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This study evaluated the genotyping by sequencing (GBS) protocol for fingerprinting Brassica rapa, and the data derived were more reliable than the re-sequencing data of B. rapa. Of the 10 enzyme solutions used to analyze the numbers of genotypes and single-nucleotide polymorphisms (SNPs) in B. rapa, five solutions showed better results, namely, A (HaeIII, 450–500 bp), E (RsaI+HaeIII, 500–550 bp), F (RsaI+HaeIII, 500–600 bp), G (RsaI+HaeIII, ‘All’ fragment), and J (RsaI+EcoRV-HF®, ‘All’ fragment). The five enzyme solutions showed less than 40% similarity in different individuals from various samples, and 90% similarity between two individuals from one sample. The E enzyme solution was the most suitable for fingerprinting B. rapa, revealing well-distributed SNPs in the whole genome. Of the 82 highly inbred lines and 18 F1 lines of B. rapa sequenced by GBS in the E enzyme solution, known parents of 10 F1 lines were verified, and male parents were discovered for 8 F1 lines that had only known female parents. This study provides a valuable method for screening parents for F1 lines in B. rapa for the efficient evaluation of GBS with varied library construction strategies.

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.

By constructing an F

BackgroundVernalization 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 rapaL.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.ResultsThere 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, includingMTHFR2,CPRD49,AAP8, endoglucanase 10,BXLs,GATLs, andWRKYs. Additionally, five genes related to flower development,BBX32(binds to theFTpromoter),SUS1(increasesFTexpression),TSF(the closest homologue ofFT),PAOandNAC029(plays a role in leaf senescence), were expressed in the two Chinese cabbage accessions.ConclusionThe 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.

Four heterotic QTL and a heterozygous segment for plant weight were identified by Graded Pool-Seq, QTL-seq and traditional genetic linkage analysis in heading Chinese cabbage. Heading Chinese cabbage (Brassica rapa L. spp. pekinensis) is a cross-pollinated leafy vegetable with significant heterosis. The use of heterosis is important for breeding high-yield Chinese cabbage hybrids. However, the formation and mechanism of heterosis have not been studied. We dissected the molecular mechanism of heterosis of yield-related traits in Chinese cabbage. An F

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.

Chinese cabbage (

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.

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.

Clubroot disease is one of the major diseases affecting Brassica crops, especially Chinese cabbage (Brassica rapa L. ssp. pekinensis), which is known to be highly susceptible to the disease. In this study, the obligate biotrophic protist Plasmodiophora brassicae Woronin was used to infect the roots of Chinese cabbage seedlings. The disease symptoms were noticeable at 28 and 35 days after inoculation (DAI) in the susceptible (CM) line. Using isobaric tags for relative and absolute quantitation (iTRAQ) analysis, a total of 5,003 proteins of differential abundance were identified in the resistant/susceptible lines, which could be quantitated by dipeptide or polypeptide segments. Gene ontology (GO) analysis indicated that the differentially expressed proteins (DEPs) between the susceptible (CM) and resistant (CCR) lines were associated with the glutathione transferase activity pathway, which could catalyze the combination of glutathione and other electrophilic compounds to protect plants from disease. In addition, the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that the DEPs may be significantly enriched cytokinin signaling or arginine biosynthesis pathways, both of which are responses to stimuli and are plant defense reactions. The cytokinins may facilitate cell division in the shoot, resulting in the hypertrophy and formation of galls and the presentation of typical clubroot symptoms. In this study, the proteomic results provide a new perspective for creating germplasm resistance to P. brassicae, as well as a genetic basis for breeding to improve Chinese cabbage.

Interspecific hybridization is an excellent way to create germplasm resources and is considered an important driver of genome evolution. We selected one Chinese cabbage variety and one red cabbage variety for hybridization. Heterologous haploid offspring were obtained by embryo rescue, and heterologous diploids were obtained by colchicine-induced chromosomal doubling. The field traits and simple sequence repeat markers of 98 F2 plants were investigated. The result showed that the parental traits were separated in the hybrids, with traits tending to shift from those of Chinese cabbage to those of red cabbage, and trait values showed normal distributions. Simple sequence repeat patterns varied, with the number of missing bands being significantly greater than that of novel bands in hybrids. Thus, the phenotypes of the early formed allopolyploids were unstable and accompanied by dramatic changes in the genome. This study not only increased the genetic resources available for B. napus but also laid a theoretical foundation for exploring trait segregation in early formed allopolyploids.

Histone methyltransferases and demethylases are two major enzymes that regulate histone methylation, which in turn influences hundreds of genes in plants. Early Flowering 6 (AtELF6), which contains a Jumonji C (JmjC) domain, is a demethylase that plays a vital role in regulating flowering time in Arabidopsis thaliana. In the present study, the BraELF6 gene of Brassica rapa cultivar ‘1510746’ was cloned to elucidate its role on flowering time. Homologous genes of BraELF6 were identified in Brassica AA/CC genomes, but not in the BB genome. Analysis of the BraELF6 gene structure identified three domains, namely, JmjN, JmjC, and ZnF C2H2. The relative expression of BraELF6 increased with plant growth and was the highest in the flowers compared to that in the roots, stems, and leaves. The BraELF6 gene was transfected into the Arabidopsis elf6-4 mutants, and Northern blot analysis confirmed its expression in the transgenic plants. The T1 plants showed the later flowering than the elf6-4 mutants, which recovered to the level of Col plants. In addition, the expression of the BraELF6 and AtFLC genes was relatively higher in the transgenic plants. In the T2 plants, the flowering phenotype segregated to some extent, which may be associated with the number of transgenic genes integrated into the genome. This present study lays the foundation for elucidating the mechanism underlying the regulation of flowering time in B. rapa by the BraELF6 gene, as well as excavating more earlier or later flowering germplasm materials in the future.

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 genomic variation of eight F2 synthetic B. napus using whole-genome sequencing. We found that there was a genetic variation in the F2 generation. Part of the variation was consistent in the F2 generation, and a small number of mutations only appeared in a single plant of the F2 generation. The analysis of copy number variation (CNV) found that most of the AA genome was lost, and most of the CC genome was obtained. In addition, there was inter-chromosomal translocation (CTX) in the F2 generation and the number of each plant was different. The above results indicate that the F2 generation showed genetic variation and there was a difference between eight plants, which may lay a molecular basis for the unique field performance of the offspring. It provides a new perspective of genomic variation and trait separation in the early stages of allopolyploid polyploid formation.

Hybrid vigor, or heterosis, means that the first-generation hybrids show the better performance than each parent, which is a biological phenomenon among crop production...

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 &ldquo, 10601&rdquo, (European turnip with swollen root, Brassica rapa ssp. rapifera, AA, 2n = 2&times, = 20) and &ldquo, 10603&rdquo, (Chinese cabbage with normal root, Brassica rapa ssp. pekinensis, AA, 2n = 2&times, = 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.

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 activationsilent 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.

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, &lsquo, Jin Wawa&rsquo, and &lsquo, Xiao Baojian&rsquo, 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. rapavernalization, as well as serve as an excellent reference for B. rapa breeding.

sInterspecific hybridization is widely observed within diverse eukaryotic taxa and is considered an important driver of genome evolution. We selected one flowering Chinese cabbage variety and one broccoli variety for hybridization. Heterologous haploid offspring were obtained by embryo rescue, and heterologous diploids were obtained by colchicine-induced chromosomal doubling. The field traits and simple sequence repeat markers of 124 F2 plants were investigated. We also analyzed the nutritional components of the parental and 10 progeny F2 plants. The parental traits were separated in the hybrids, with traits tending to shift from those of flowering Chinese cabbage to those of broccoli, and trait values showed normal distributions. Simple sequence repeat patterns varied, with the number of missing bands being significantly greater than that of novel bands in hybrids. Additionally, there were higher levels of some nutritional components in the hybrids compared with in the parents. Thus, the phenotypes of the early formed allopolyploids were unstable and accompanied by dramatic changes in the genome. Hybrids showed new traits and high levels of nutritional components. This study not only increased the genetic resources available for flowering Chinese cabbage but also laid a theoretical foundation for exploring trait segregation in early formed allopolyploids.

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.

Anthocyanins are the major pigments responsible for purple coloration in flowers, fruits and leaves, and the genes involved in their biosynthetic pathway have been identified in many plants. A purple-leaf Chinese cabbage (Brassica rapa L. ssp. pekinensis) was bred by interspecies crossing between Chinese cabbage and purple-leaf mustard [Brassica juncea (L.) Coss. var. foliosa L. H. Bailey]. In this study, high-performance liquid chromatographic analysis indicated purple coloration in Chinese cabbage is due to the accumulation of the same kind of cyaninin as in purple mustard. To elucidate the genetic factors controlling anthocyanin accumulation in this purple-leaf Chinese cabbage, we mapped the anthocyanin gene from the mustard (Anm) locus in an F2 population and performed expression profiling of anthocyanin-related genes. A genetic analysis revealed that the purple-leaf phenotype is a qualitative trait and that its inheritance is unstable in purple-leaf Chinese cabbage. Mapping insertion/deletion markers from 288 individuals of the F2 population located the Anm locus within a 2.5-cM interval on B. rapa chromosome A02. The sequencing and alignment of the amplified fragments demonstrated that purple Chinese cabbage contains fragments of purple mustard on chromosome A02. We evaluated the expression profiles of 12 anthocyanin-related genes on A02 by reverse-transcription and quantitative real-time PCR methods, which revealed that the expression levels of five genes were higher in purple Chinese cabbage than in the non-purple variety. These results offer insights into the molecular mechanism of anthocyanin biosynthesis and improve the knowledge on molecular breeding of purple-type Chinese cabbage.

Turnip mosaic virus (TuMV) is responsible for a serious disease that affects the production of Chinese cabbage. Previous studies have cloned a series of TuMV resistance genes and developed molecular markers. In this study, a derived cleaved amplified polymorphism sequence (dCAPS) marker and a Kompetitive Allele Specific PCR (KASP) marker were developed based on a single recessive gene, retr02, which confers broad-spectrum TuMV resistance in Chinese cabbage by means of an additional G at the junction of exon 1 and intron 1. The two markers were able to detect the retr02 allele in Chinese cabbage accessions used in breeding programmes. Compared with the dCAPS marker, the KASP marker was flexible, cost-effective and quick to process, which is likely to be beneficial in establishing high-throughput assays for marker-assisted selection.

Plant potyviruses require eukaryotic translation initiation factors (eIFs) such as eIF4E and eIF(iso)4E to replicate and spread. When Turnip mosaic virus (TuMV) infects a host plant, its viral protein linked to the genome (VPg) needs to interact with eIF4E or eIF(iso)4E to initiate translation. TuMV utilizes BraA.eIF4E.a, BraA.eIF4E.c, BraA.eIF(iso)4E.a, and BraA.eIF(iso)4E.c of Brassica rapa to initiate translation in Arabidopsis thaliana. In this study, the BraA.eIF4E.a, BraA.eIF4E.c, BraA.eIF(iso)4E.a, and BraA.eIF(iso)4E.c genes were cloned and sequenced from eight B. rapa lines, namely, two BraA.eIF4E.a alleles, four BraA.eIF4E.c alleles, four BraA.eIF(iso)4E.a alleles, and two BraA.eIF(iso)4E.c alleles. Yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) analyses indicated that TuMV VPg could not interact with eIF4E, but only with eIF(iso)4E of B. rapa. In addition, the VPgs of the different TuMV isolates interacted with various eIF(iso)4E copies in B. rapa. In particular, TuMV-UK1/CDN1 VPg only interacted with BraA.eIF(iso)4E.c, not with BraA.eIF(iso)4E.a. Some single nucleotide polymorphisms (SNPs) were identified that may have affected the interaction between eIF(iso)4E and VPg such as the SNP T106C in BraA.eIF(iso)4E.c and the SNP A154C in VPg. Furthermore, a three-dimensional structural model of the BraA.eIF(iso)4E.c-1 protein was constructed to identify the specific conformation of the variable amino acids from BraA.eIF(iso)4E.c. The 36th amino acid in BraA.eIF(iso)4E.c is highly conserved and may play an important role in establishing protein structural stability. The findings of the present study may lay the foundation for future investigations on the co-evolution of TuMV and eIF(iso)4E.

Clubroot disease, caused by Plasmodiophora brassicae Woronin, has become a major problem in cruciferous crops worldwide. Chinese cabbage (Brassica rapa), pak choi (B. rapa), and mustard (B. juncea) are important vegetable crops in China. Development of clubroot-resistant cultivars of these crops is urgently needed. In this study, 71 B. rapa and B. juncea genotypes from China, including cultivars and inbred lines, were evaluated for resistance to three P. brassicae pathotypes. A significant interaction was observed between the P. brassicae pathotypes and the Brassica genotypes. Pathotype 3, as defined on the differentials of Williams, exhibited the weakest virulence on all plant material. By contrast, pathotypes 5 and 6 were both highly pathogenic on most of the tested genotypes. In all, 10 of the 14 Chinese cabbage cultivars were resistant to all three pathotypes, while 4 were resistant only to a specific pathotype. Seven of eight progenies obtained from the selfing of Chinese cabbage cultivars were resistant to pathotype 3 but most were susceptible to pathotypes 5 and 6. Most inbred lines of Chinese cabbage and all inbred lines of pak choi and mustard were susceptible to all three pathotypes but their susceptibility was lower to pathotype 3 than to pathotypes 5 and 6.

The Brassica genus comprises many economically important worldwide cultivated crops. The well-established model of the Brassica genus, U’s triangle, consists of three basic diploid plant species (Brassica rapa, Brassica oleracea and Brassica nigra) and three amphidiploid species (Brassica napus, Brassica juncea and Brassica carinata) that arose through interspecific hybridizations. Despite being extensively studied because of its commercial relevance, several aspects of the origin of the Brassica species and the relationships within and among these six species still remain open questions. Here, we successfully de novo assembled 60 complete chloroplast genomes of Brassica genotypes of all six species. A complete map of the Single Nucleotide Variants (SNVs) and Insertions and Deletions (InDels) in the chloroplast genomes of different Brassica species was produced. The chloroplast genome consists of a Large and a Small Single Copy (LSC and SSC) region between two Inverted Repeats (IRs), and while these regions of chloroplast genomes have very different molecular evolutionary rates, phylogenetic analyses of different regions yielded no contradicting topologies and separated the Brassica genus into four clades. B. carinata and B. juncea share their chloroplast genome with one of their hybridization donors B. nigra and B. rapa, respectively, which fits the U model. B. rapa, surprisingly, shows evidence of two types of chloroplast genomes, with one type specific to some Italian broccoletto accessions. B. napus clearly has evidence for two independent hybridization events, as it contains either B. rapa chloroplast genomes. The divergence estimation suggests that B. nigra and B. carinata diverged from the main Brassica clade 13.7 million years ago (Mya), while B. rapa and B. oleracea diverged at 2.18 Mya. The use of the complete chloroplast DNA sequence not only provides insights into comparative genome analysis but also paves the way for a better understanding of the phylogenetic relationships within the Brassica genus.

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.

Polyketide 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

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.

Bitterness in cucumber fruit and foliage is due to the presence of cucurbitacins. Several genes have been described that control the trait, with bi (bi-1) making fruit and foliage bitter free and Bt (Bt-1) making the fruit highly bitter. Previous studies have reported the inheritance and molecular markers linked to bi-1 or Bt-1, but we were interested in studying the inheritance of fruit bitterness in the progeny of 2 nonbitter fruit inbred lines. The objective was to determine the inheritance of cucumber fruit and foliage bitterness and to locate them on a current linkage map using a recombinant inbred lines (RILs) population derived by crossing 9110Gt and 9930. It was concluded from the inheritance analysis that there were 2 loci controlling fruit bitterness in the population. One locus was in the same position as the location previously identified for bi-1, and another locus was for bi-3. Using a simple sequence repeat (SSR) linkage map, 2 loci for fruit bitterness in this RILs population were mapped. The locus of bi-1 was located at the region between SSR0004 and SSR02309 within the genetic distance of 5.2 cM on chromosome 6. The locus of bi-3 was placed in the region of SSR00116-SSR05321 within the genetic distance of 6.3 cM on chromosome 5. The physical distances for the regions of bi-1 and bi-3 were 11,430.94 Kb with 160 predicted genes and 1528.23 Kb with 198 predicted genes, respectively. Among 160 predicted genes for bi-1, there is a terpene synthase gene named Csa008595, which was speculated as the candidate gene of bi-1.

The extreme resistance to Turnip mosaic virus observed in the Chinese cabbage (Brassica rapa) line, BP8407, is monogenic and recessive. Bulked segregant analysis was carried out to identify simple sequence repeat and Indel markers linked to this recessive resistance gene, termed recessive Turnip mosaic virus resistance 02 (retr02). Mapping of PCR-specific Indel markers on 239 individuals of a BP8407 × Ji Zao Chun F(2) population, located this resistance gene to a 0.9-cM interval between two Indel markers (BrID10694 and BrID101309) and in scaffold000060 or scaffold000104 on chromosome A04 of the B. rapa genome. Eleven eukaryotic initiation factor 4E (eIF4E) and 14 eukaryotic initiation factor 4G (eIF4G) genes are predicted in the B. rapa genome. A candidate gene, Bra035393 on scaffold000104, was predicted within the mapped resistance locus. The gene encodes the eIF(iso)4E protein. Bra035393 was sequenced in BP8407 and Ji Zao Chun. A polymorphism (A/G) was found in exon 3 between BP8407 and Ji Zao Chun. This gene was analysed in four resistant and three susceptible lines. A correlation was observed between the amino acid substitution (Gly/Asp) in the eIF(iso)4E protein and resistance/susceptibility. eIF(iso)4E has been shown previously to interact with the TuMV genome-linked protein, VPg.