Mapping-based localization of blackleg-resistant candidate genes of Chinese cabbage (Brassica rapa).

Recently, blackleg disease has seriously impacted the cultivation and development of Brassica crops. In this study, we conducted mapping-based localization of blackleg-resistant candidate genes in Chinese cabbage. Through phenotype evaluation, Chinese cabbage materials 15S414 and 15S420 were selected as blackleg-resistant and blackleg-susceptible parents, respectively. Inheritance pattern analysis suggested that the dominant major genes mainly determined the blackleg resistance of Chinese cabbage. Upon bulked segregation analysis , the blackleg-resistant candidate genes were initially located within a 4.3 Mb interval on chromosome A06. Through construction of the genetic linkage map, blackleg-resistant candidate genes were further limited to a region of 160 kb containing seven resistance-related genes. Coding sequence variation analysis revealed that all seven resistance-related genes displayed various degrees of single nucleotide polymorphism variations between parent materials 15S414 and 15S420.

Effects of different light intensity on leaf color changes in a Chinese cabbage yellow cotyledon mutant.

Leaf color is one of the most important phenotypic features in horticultural crops and directly related to the contents of photosynthetic pigments. Most leaf color mutants are determined by the altered chlorophyll or carotenoid, which can be affected by light quality and intensity. Our previous study obtained a Chinese cabbage yellow cotyledon mutant that exhibited obvious yellow phenotypes in the cotyledons and the new leaves. However, the underlying mechanisms in the formation of yellow cotyledons and leaves remain unclear. In this study, the Chinese cabbage yellow cotyledon mutant 19YC-2 exhibited obvious difference in leaf color and abnormal chloroplast ultrastructure compared to the normal green cotyledon line 19GC-2. Remarkably, low-intensity light treatment caused turn-green leaves and a significant decrease in carotenoid content in 19YC-2. RNA-seq analysis revealed that the pathways of photosynthesis antenna proteins and carotenoid biosynthesis were significantly enriched during the process of leaf color changes, and many differentially expressed genes related to the two pathways were identified to respond to different light intensities. Remarkably, BrPDS and BrLCYE genes related to carotenoid biosynthesis showed significantly higher expression in 19YC-2 than that in 19GC-2, which was positively related to the higher carotenoid content in 19YC-2. In addition, several differentially expressed transcription factors were also identified and highly correlated to the changes in carotenoid content, suggesting that they may participate in the regulatory pathway of carotenoid biosynthesis. These findings provide insights into the molecular mechanisms of leaf color changes in yellow cotyledon mutant 19YC-2 of Chinese cabbage.

Effects of long-term blue light irradiation on carotenoid biosynthesis and antioxidant activities in Chinese cabbage (Brassica rapa L. ssp. pekinensis).

The aim of this study was to investigate the impact of long-term exposure to blue light-emitting diodes (LEDs) on the accumulation of indolic glucosinolates and carotenoids, as well as the plant growth and antioxidant activities in both orange and common Chinese cabbage (Brassica rapa L. ssp. pekinensis). Blue light treatment also induced higher ferric-reducing antioxidant power and 2,2-diphenyl-1-picrylhydrazyl by 20.66 % and 30.82 % and antioxidant enzyme activities catalase, peroxidase, superoxide dismutase, and the accumulation of non-enzymatic antioxidant substances (total phenols and total flavonoids) in the orange Chinese cabbage. Furthermore, long-term exposure to blue light had negative effects on the net photosynthetic rate and chlorophyll fluorescence levels. Meanwhile, blue light promoted accumulation of Indol-3-ylmethyl glucosinolate (I3M), ß-carotene, lutein and zeaxanthin due to the high expression of regulatory and biosynthetic genes of the above metabolic pathways. In particular, lycopene and ß-carotene content in orange Chinese cabbage increased by 60.14 % and 65.33 % compared to the ones in common line. The accumulation of carotenoid and increasing antioxidant levels in the orange cabbage line was influenced by long-term blue light irradiation, leading to better tolerance to low temperature and drought stresses. The up-regulation of transcription factors such as BrHY5-2, BrPIF4 and BrMYB12 may also contribute to the increased tolerance in orange Chinese cabbage to extreme environmental stresses. The BrHY5-2 gene could activate carotenoid biosynthetic genes and induce the accumulation of carotenoids. These findings suggested that long-term blue light irradiation could be a promising technique for increasing the nutrition value and enhancing tolerance to low temperature and drought stresses in Chinese cabbage.

Genome-Wide Identification of BrCAX Genes and Functional Analysis of BrCAX1 Involved in Ca2+ Transport and Ca2+ Deficiency-Induced Tip-Burn in Chinese Cabbage (Brassica rapa L. ssp. pekinensis).

Calcium (Ca2+) plays essential roles in plant growth and development. Ca2+ deficiency causes a physiological disorder of tip-burn in Brassiceae crops and is involved in the regulation of cellular Ca2+ homeostasis. Although the functions of Ca2+/H+ exchanger antiporters (CAXs) in mediating transmembrane transport of Ca2+ have been extensively characterized in multiple plant species, the potential roles of BrCAX genes remain unclear in Chinese cabbage. In this study, eight genes of the BrCAX family were genome-widely identified in Chinese cabbage. These BrCAX proteins contained conserved Na_Ca_ex domain and belonged to five members of the CAX family. Molecular evolutionary analysis and sequence alignment revealed the evolutionary conservation of BrCAX family genes. Expression profiling demonstrated that eight BrCAX genes exhibited differential expression in different tissues and under heat stress. Furthermore, Ca2+ deficiency treatment induced the typical symptoms of tip-burn in Chinese cabbage seedlings and a significant decrease in total Ca2+ content in both roots and leaves. The expression changes in BrCAX genes were related to the response to Ca2+ deficiency-induced tip-burn of Chinese cabbage. Specially, BrCAX1-1 and BrCAX1-2 genes were highly expressed gene members of the BrCAX family in the leaves and were significantly differentially expressed under Ca2+ deficiency stress. Moreover, overexpression of BrCAX1-1 and BrCAX1-2 genes in yeast and Chinese cabbage cotyledons exhibited a higher Ca2+ tolerance, indicating the Ca2+ transport capacity of BrCAX1-1 and BrCAX1-2. In addition, suppression expression of BrCAX1-1 and BrCAX1-2 genes reduced cytosolic Ca2+ levels in the root tips of Chinese cabbage. These results provide references for functional studies of BrCAX genes and to investigate the regulatory mechanisms underlying Ca2+ deficiency disorder in Brassiceae vegetables.

The Accumulation of Health-Promoting Nutrients from Representative Organs across Multiple Developmental Stages in Orange Chinese Cabbage.

Orange Chinese cabbage (Brassica rapa L. ssp. pekinensis) is an excellent source of health-promoting nutrients that could reduce the risk of chronic diseases. This study mainly investigated the accumulation patterns of eight lines of orange Chinese cabbage for indolic glucosinolates (GLSs) and pigment content from representative plant organs across multiple developmental stages. The indolic GLSs were highly accumulated at the rosette stage (S2), especially in inner and middle leaves, and the order of indolic GLSs accumulation in non-edible organs was flower > seed > stem > silique. The expression levels of biosynthetic genes in light signaling, MEP, carotenoids, and GLS pathways were consistent with the metabolic accumulation patterns. The results of a principal component analysis show a clear separation of high indolic GLS lines (15S1094 and 18BC6) from low indolic GLS lines (20S530). A negative correlation between the accumulation of indolic GLS and carotenoids was identified in our study. Our work contributes to providing valuable knowledge required to breed, grow, and select orange Chinese cabbage varieties and their eatable organs with higher nutritional value.

Correlation between Parental Transcriptome and Field Data for the Characterization of Heterosis in Chinese Cabbage.

In Chinese cabbage breeding, hybrids have made a terrific contribution due to heterosis, the superior performance of offspring compared to their inbred parents. Since the development of new, top-performing hybrids requires a large scale of human and material resources, the prediction of hybrid performance is of utmost interest to plant breeders. In our research, leaf transcriptome data from eight parents were used to investigate if they might be employed as markers to predict hybrid performance and heterosis. In Chinese cabbage, heterosis of plant growth weight (PGW) and heterosis of head weight (HW) were more obvious than other traits. The number of differential expression genes (DEGs) between parents was related to the PGW, length of the biggest outer leaf (LOL), leaf head height (LHH), leaf head width (LHW), HW, leaf number of head (LNH) and plant height (PH) of hybrids, and up-regulated DEGs number was also associated with these traits. Euclidean and binary distances of parental gene expression levels were significantly correlated with the PGW, LOL, LHH, LHW, HW and PH of hybrids. Additionally, there was a significant correlation between the parental expression levels of multiple genes involved in the ribosomal metabolic pathway and hybrid observations and heterosis in PGW, with the BrRPL23A gene showing the highest correlation with the MPH of PGW(r = 0.75). Therefore, leaf transcriptome data can preliminarily predict the hybrid performance and select parents in Chinese cabbage.

Transcriptome Analysis Reveals a Major Gene Expression Pattern and Important Metabolic Pathways in the Control of Heterosis in Chinese Cabbage.

Although heterosis is commonly used in Chinese cabbage, its molecular basis is poorly understood. In this study, 16Chinese cabbage hybrids were utilized as test subjects to explore the potential molecular mechanism of heterosis. RNA sequencing revealed 5815-10,252 differentially expressed genes (DEGs) (female parent vs. male parent), 1796-5990 DEGs (female parent-vs-hybrid), and 2244-7063 DEGs (male parent vs. hybrid) in 16 cross combinations at the middle stage of heading. Among of them, 72.83-84.20% DEGs conformed to the dominant expression pattern, which is the predominant expression pattern in hybrids. There were 13 pathways in which DEGs were significantly enriched in most cross combinations. Among them, the plant-pathogen interaction (ko04626) and circadian rhythm-plant (ko04712)were significantly enriched by DEGs in strong heterosis hybrids. WGCNA also proved that the two pathways were significantly related to heterosis in Chinese cabbage.

The effects of short term blue light treatment on promoting nutrition value in Chinese cabbage.

Chinese cabbage is a nutrients-rich vegetable with diverse leaf colors. Here, we used widely-targeted metabolomics technology to study the metabolic responses of three Chinese cabbage varieties with representative leaf colors after blue light treatment. The inner leaf color of orange varieties 20S530 and 15S1094 changed from yellow to golden yellow, while no visible color change occurred in the common variety 14S23 after the treatment. A total of 844 metabolites were measured from the leaf samples of these three varieties in a time course study after short term blue light treatment, with kaempferol-4'-O-glucoside, isoquercitrin, hyperin, arbutin, sulforaphane as enriched nutritional metabolites. Orange Chinese cabbage varieties showed additional nutrition enhancement after the treatment. This study is the first to explore the global metabolic responses of Chinese cabbage after blue light treatment, and our findings provided valuable insights on how to effectively use lighting conditions to enhance specific groups of nutrients in vegetables.

Comparative Metabolic Study of Two Contrasting Chinese Cabbage Genotypes under Mild and Severe Drought Stress.

Chinese cabbage (Brassica rapa L. ssp. pekinensis) is an important leafy vegetable crop cultivated worldwide. Drought is one of the most important limiting factors for the growth, production and quality of Chinese cabbage due to its weak drought tolerance. In order to deepen the understanding of drought stress response in Chinese cabbage, metabolomics studies were conducted in drought-tolerant (DT) and drought-susceptible (DS) genotypes of Chinese cabbage under water deficit-simulated mild and severe drought stress conditions. A total of 777 metabolites were detected, wherein 90 of them were proposed as the drought-responsive metabolites in Chinese cabbage, with abscisic acid (ABA), serine, choline alfoscerate, and sphingosine as potential representative drought stress biomarkers. We also found that drought-tolerant and drought-susceptible genotypes showed differential metabolic accumulation patterns with contrasting drought response mechanisms. Notably, constitutively high levels of ABA and glutathione were detected in drought-tolerant genotype in all tested and control conditions. In addition, proline, sucrose, γ-aminobutyric acid, and glutathione were also found to be highly correlated to drought tolerance. This study is the first metabolomic study on how Chinese cabbage responds to drought stress, and could provide insights on how to develop and cultivate new drought-resistant varieties.

Comparative transcriptome and flavonoids components analysis reveal the structural genes responsible for the yellow seed coat color of Brassica rapa L.

BACKGROUND: Seed coat color is an important horticultural trait in Brassica crops, which is divided into two categories: brown/black and yellow. Seeds with yellow seed coat color have higher oil quality, higher protein content and lower fiber content. Yellow seed coat color is therefore considered a desirable trait in hybrid breeding of Brassica rapa, Brassica juncea and Brassica napus. METHODS: Comprehensive analysis of the abundance transcripts for seed coat color at three development stages by RNA-sequencing (RNA-seq) and corresponding flavonoids compounds by liquid chromatography-tandem mass spectrometry (LC-MS/MS) were carried out in B. rapa. RESULTS: We identified 41,286 unigenes with 4,989 differentially expressed genes between brown seeds (B147) and yellow seeds (B80) at the same development stage. Kyoto Encyclopedia of Genes and Genomes enrichment analysis identified 19 unigenes associated with the phenylpropanoid, flavonoid, flavone and flavonol biosynthetic pathways as involved in seed coat color formation. Interestingly, expression levels of early biosynthetic genes (BrCHS, BrCHI, BrF3H, BrF3'H and BrFLS) in the flavonoid biosynthetic pathway were down-regulated while late biosynthetic genes (BrDFR, BrLDOX and BrBAN) were hardly or not expressed in seeds of B80. At the same time, BrTT8 and BrMYB5 were down-regulated in B80. Results of LC-MS also showed that epicatechin was not detected in seeds of B80. We validated the accuracy of our RNA-seq data by RT-qPCR of nine critical genes. Epicatechin was not detected in seeds of B80 by LC-MS/MS. CONCLUSIONS: The expression levels of flavonoid biosynthetic pathway genes and the relative content of flavonoid biosynthetic pathway metabolites clearly explained yellow seed color formation in B. rapa. This study provides a foundation for further research on the molecular mechanism of seed coat color formation.

Transcriptome analysis of biological pathways associated with heterosis in Chinese cabbage.

Chinese cabbage is an important vegetable in Asia, and high-yielding hybrids are needed to cope with the growing demand. A comparative transcriptome profiling was conducted to reveal the differentially expressed genes (DEGs) associated with heterosis in two hybrids relative to their parents. Our data suggests that heterosis is underlined by a significant upregulation of gene expression. High expression of DEGs in glycolysis and photosynthesis pathways in hybrids depicted their relation with growth and hybrid vigor. Besides, DEGs related to auxin, abscisic acid, ethylene and gibberellin were identified, implying that these hormones may boost the mechanisms of growth and developmental processes in the hybrids. Furthermore, transcription factors, including bHLH, ERF, MYB and WRKY were predicted to regulate downstream genes linked to hybrid vigor. Collectively, the present study will be helpful for a better understanding of the regulation mechanisms of heterosis to aid cabbage yield improvement.

The novel gene BrMYB2, located on chromosome A07, with a short intron 1 controls the purple-head trait of Chinese cabbage (Brassica rapa L.).

Anthocyanins are important secondary metabolites in plants, but information on anthocyanin biosynthesis mechanisms in Chinese cabbage is limited. The new purple head Chinese cabbage cultivar 11S91 was analyzed, and an R2R3-MYB regulatory gene BrMYB2, located on chromosome A07, controlling the dominant purple-head trait was isolated. High expression of BrMYB2 generated a large accumulation of anthocyanins in 11S91, accompanied by highly upregulated BrTT8, BrF3'H, BrDFR1, BrANS1, BrUGTs, BrATs, and BrGSTs. 11S91 inherited the purple locus from purple trait donor 95T2-5, and they shared consensus CDSs and gDNAs with those of BrMYB2 (cBrMYB2 and gBrMYB2). Two SNPs in cBrMYB2 in 11S91 did not cause loss of function; in addition to several SNPs at both ends of intron 1, a large deletion had occurred in intron 1 of gBrMYB2 in 11S91. Genetic transformation of Arabidopsis showed that gBrMYB2 overexpression lines presented deeper purple color and higher expression than did the cBrMYB2 and cBrmyb2 lines, whereas gBrmyb2 with a long intron 1 did not cause the purple phenotype. We first show that BrMYB2 promotes anthocyanin biosynthesis under the control of the short intron 1 of gBrMYB2 in purple head Chinese cabbage, and gBrmyb2 with a long intron 1 represses anthocyanin production in white head Chinese cabbage. This evidence provides a new understanding of anthocyanin biosynthesis and purple germplasm generation in Brassica vegetables.

Fine mapping and candidate gene analysis of the white flower gene Brwf in Chinese cabbage (Brassica rapa L.).

Flower color can be applied to landscaping and identification of the purity of seeds in hybrid production. However, the molecular basis of white flower trait remains largely unknown in Brassica rapa. In this study, an F2 population was constructed from the cross between 15S1040 (white flower) and 92S105 (yellow flower) for fine mapping of white flower genes in B. rapa. Genetic analysis indicated that white flower trait is controlled by two recessive loci, Brwf1 and Brwf2. Using InDel and SNP markers, Brwf1 was mapped to a 49.6-kb region on chromosome A01 containing 9 annotated genes, and among them, Bra013602 encodes a plastid-lipid associated protein (PAP); Brwf2 was located in a 59.3-kb interval on chromosome A09 harboring 12 annotated genes, in which Bra031539 was annotated as a carotenoid isomerase gene (CRTISO). The amino acid sequences of BrPAP and BrCRTISO were compared between two yellow-flowered and three white-flowered lines and critical amino acid mutations of BrPAP and BrCRTISO were identified between yellow-flowered and white-flowered lines. Therefore, Bra013602 and Bra031539 were predicted as potential candidates for white flower trait. Our results provide a foundation for further identification of Brwf and increase understanding of the molecular mechanisms underlying white flower formation in Chinese cabbage.

Low Temperature Promotes Anthocyanin Biosynthesis and Related Gene Expression in the Seedlings of Purple Head Chinese Cabbage (Brassica rapa L.).

To elucidate the effect of low temperature on anthocyanin biosynthesis in purple head Chinese cabbage, we analyzed anthocyanin accumulation and related gene expression in the seedlings of purple head Chinese cabbage, white head parent Chinese cabbage, and its purple male parent under a normal 25 °C temperature and a low 12 °C temperature. Anthocyanin accumulation in purple lines was strongly induced by low temperature, and the total anthocyanin content of seedlings was significantly enhanced. In addition, nearly all phenylpropanoid metabolic pathway genes (PMPGs) were down-regulated, some early biosynthesis genes (EBGs) were up-regulated, and nearly all late biosynthesis genes (LBGs) directly involved in anthocyanin biosynthesis showed higher expression levels in purple lines after low-temperature induction. Interestingly, a R2R3-MYB transcription factor (TF) gene 'BrMYB2' and a basic-helix-loop-helix (bHLH) regulatory gene 'BrTT8' were highly up-regulated in purple lines after low temperature induction, and two negative regulatory genes 'BrMYBL2.1' and 'BrLBD38.2' were up-regulated in the white line. BrMYB2 and BrTT8 may play important roles in co-activating the anthocyanin structural genes in purple head Chinese cabbage after low-temperature induction, whereas down-regulation of BrMYB2 and up-regulation of some negative regulators might be responsible for white head phenotype formation. Data presented here provide new understanding into the anthocyanin biosynthesis mechanism during low temperature exposure in Brassica crops.

Dynamic Changes of the Anthocyanin Biosynthesis Mechanism During the Development of Heading Chinese Cabbage (Brassica rapa L.) and Arabidopsis Under the Control of BrMYB2.

Chinese cabbage is an important vegetable mainly planted in Asian countries, and mining the molecular mechanism responsible for purple coloration in Brassica crops is fast becoming a research hotspot. In particular, the anthocyanin accumulation characteristic of purple heading Chinese cabbage, along with the plant's growth and head developing, is still largely unknown. To elucidate the dynamic anthocyanin biosynthesis mechanism of Chinese cabbage during its development processes, here we investigated the expression profiles of 86 anthocyanin biosynthesis genes and corresponding anthocyanin accumulation characteristics of plants as they grew and their heads developed, between purple heading Chinese cabbage 11S91 and its breeding parents. Anthocyanin accumulation of 11S91 increased from the early head formation period onward, whereas the purple trait donor 95T2-5 constantly accumulated anthocyanin throughout its whole plant development. Increasing expression levels of BrMYB2 and BrTT8 together with the downregulation of BrMYBL2.1, BrMYBL2.2, and BrLBD39.1 occurred in both 11S91 and 95T2-5 plants during their growth, accompanied by the significantly continuous upregulation of a phenylpropanoid metabolic gene, BrPAL3.1; a series of early biosynthesis genes, such as BrCHSs, BrCHIs, BrF3Hs, and BrF3'H; as well as some key late biosynthesis genes, such as BrDFR1, BrANS1, BrUF3GT2, BrUF5GT, Br5MAT, and Brp-Cout; in addition to the transport genes BrGST1 and BrGST2. Dynamic expression profiles of these upregulated genes correlated well with the total anthocyanin contents during the processes of plant growth and leaf head development, and results supported by similar evidence for structural genes were also found in the BrMYB2 transgenic Arabidopsis. After intersubspecific hybridization breeding, the purple interior heading leaves of 11S91 inherited the partial purple phenotypes from 95T2-5 while the phenotypes of seedlings and heads were mainly acquired from white 94S17; comparatively in expression patterns of investigated anthocyanin biosynthesis genes, cotyledons of 11S91 might inherit the majority of genetic information from the white type parent, whereas the growth seedlings and developing heading tissues of 11S91 featured expression patterns of these genes more similar to 95T2-5. This comprehensive set of results provides new evidence for a better understanding of the anthocyanin biosynthesis mechanism and future breeding of new purple Brassica vegetables.

Transcriptome-based analysis of carotenoid accumulation-related gene expression in petals of Chinese cabbage (Brassica rapa L.).

To identify genes associated with carotenoid accumulation in petals of Chinese cabbage, the composition and content of carotenoids were analyzed, and comparative transcriptome sequencing was performed between the yellow flower line, 92S105, and the orange flower line, 94C9. High-performance liquid chromatography (HPLC) revealed that petals of 92S105 were high in violaxanthin as well as lutein, whereas petals of 94C9 showed considerable levels of lutein and ß-carotene. Transcriptome analysis showed that 3534 and 3833 genes were up- and down-regulated in 94C9, respectively. Among these differentially expressed genes (DEGs), many related to carotenoid accumulation were identified, including 12 carotenoid biosynthesis pathway genes, 4 transcription factor genes, and 1028 specifically expressed genes. ß-carotene hydroxylase 1 (BrBCH1), BrBCH2, zeaxanthin epoxidase (BrZEP), and MYB transcription factor gene (BrGAMYB) were down-regulated in petals of 94C9 when compared with petals of 92S105, which caused ß-carotene accumulation and may lead to orange petal color in 94C9. Expression levels of 20 DEGs were verified by qPCR and the results were highly consistent with those of transcriptome sequencing. Moreover, Gene Ontology (GO) enrichment analysis revealed that membrane, binding, and metabolic processes were the most significantly enriched GO terms in cellular component, molecular function, and biological process ontologies, respectively. In conclusion, our study analyzed the differences in composition and content of carotenoids between 92S105 and 94C9 and identified potential candidate genes related to carotenoid accumulation in petals, thereby creating a solid foundation for future studies on the mechanism regulating carotenoid accumulation in petals of Chinese cabbage.

Genome-wide identification and expression analysis of calmodulin-like (CML) genes in Chinese cabbage (Brassica rapa L. ssp. pekinensis).

BACKGROUND: Calmodulin-like (CML) proteins are a primary family of plant-specific Ca2+ sensors that specifically bind to Ca2+ and deliver a Ca2+ signal. CML proteins have been identified and characterized in many plant species, such as the model plant Arabidopsis and rice. Based on considerable evidence, the roles of CML proteins are crucial in plant growth and development and in the response to various external stimuli. Nevertheless, the characterization and expression profiling of CML genes in Chinese cabbage (Brassica rapa L. ssp. pekinensis) remain limited. RESULTS: In this study, a genome-wide search and comprehensive analysis were performed, and a total of 79 BrCML genes were identified in Chinese cabbage. Gene structure analysis revealed that these BrCML genes contained two to four conserved EF-hand motifs. Phylogenetic analysis showed that CML homologs between Chinese cabbage and Arabidopsis shared close relationships. The identified BrCML genes were located across ten chromosomes and three different subgenomes of Chinese cabbage. Moreover, 126 pairs of orthologous CML genes were found among Chinese cabbage, Arabidopsis and Brassica oleracea. Expression analysis revealed that the expression of some BrCML genes was tissue-specific and that of some was susceptible to temperature stress. A putative interaction network of BrCML proteins was proposed, which suggested that BrCML2, BrCML6, BrCML15 and BrCML25 were co-expressed and might play roles in flower development and other relevant biological processes of Chinese cabbage. CONCLUSIONS: The results of this study increased the understanding and characterization of BrCML genes in Chinese cabbage, and will be a rich resource for further studies to investigate BrCML protein function in various developmental processes of Chinese cabbage.

Characteristics of Color Development in Seeds of Brown- and Yellow-Seeded Heading Chinese Cabbage and Molecular Analysis of Brsc, the Candidate Gene Controlling Seed Coat Color.

The proanthocyanidin (PA) is the main flavonoids which affect the seed coat color in Brassica species. In this paper, characteristics of color development and accumulation of flavonoids were analyzed in the seeds of brown-seeded (B147) and yellow-seeded (B80) heading Chinese cabbage (Brassica rapa L. ssp. Pekinensis). It is found that the content of phenolic compounds in B147 were significantly more than that of B80 by using dimethylaminocinnamaldehyde (DMACA) staining and toluidine blue O (TBO) staining. In previous studies, the locus associated with seed coat color has been mapped. The results of whole genome re-sequencing showed that there are large fragment deletions variation in the mapping region between the brown-seeded parent '92S105' and the yellow-seeded parent '91-125.' Based on the B. rapa genome annotation information, the TRANSPARENT TESTA GLABRA 1 (TTG1), is likely to be the candidate gene controlling seed coat color. A 94-base deletion was found in the 96th base downstream of the initiation codon in the TTG1 of yellow seed, thus, the termination codon TGA was occurred in the 297th base which makes the full length of TTG1 of yellow seed is 300 bp. Based on the differential sequences of TTG1 of brown and yellow seed, a functional marker, Brsc-yettg1, was developed to detect the variation of TTG1. Quantitative real-time PCR analysis of BrTTG1 in different tissues showed that expression levels of BrTTG1 was not tissue-specific. During the whole seed development period, the expression of BrTTG1 in B147 was higher than that of B80. The expression levels of four structural genes, BrDFR, BrANS, BrANR1, and BrANR2 in B147 were also higher than those in B80. The co-segregation molecular markers obtained in this report and TTG1 related information provide a basis for further understanding of the molecular mechanism of seed coat color in heading Chinese cabbage.

Identification of SSR markers closely linked to the yellow seed coat color gene in heading Chinese cabbage (Brassica rapa L. ssp. pekinensis).

Research on the yellow-seeded variety of heading Chinese cabbage will aid in broadening its germplasm resources and lay a foundation for AA genome research in Brassica crops. Here, an F2 segregating population of 1575 individuals was constructed from two inbred lines (brown-seeded '92S105' and yellow-seeded '91-125'). This population was used to identify the linkage molecular markers of the yellow seed coat trait using simple sequence repeat (SSR) techniques combined with a bulk segregant analysis (BSA). Of the 144 SSR primer pairs on the A01-A10 chromosomes from the Brassica database (http://brassicadb.org/brad/), two pairs located on the A06 chromosome showed polymorphic bands between the bulk DNA pools of eight brown-seeded and eight yellow-seeded F2 progeny. Based on the genome sequence, 454 SSR markers were designed to A06 to detect these polymorphic bands and were synthesized. Six SSR markers linked to the seed coat color gene were successfully selected for fine linkage genetic map construction, in which the two closest flanking markers, SSR449a and SSR317, mapped the Brsc-ye gene to a 40.2 kb region with distances of 0.07 and 0.06 cM, respectively. The molecular markers obtained in this report will assist in the marker-assisted selection and breeding of yellow-seeded lines in Brassica rapa L. and other close species.

Allelism analysis of BrRfp locus in different restorer lines and map-based cloning of a fertility restorer gene, BrRfp1, for pol CMS in Chinese cabbage (Brassica rapa L.).

KEY MESSAGE: In Chinese cabbage, there are two Rf loci for pol CMS and one of them was mapped to a 12.6-kb region containing a potential candidate gene encoding PPR protein. In Chinese cabbage (Brassica rapa), polima cytoplasmic male sterility (pol CMS) is an important CMS type and is widely used for hybrid breeding. By extensive test crossing in Chinese cabbage, four restorer lines (92s105, 01s325, 00s109, and 88s148) for pol CMS were screened. By analyzing the allelism of the four restorer lines, it was found that 92s105, 01s325, and 00s109 had the same "restorers of fertility" (Rf) locus (designated as BrRfp1), but 88s148 had a different Rf locus (designated as BrRfp2). For fine mapping the BrRfp1 locus of 92s105, a BC1F1 population with 487 individuals and a BC1F2 population with 2485 individuals were successively constructed. Using simple sequence repeat (SSR) markers developed from Brassica rapa reference genome and InDel markers derived from whole-genome resequencing data of 94c9 and 92s105, BrRfp1 was mapped to a 12.6-kb region containing a potential candidate gene encoding pentatricopeptide repeat-containing protein. Based on the nucleotide polymorphisms of the candidate gene sequence between the restoring and nonrestoring alleles, a co-segregating marker SC718 was developed, which would be helpful for hybrid breeding by marker-assisted screening and for detecting new restorer lines.