plasmodia. The defense mechanisms and expression of resistance appears to differ between Chinese cabbage cultivars carrying different sources of resistance.

Chinese cabbage, which is a cold season crop, can still be damaged at an overly low temperature. It is crucial to study the mechanism of the resistance to low temperature of Chinese cabbage. In this study, the Chinese cabbage ‘XBJ’ was used as the material, and nine different low temperatures and control samples were treated. Using RNA-seq and lignin content determination, we analyzed 27 samples, and the stained sections of them were observed. A total of 8845 genes were screened for the WGCNA analysis, yielding 17 modules. The GO and KEGG analyses of the modules was highly associated with a low-temperature treatment. The pathways such as ‘starch and sucrose metabolism’ and ‘plant hormone signal transduction’ were enriched in modules related to low temperature. Interestingly, L-15DAT-associated MEcoral2 was found to have 14 genes related to the ‘lignin biosynthetic process’ in the GO annotation. The combination of the determination of the lignin content and the treatment of the stained sections showed that the lignin content of the low-temperatures samples were indeed higher than that of the control. We further explored the expression changes of the lignin synthesis pathway and various genes and found that low temperature affects the expression changes of most genes in the lignin synthesis pathway, leading to the speculation that the lignin changes at low temperature are a defense mechanism against low temperatures. The 29 BrCOMT gene sequence derived from the RNA-seq was non-conserved, and eight BrCOMT genes were differentially expressed. This study provides a new insight into how lignin is affected by low temperature.

In plants, the accumulation of carotenoids can maintain the balance of the photosystem and improve crop nutritional quality. Therefore, the molecular mechanisms underlying carotenoid synthesis and accumulation should be further explored. In this study, carotenoid accumulation differed significantly among parental Brassica rapa. Genetic analysis was carried out using the golden inner leaf ‘1900264′ line and the light−yellow inner leaf ‘1900262′ line, showing that the golden inner leaf phenotype was controlled by a single dominant gene. Using bulked−segregant analysis sequencing, BraA09g007080.3C encoding the ORANGE protein was selected as a candidate gene. Sequence alignment revealed that a 4.67 kb long terminal repeat insertion in the third exon of the BrGOLDEN resulted in three alternatively spliced transcripts. The spatiotemporal expression results indicated that BrGOLDEN might regulate the expression levels of carotenoid−synthesis−related genes. After transforming BrGOLDEN into Arabidopsis thaliana, the seed−derived callus showed that BrGOLDENIns and BrGOLDENDel lines presented a yellow color and the BrGOLDENLdel line presented a transparent phenotype. In addition, using the yeast two−hybrid assay, BrGOLDENIns, BrGOLDENLdel, and Brgoldenwt exhibited strong interactions with BrPSY1, but BrGOLDENDel did not interact with BrPSY1 in the split−ubiquitin membrane system. In the secondary and 3D structure analysis, BrGOLDENDel was shown to have lost the PNFPSFIPFLPPL sequences at the 125 amino acid position, which resulted in the α−helices of BrGOLDENDel being disrupted, restricting the formation of the 3D structure and affecting the functions of the protein. These findings may provide new insights into the regulation of carotenoid synthesis in B. rapa.

Brassica rapa is one of the most important leafy vegetables worldwide, and has a long history of cultivation. However, it has not been possible to completely control the damage of turnip mosaic virus (TuMV), a serious virus in B. rapa, to production. In this study, the genome-wide identification and expression detection of eIF family genes from B. rapa in response to TuMV resistance were analyzed, including the identification of eIF family genes, chromosomal distribution, three-dimensional (3D) structure and sequence logo analyses, and the expression characterization as well as differential metabolite analysis of eIF family genes in resistant/susceptible lines, which may further prove the whole-genome tripling (WGT) event in B. rapa evolution and provide evidence for the functional redundancy and functional loss of multicopy eIF genes in evolution. A qRT-PCR analysis revealed that the relative expressions of eIF genes in a susceptible line (80461) were higher than those in a resistant line (80124), which may prove that, when TuMV infects host plants, the eIF genes can combine with the virus mRNA 5′ end cap structure and promote the initiation of virus mRNA translation in the susceptible B. rapa line. In addition, the metabolite substances were detected, the differences in metabolites between disease-resistant and disease-susceptible plants were mainly manifested by altered compounds such as flavonoids, jasmonic acid, salicylic acid, ketones, esters, etc., which inferred that the different metabolite regulations of eIF family genes and reveal the resistance mechanisms of eIF genes against TuMV in brassica crops. This study may lay a new theoretical foundation for revealing eIF family gene resistance to TuMV in B. rapa, as well as advancing our understanding of virus–host interactions.

Turnip mosaic virus (TuMV) is an important threat to the yield and quality of brassica crops in China, and has brought serious losses to brassica crops in the Far East, including China and the north. Aphids (Hemiptera, Aphidoidea) are the main mediators of TuMV transmission in field production, and not only have strong virus transmission ability (small individuals, strong concealment, and strong fecundity), but are also influenced by the environment, making them difficult to control. Till now, there have been few studies on the resistance to aphids in brassica crops, which depended mainly on pesticide control in agriculture production. However, the control effect was temporarily effective, which also brought environmental pollution, pesticide residues in food products, and destroyed the ecological balance. This study reviews the relationship among brassica crop–TuMV, TuMV–aphid, and brassica crop–aphid interactions, and reveals the influence factors (light, temperature, and CO2 concentration) on brassica crop–TuMV–aphid interactions, summarizing the current research status and main scientific problems about brassica crop–TuMV–aphid interactions. It may provide theoretical guidance for opening up new ways of aphid and TuMV management in brassica crops.

Turnip mosaic virus (TuMV), which is distributed almost all over the world and has a wide range of hosts, mainly brassica crops, was first described in Brassica rapa in the USA. Plant volatile compounds play an important role in the host searching behavior of natural enemies of herbivorous insects. In this study, TuMV-inoculated resistant and susceptible B. rapa lines were tested using volatile metabolome and transcriptome analyses. In volatile metabolome analysis, the volatile organic compounds (VOCs) were different after inoculation with TuMV in resistant B80124 and susceptible B80461, and the degree of downregulation of differentially expressed metabolites was more obvious than the degree of upregulation. Through transcriptome analysis, 70% of differentially expressed genes were in biological process, especially focusing on defense response, flavonoid biosynthetic process, and toxin metabolic process, which indicates that TuMV stress maybe accelerate the increase of VOCs. Integrating the metabolome and transcriptome analyses, after inoculating with TuMV, auxin regulation was upregulated, and ARF, IAA and GH3 were also upregulated, which accelerated cell enlargement and plant growth in tryptophan metabolism. The different genes in zeatin biosynthesis pathways were downregulated, which reduced cell division and shoot initiation. However, the metabolite pathways showed upregulation in brassinosteroid biosynthesis and α-linolenic acid metabolism, which could cause cell enlargement and a stress response. This study determined the difference in volatiles between normal plants and infected plants and may lay a foundation for anti-TuMV research in B. rapa.

European fodder turnips (Brassica rapa ssp. rapifera) were identified as sources of clubroot resistance (CR) and have been widely used in Brassica resistance breeding. An F2 population derived from a cross between a resistant turnip and a susceptible Chinese cabbage was used to determine the inheritance and locating the resistance Quantitative Trait Loci (QTLs). The parents showed to be very resistant/susceptible to the field isolates (pathotype 4) of clubroot from Henan in China. After inoculation, 27 very resistant or susceptible individuals were selected to construct bulks, respectively. Next-generation-sequencing-based Bulk Segregant Analysis Sequencing (BSA-Seq) was used and located resistance QTL on chromosome A03 (3.3–7.5 Mb) and A08 (0.01–6.5 Mb), named Bcr1 and Bcr2, respectively. Furthermore, an F3 population including 180 families derived from F2 individuals was phenotyped and used to verify and narrow candidate regions. Ten and seven Kompetitive Allele-Specific PCR (KASP) markers narrowed the target regions to 4.3–4.78 Mb (A03) and 0.02–0.79 Mb (A08), respectively. The phenotypic variation explained (PVE) of the two QTLs were 33.3% and 13.3% respectively. The two candidate regions contained 99 and 109 genes. In the A03 candidate region, there were three candidate R genes, namely Bra006630, Bra006631 and Bra006632. In the A08 candidate region, there were two candidate R genes, namely Bra030815 and Bra030846.

Chinese cabbage (Brassica rapa) is a major vegetable crop in China. The accumulation of anthocyanins improves the quality and flavor of Brassica crops and is beneficial for human health. There has been great research interest in breeding purple Chinese cabbage, for which it is necessary to study the key genes and mechanisms of anthocyanin accumulation. Through distant hybridization between purple mustard (Brassica juncea) and green Chinese cabbage (B. rapa), purple Chinese cabbage plants were obtained. Furthermore, the Dark_Pur gene was cloned in the purple Chinese cabbage plants, which came from purple mustard and may be responsible for the purple phenotype in purple Chinese cabbage plants. Through particle bombardment of isolated microspores from Chinese cabbage to transform the Dark_Pur gene, the transformed purple Chinese cabbage plant was obtained, thus verifying the function of the Dark_Pur gene. To further study the Dark_Pur gene regulatory mechanism of anthocyanin accumulation in Chinese cabbage, the purple/green Chinese cabbage lines and purple/green mustard lines were subjected to transcriptome–metabolome analysis. Three stages (cotyledon, seedling, and large-leaf stages) of the purple/green Chinese cabbage lines and purple/green mustard lines were selected for analysis. The results indicated that the expression level of the transcription factor genes BraA09g028560.3C, BraA03g019460.3C, and BraA07g035710.3C may be induced by the Dark_Pur gene and they play an important role in purple Chinese cabbage, and BjuB010898 and BjuO006089 may be responsible for anthocyanin accumulation in mustard. Studying the structural genes of the purple Chinese cabbage showed that PAL, C4H, 4CL, CHS, CHI, F3H, F3’H, FLS, DFR, ANS, and UGT were up-regulated in three growth periods. There were 22 and 10 differentially expressed metabolites (DEMs) in seedling and large-leaf stages between purple/green Chinese cabbage, respectively, and 12 and 14 differentially expressed metabolites (DEMs) in seedling and large-leaf stages between purple/green mustard, respectively, which may indicate that the Dark_Pur gene from purple mustard greatly regulates anthocyanin accumulation in purple Chinese cabbage. This study provides a foundation for further elucidating anthocyanin regulation.

Brassica rapa L. is cultivated globally and consumed in many areas worldwide. Using the transgenic Agrobacterium-mediated transformation method, which is a reproducible and efficient technique, genes can be transferred into various B. rapa species. This review summarizes the processes involved in Agrobacterium-mediated transformation of B. rapa, including surface seed sterilization, co-cultivation with A. tumefaciens, induction of callus/shoot/root formation, and confirmation of transgenic plants. In addition, factors such as the Agrobacterium strain, plant genotype, explant age, transformation efficiency of the hybrid or inbred line, and the concentrations of N6-benzyl amino purine and naphthalene acetic acid, are discussed. And this review shows clearly how to do it, what to do, and what not to do in the transgenic Agrobacterium-mediated in Brassica rapa. The information presented here lays the foundation for a simple and efficient method that resolves existing problems and improves overall transgenic B. rapa production, thereby benefiting both basic and applied research. Keywords: Brassica rapa, Agrobacterium tumefaciens, transgenic, transformation

Allopolyploidy is an evolutionary and mechanisticaly intriguing process involving the reconciliation of two or more sets of diverged genomes and regulatory interactions, resulting in new phenotypes. In this study, we explored the small RNA changes of eight F2 synthetic B. napus using small RNA sequencing. We found that a part of miRNAs and siRNAs were non-additively expressed in the synthesized B. napus allotetraploid. Differentially expressed miRNAs and siRNAs differed among eight F2 individuals, and the differential expression of miR159 and miR172 was consistent with that of flowering time trait. The GO enrichment analysis of differential expression miRNA target genes found that most of them were concentrated in ATP-related pathways, which might be a potential regulatory process contributing to heterosis. In addition, the number of siRNAs present in the offspring was significantly higher than that of the parent, and the number of high parents was significantly higher than the number of low parents. The results have shown that the differential expression of miRNA lays the foundation for explaining the trait separation phenomenon, and the significant increase of siRNA alleviates the shock of the newly synthesized allopolyploidy. It provides a new perspective between small RNA changes and trait separation in the early stages of allopolyploid polyploid formation.

Allopolyploidy is an evolutionary and mechanistically intriguing process involving the reconciliation of two or more sets of diverged genomes and regulatory interactions, resulting in new phenotypes. In this study, we explored the gene expression patterns of eight F2 synthetic Brassica napus using RNA sequencing. We found that B. napus allopolyploid formation was accompanied by extensive changes in gene expression. A comparison between F2 and the parent shows a certain proportion of differentially expressed genes (DEG) and activation\silent gene, and the two genomes (female parent (AA)\male parent (CC) genomes) showed significant differences in response to whole-genome duplication (WGD); non-additively expressed genes represented a small portion, while Gene Ontology (GO) enrichment analysis showed that it played an important role in responding to WGD. Besides, genome-wide expression level dominance (ELD) was biased toward the AA genome, and the parental expression pattern of most genes showed a high degree of conservation. Moreover, gene expression showed differences among eight individuals and was consistent with the results of a cluster analysis of traits. Furthermore, the differential expression of waxy synthetic pathways and flowering pathway genes could explain the performance of traits. Collectively, gene expression of the newly formed allopolyploid changed dramatically, and this was different among the selfing offspring, which could be a prominent cause of the trait separation. Our data provide novel insights into the relationship between the expression of differentially expressed genes and trait segregation and provide clues into the evolution of allopolyploids.

The swollen root is an important agronomic trait and is a determinant of yield for turnips, which are cultivated as both vegetables and fodder. However, the genetic mechanism of swollen root formation is poorly understood. In this study, we analyzed the F2 and BC1P2 populations derived from a cross between “10601” (European turnip with swollen root, Brassica rapa ssp. rapifera, AA, 2n = 2× = 20) and “10603” (Chinese cabbage with normal root, Brassica rapa ssp. pekinensis, AA, 2n = 2× = 20), and suggested that the swollen root is a quantitative trait. Two major quantitative trait loci (QTLs), FR1.1 (Fleshy root 1.1) and FR7.1 (Fleshy root 7.1), were identified by QTL-seq analysis and further confirmed by QTL mapping in F2 and BC1P2 populations. The QTL FR1.1 with a likelihood of odd (LOD) of 7.01 explained 17.2% of the total phenotypic variations for root diameter and the QTL FR7.1 explained 23.0% (LOD = 9.38) and 31.0% (LOD = 13.27) of the total phenotypic variations in root diameter and root weight, respectively. After a recombinant screening, the major QTL FR7.1 was further narrowed down to a 220 kb region containing 47 putative genes. A candidate gene, Bra003652, which is a homolog of AT1G78240 that plays an essential role in cell adhesion and disorganized tumor-like formation in Arabidopsis thaliana, was identified in this region. In addition, expression and parental allele analysis supported that Bra003652 was a possible candidate gene of QTL FR7.1 for swollen root formation in turnip. Our research may provide new insight into the molecular mechanism of swollen root formation in root crops.

Brassica rapa is an important Chinese vegetable crop that is beneficial to human health. The primary factor affecting B. rapa yield is low temperature, which promotes bolting and flowering, thereby lowering its commercial value. However, quickened bolting and flowering can be used for rapid breeding. Therefore, studying the underlying molecular mechanism of vernalization in B. rapa is crucial for solving production-related problems. Here, the transcriptome of two B. rapa accessions were comprehensively analyzed during different vernalization periods. During vernalization, a total of 974,584,022 clean reads and 291.28 Gb of clean data were obtained. Compared to the reference genome of B. rapa, 44,799 known genes and 2280 new genes were identified. A self-organizing feature map analysis of 21,035 differentially expressed genes was screened in two B. rapa accessions, ‘Jin Wawa’ and ‘Xiao Baojian’. The analysis indicated that transcripts related to the plant hormone signal transduction, starch and sucrose metabolism, photoperiod and circadian clock, and vernalization pathways changed notably at different vernalization periods. Moreover, different expression patterns of TPS, UGP, CDF, VIN1, and seven hormone pathway genes were observed during vernalization between the two accessions. The transcriptome results of this study provide a new perspective on the changes that occur during B. rapa vernalization, as well as serve as an excellent reference for B. rapa breeding.

Wucai is a biannual leafy vegetable that is more sensitive to cold than most Brassicaceae vegetables. Here, phenotypic differences in wucai were investigated to identify those genotypes with high cold tolerance at low temperature, which could allow their cultivation in cold climates. In all, on the basis of PIabs, 20 wucai genotypes (10 lower PIabs and 10 higher PIabs) were selected from a collection of 124 genotypes. Both W16-13 and SW-3 (higher PIabs) and the LS-6 and W15-16 (lower PIabs) were screened from the 20 wucai genotypes according to several key plant physiological traits: net photosynthetic rate, electrolyte leakage, chilling injury index, specific leaf area, malondialdehyde contents, and PIabs. To further verify the reliability of PIabs, four genotypes were grown under low temperature gradient conditions and their morphological indexes, chlorophyll content, total antioxidant capacity, fluorescence transients (OJIP transients), leaf cell ultrastructure, and gene expression were measured. These indicators strongly demonstrated that the W16-13 and SW-3, which had higher PIabs, possessed higher resistance to cold stress, while both LS-6 and W15-16 were sensitive to cold. As this study shows, the easily measured performance index, PIabs, could be applied to wucai genotypes to screen for one or more varieties characterized by higher cold tolerance.

Interspecific hybridization is considered to be an important driving force in the evolution, diversification, and formation of plant species. We selected one flowering Chinese cabbage variety and three Chinese kale varieties to make hybrids. Heterologous haploid offspring were obtained by embryo rescue and heterologous diploids were obtained by colchicine doubling. A total of 108 individuals of the F2, F3 and F4 generations from three parental combinations were investigated for field traits and SSR (simple sequence repeats) markers. The results showed trait separation and the appearance and disappearance of SSR bands in the hybrids, showing significant differences among parental combinations and among the different generations. This proved that the phenotypes of the initial generations of allopolyploids were not stable. This study not only enriches the genetic resources available for breeding flowering Chinese cabbage and Chinese kale, but lays a theoretical foundation for exploring the segregation of traits in distant hybrids and in different generations.

Introgression breeding is a widely used method for the genetic improvement of crop plants; however, the mechanism underlying candidate gene flow patterns during hybridization is poorly understood. In this study, we used a powerful pipeline to investigate a Chinese cabbage (Brassica rapa L. ssp. pekinensis) introgression line with the anthocyanin overaccumulation phenotype. Our purpose was to analyze the gene flow patterns during hybridization and elucidate the genetic factors responsible for the accumulation of this important pigment compound. We performed RNA-seq analysis by using two pipelines, one with and one without a reference sequence, to obtain transcriptome data. We identified 930 significantly differentially expressed genes (DEGs) between the purple-leaf introgression line and B. rapa green cultivar, namely, 389 up-regulated and 541 down-regulated DEGs that mapped to the B. rapa reference genome. Since only one anthocyanin pathway regulatory gene was identified, i.e., Bra037887 (bHLH), we mined unmapped reads, revealing 2,031 de novo assembled unigenes, including c3563g1i2. Phylogenetic analysis suggested that c3563g1i2, which was transferred from the Brassica B genome of the donor parental line Brassica juncea, may represent an R2R3-MYB transcription factor that participates in the ternary transcriptional activation complex responsible for the anthocyanin overaccumulation phenotype of the B. rapa introgression line. We also identified genes involved in cold and light reaction pathways that were highly upregulated in the introgression line, as confirmed using quantitative real-time PCR analysis. The results of this study shed light on the mechanisms underlying the purple leaf trait in Brassica plants and may facilitate the use of introgressive hybridization for many traits of interest.

AbstractPolyketide synthases (PKSs) utilize the products of primary metabolism to synthesize a wide array of secondary metabolites in both prokaryotic and eukaryotic organisms. PKSs can be grouped into three distinct classes, type I, II, and III, based on enzyme structure, substrate specificity, and catalytic mechanisms. The type III PKS enzymes function as homodimers, and are the only class of PKS that do not require acyl carrier protein. Plant type III PKS enzymes, also known as chalcone synthase (CHS)-like enzymes, are of particular interest due to their functional diversity. In this study, we mined type III PKS gene sequences from the genomes of six aquatic algae and twenty-five land plants (one bryophyte, one lycophyte, two basal angiosperms, sixteen core eudicots, and five monocots). PKS III sequences were found relatively conserved in all embryophytes, but not exist in algae. We also examined gene expression patterns by analyzing available transcriptome data, and identified potential cis regulatory elements in upstream sequences. Phylogenetic trees of dicots angiosperms showed that plant type III PKS proteins fall into three clades. Clade A contains CHS/STS-type enzymes coding genes with diverse transcriptional expression patterns and enzymatic functions, while clade B is further divided into subclades b1 and b2, which consist of anther-specific CHS-like enzymes. Differentiation regions, such as amino acids 196-207 between clades A and B, and predicted positive selected sites within α-helixes in late appeared branches of clade A, account for the major diversification in substrate choice and catalytic reaction. The integrity and location of conserved cis-elements containing MYB and bHLH binding sites can affect transcription levels. Potential binding sites for transcription factors such as WRKY, SPL or AP2/EREBP may contribute to tissue- or taxon-specific differences in gene expression. Our data shows that gene duplications and functional diversification of plant type III PKS enzymes played a critical role in the ancient conquest of the land by early plants and angiosperm diversification.Keywords PKS III multigene family, CHS, STS, phylogenetic reconstruction, functional diversification, gene expression, cis-elements