Gene silencing of BnaA09.ZEP and BnaC09.ZEP confers orange color in Brassica napus flowers.

Brassica napus is currently cultivated as an important ornamental crop in China. Flower color has attracted much attention in rapeseed genetics and breeding. Here, we characterize an orange-flowered mutant of B. napus that exhibits an altered carotenoid profile in its petals. As revealed by map-based cloning, the change in color from yellow to orange is attributed to the loss of BnaC09.ZEP (zeaxanthin epoxidase) and a 1695-bp deletion in BnaA09.ZEP. HPLC analysis, genetic complementation and CRISPR/Cas9 experiments demonstrated that BnaA09.ZEP and BnaC09.ZEP have similar functions, and the abolishment of both genes led to a substantial increase in lutein content and a sharp decline in violaxanthin content in petals but not leaves. BnaA09.ZEP and BnaC09.ZEP are predominantly expressed in floral tissues, whereas their homologs, BnaA07.ZEP and BnaC07.ZEP, mainly function in leaves, indicating redundancy and tissue-specific diversification of BnaZEP function. Transcriptome analysis in petals revealed differences in the expression of carotenoid and flavonoid biosynthesis-related genes between the mutant and its complementary lines. Flavonoid profiles in the petals of complementary lines were greatly altered compared to the mutant, indicating potential cross-talk between the regulatory networks underlying the carotenoid and flavonoid pathways. Additionally, our results indicate that there is functional compensation by BnaA07.ZEP and BnaC07.ZEP in the absence of BnaA09.ZEP and BnaC09.ZEP. Cloning and characterization of BnaZEPs provide insights into the molecular mechanisms underlying flower pigmentation in B. napus and would facilitate breeding of B. napus varieties with higher ornamental value.

Genetic mapping reveals BjRf as a candidate gene controlling fertility restoration of the oxa CMS in Brassica juncea.

KEY MESSAGE: A candidate gene for male fertility restoration in Brassica juncea, BjRf, was isolated from a 23-kb interval on chromosome A05 using map-based cloning and BSA methods. The cytoplasmic male sterility/fertility restoration (CMS/Rf) system has been extensively used for heterosis in plants. It also provides valuable resources for studying mitochondrial-nuclear coevolution and interaction. The oxa CMS, which is a new CMS type reported in Brassica juncea (B. juncea), has been broadly used in the exploitation and application of heterosis in this species. However, the oxa CMS fertility restorer gene BjRf has not been reported. In this study, a stable restorer line was successfully constructed via continuous testcross and artificial selection. Besides, a new Rf gene was mapped in a 23-kb region on chromosome A05 in B. juncea with a genetic distance of 0.5 cM by the method incorporating bulk segregant analysis (BSA) and conventional map-based cloning. Finally, BjuA017917, a non-PPR Rf gene encoding a guanosine nucleotide diphosphate dissociation inhibitor (GDI), is proposed to be the candidate gene for fertility restoration of the oxa CMS line in B. juncea. Moreover, a functional marker, CRY3, was developed for marker-assisted selection for Brassica juncea breeding.

Fine-mapping of the BjPur gene for purple leaf color in Brassica juncea.

Purple leaves are rich in health-protecting anthocyanins and food colorants in Brassica juncea. But the causal gene, which is related to leaf color formation, have not been reported in B. juncea. Anthocyanins mainly accumulated throughout the adaxial and abaxial epidermal leaf cells of purple leaves. A genetic analysis indicated that an incompletely dominant gene controls the purple leaf trait in B. juncea. Furthermore, the BjPur gene, which increased anthocyanin accumulation in purple-leaf mustard, was cloned. Blast and phylogenetic analyses revealed that BjPur encodes a new R2R3-MYB transcription factor. Sequence analysis of two alleles revealed a DNA sequence insertion in the first intron of BjPur in green leaves parent line (LY) when compared with the BjPur gene in the purple-leaf parent line (ZY). And this insertion greatly reduced the transcription of BjPur in green leaves. In purple-leaf plants, the transcript level of BjPur was significantly higher in leaves than in roots, stems, siliques, and flower buds. Additionally, molecular markers linked to leaf color were developed to distinguish different genotypes of B. juncea. These results will be helpful for the genetic improvement of the purple leaf color in B. juncea.

Construction of restorer lines and molecular mapping for restorer gene of hau cytoplasmic male sterility in Brassica napus.

KEY MESSAGE: Successfully constructing restorer lines for the hau CMS line and molecular mapping of Rfh to a 94 kb candidate region on chromosome A03 in Brassica napus. Cytoplasmic male sterility is a general phenomenon in almost 200 species, and the interaction between chimeric genes in mitochondria and restorer genes in nucleus may be responsible for restoration of male fertility. Orf288 has been identified as a CMS-associated gene in the hau CMS line of Brassica napus and Brassica juncea; however, the restorer lines/genes have not been found yet. We therefore have successfully constructed two restorer lines in B. napus by extensive testcrossing and have mapped a major restorer gene Rfh to a physical distance of 94 kb on chromosome A03 by whole-genome resequencing and molecular markers. We found that the restorer line is indeed restored to male fertility at histological level. Comparative genomics and collinearity analysis between close relatives revealed that rearrangements and recombination may have happened and thus caused the production of Rfh or components of the restoration of fertility complex. Meanwhile, nuclear backgrounds with multiple loci and temperature were related to the variation and instability of restoration of fertility in three different populations. Our study provides new sights into the coevolution between restorer genes and CMS-associated genes as well as the cultivation of superior hybrids via molecular breeding.

Transcript levels of orf288 are associated with the hau cytoplasmic male sterility system and altered nuclear gene expression in Brassica juncea.

Cytoplasmic male sterility (CMS) is primarily caused by chimeric genes located in the mitochondrial genomes. In Brassica juncea, orf288 has been identified as a CMS-associated gene in the hau CMS line; however, neither the specific abortive stage nor the molecular function of the gene have been determined. We therefore characterized the hau CMS line, and found that defective mitochondria affect the development of archesporial cells during the L2 stage, leading to male sterility. The expression level of the orf288 transcript was higher in the male-sterility line than in the fertility-restorer line, although no significant differences were apparent at the protein level. The toxicity region of ORF288 was found to be located near the N-terminus and repressed growth of Escherichia coli. However, transgenic expression of different portions of ORF288 indicated that the region that causes male sterility resides between amino acids 73 and 288, the expression of which in E. coli did not result in growth inhibition. Transcriptome analysis revealed a wide range of genes involved in anther development and mitochondrial function that were differentially expressed in the hau CMS line. This study provides new insights into the hau CMS mechanism by which orf288 affects the fertility of Brassica juncea.

Morphological and genetic characterization of a new cytoplasmic male sterility system (oxa CMS) in stem mustard (Brassica juncea).

KEY MESSAGE: oxa CMS is a new cytoplasmic male sterility type in Brassica juncea. oxa CMS is a cytoplasmic male sterility (CMS) line that has been widely used in the production and cultivation of stem mustard in the southwestern China. In this study, different CMS-type specific mitochondrial markers were used to confirm that oxa CMS is distinct from the pol CMS, ogu CMS, nap CMS, hau CMS, tour CMS, Moricandia arvensis CMS, orf220-type CMS, etc., that have been previously reported in Brassica crops. Pollen grains of the oxa CMS line are sterile with a self-fertility rate of almost 0% and the sterility strain rate and sterility degree of oxa CMS is 100% due to a specific flower structure and flowering habit. Scanning electron microscopy revealed that most pollen grains in mature anthers of the oxa CMS line are empty, flat and deflated. Semi-thin section further showed that the abortive stage of anther development in oxa CMS is initiated at the late uninucleate stage. Abnormally vacuolated microspores caused male sterility in the oxa CMS line. This cytological study combined with marker-assisted selection showed that oxa CMS is a novel CMS type in stem mustard (Brassica juncea). Interestingly, the abortive stage of oxa CMS is later than those in other CMS types reported in Brassica crops, and there is no negative effect on the oxa CMS line growth period. This study demonstrated that this novel oxa CMS has a unique flower structure with sterile pollen grains at the late uninucleate stage. Our results may help to uncover the mechanism of oxa CMS in Brassica juncea.

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

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

Fine mapping and candidate gene analysis of an anthocyanin-rich gene, BnaA.PL1, conferring purple leaves in Brassica napus L.

Because of the advantages of anthocyanins, the genetics and breeding of crops rich in anthocyanins has become a hot research topic. However, due to the lack of anthocyanin-related mutants, no regulatory genes have been mapped in Brassica napus. In this study, we first report the characterization of a B. napus line with purple leaves and the fine mapping and candidate screening of the BnaA.PL1 gene. The amount of anthocyanins in the purple leaf line was six times higher than that in a green leaf line. A genetic analysis indicated that the purple character was controlled by an incomplete dominant gene. Through map-based cloning, we localized the BnaA.PL1 gene to a 99-kb region at the end of B. napus chromosome A03. Transcriptional analysis of 11 genes located in the target region revealed that the expression level of only the BnAPR2 gene in seedling leaves decreased from purple to reddish green to green individuals, a finding that was consistent with the measured anthocyanin accumulation levels. Molecular cloning and sequence analysis of BnAPR2 showed that the purple individual-derived allele contained 17 variants. Markers co-segregating with BnaA.PL1 were developed from the sequence of BnAPR2 and were validated in the BC4P2 population. These results suggested that BnAPR2, which encodes adenosine 5'-phosphosulfate reductase, is likely to be a valuable candidate gene. This work may lay the foundation for the marker-assisted selection of B. napus vegetables that are rich in anthocyanins and for an improved understanding of the molecular mechanisms controlling anthocyanin accumulation in Brassica.

Characterization and classification of one new cytoplasmic male sterility (CMS) line based on morphological, cytological and molecular markers in non-heading Chinese cabbage (Brassica rapa L.).

KEY MESSAGE: A new non-heading Chinese cabbage CMS line M119A was characterized and specific molecular markers were developed to classify different CMS types. One new non-heading Chinese cabbage (Brassica rapa L.) cytoplasmic male sterile (CMS) line M119A was obtained by interspecific crosses between the recently discovered hau CMS line of Brassica juncea and B. rapa. Furthermore, the line was characterized and compared with other five isonuclear-alloplasmic CMS lines. The M119A line produced six stamens without pollen and only two stamen fused together in fewer flowers. Tissue section indicated that anther abortion in M119A may have occurred during differentiation of the archesporial cells without pollen sac. All the six CMS lines were grouped into three types based on the presence of three PCR fragments of 825, 465 and 772 bp amplified with different mitochondrial genes specific primers. The 825-bp fragment was amplified both in 09-10A and H201A using the specific primer pair P-orf224-atp6, and showed 100 % identity with the mitochondrial gene of pol CMS. The 465-bp fragment was amplified in 30A and 105A using the primer pair P-orf138 and shared 100 % identity with the mitochondrial gene of ogu CMS. The 772-bp fragment was amplified in M119A and H203A using the primer pair P-orf288 and showed 100 % identity with the mitochondrial gene of hau CMS. Therefore, these markers could efficiently distinguish different types of isonuclear-alloplasmic CMS lines of non-heading Chinese cabbage, which were useful for improving the efficiency of cross-breeding and heterosis utilization in cruciferous vegetables.

Comparative analysis of mitochondrial genomes between the hau cytoplasmic male sterility (CMS) line and its iso-nuclear maintainer line in Brassica juncea to reveal the origin of the CMS-associated gene orf288.

BACKGROUND: Cytoplasmic male sterility (CMS) is not only important for exploiting heterosis in crop plants, but also as a model for investigating nuclear-cytoplasmic interaction. CMS may be caused by mutations, rearrangement or recombination in the mitochondrial genome. Understanding the mitochondrial genome is often the first and key step in unraveling the molecular and genetic basis of CMS in plants. Comparative analysis of the mitochondrial genome of the hau CMS line and its maintainer line in B. juneca (Brassica juncea) may help show the origin of the CMS-associated gene orf288. RESULTS: Through next-generation sequencing, the B. juncea hau CMS mitochondrial genome was assembled into a single, circular-mapping molecule that is 247,903 bp in size and 45.08% in GC content. In addition to the CMS associated gene orf288, the genome contains 35 protein-encoding genes, 3 rRNAs, 25 tRNA genes and 29 ORFs of unknown function. The mitochondrial genome sizes of the maintainer line and another normal type line "J163-4" are both 219,863 bp and with GC content at 45.23%. The maintainer line has 36 genes with protein products, 3 rRNAs, 22 tRNA genes and 31 unidentified ORFs. Comparative analysis the mitochondrial genomes of the hau CMS line and its maintainer line allowed us to develop specific markers to separate the two lines at the seedling stage. We also confirmed that different mitotypes coexist substoichiometrically in hau CMS lines and its maintainer lines in B. juncea. The number of repeats larger than 100 bp in the hau CMS line (16 repeats) are nearly twice of those found in the maintainer line (9 repeats). Phylogenetic analysis of the CMS-associated gene orf288 and four other homologous sequences in Brassicaceae show that orf288 was clearly different from orf263 in Brassica tournefortii despite of strong similarity. CONCLUSION: The hau CMS mitochondrial genome was highly rearranged when compared with its iso-nuclear maintainer line mitochondrial genome. This study may be useful for studying the mechanism of natural CMS in B. juncea, performing comparative analysis on sequenced mitochondrial genomes in Brassicas, and uncovering the origin of the hau CMS mitotype and structural and evolutionary differences between different mitotypes.

A male sterility-associated cytotoxic protein ORF288 in Brassica juncea causes aborted pollen development.

Cytoplasmic male sterility (CMS) is a widespread phenomenon in higher plants, and several studies have established that this maternally inherited defect is often associated with a mitochondrial mutant. Approximately 10 chimeric genes have been identified as being associated with corresponding CMS systems in the family Brassicaceae, but there is little direct evidence that these genes cause male sterility. In this study, a novel chimeric gene (named orf288) was found to be located downstream of the atp6 gene and co-transcribed with this gene in the hau CMS sterile line. Western blotting analysis showed that this predicted open reading frame (ORF) was translated in the mitochondria of male-sterile plants. Furthermore, the growth of Escherichia coli was significantly repressed in the presence of ORF288, which indicated that this protein is toxic to the E. coli host cells. To confirm further the function of orf288 in male sterility, the gene was fused to a mitochondrial-targeting pre-sequence under the control of the Arabidopsis APETALA3 promoter and introduced into Arabidopsis thaliana. Almost 80% of transgenic plants with orf288 failed to develop anthers. It was also found that the independent expression of orf288 caused male sterility in transgenic plants, even without the transit pre-sequence. Furthermore, transient expression of orf288 and green fluorescent protein (GFP) as a fused protein in A. thaliana protoplasts showed that ORF288 was able to anchor to mitochondria even without the external mitochondrial-targeting peptide. These observations provide important evidence that orf288 is responsible for the male sterility of hau CMS in Brassica juncea.

Metabolic and transcriptome analysis of dark red taproot in radish (Raphanus sativus L.).

The red color in radish taproots is an important quality index and is mainly affected by anthocyanins. However, the metabolite components and gene expression underlying dark red taproot color formation in radish remain elusive. In this study, the metabolites and gene expression patterns affecting anthocyanin biosynthesis were monitored in the dark red taproots. Comparative analysis of anthocyanin metabolites between dark red taproots and white taproots indicated that pelargonin and pelargonidin 3-O-beta-D-glucoside were the most promising dark red pigments responsible for the coloration of the taproots. Transcriptomic analysis of gene expression between dark red taproots and white taproots revealed that most of genes involved in the anthocyanin biosynthesis pathway were up-regulated in dark red taproots. In particular, RsCHS and RsDFR were the two most up-regulated genes in the dark red taproots. Moreover, the higher coexpression of two R2R3-Myb transcription factors, RsMYB1 and RsMYB2, may contribute to dark red color formation. Our work documents metabolomic and transcriptomic changes related to the dark red color formation in taproots radish and provides valuable data for anthocyanin-rich radish breeding.

Genetic and Comparative Transcriptome Analysis Revealed DEGs Involved in the Purple Leaf Formation in Brassica juncea.

Brassica juncea is an important dietary vegetable cultivated and consumed in China for its edible stalks and leaves. The purple leaf mustard, which is rich in anthocyanins, is eye-catching and delivers valuable nutrition. However, the molecular mechanism involved in anthocyanin biosynthesis has not been well studied in B. juncea. Here, histological and transcriptome analyses were used to characterize the purple leaf color and gene expression profiles. Free-hand section analysis showed that the anthocyanin was mainly accumulated in the adaxial epidermal leaf cells. The anthocyanin content in the purple leaves was significantly higher than that in the green leaves. To investigate the critical genes and pathways involved in anthocyanin biosynthesis and accumulation, the transcriptome analysis was used to identify the differentially expressed genes (DEGs) between the purple and green leaves from the backcrossed BC3 segregation population in B. juncea. A total of 2,286 different expressed genes were identified between the purple and green leaves. Among them, 1,593 DEGs were up-regulated and 693 DEGs were down-regulated. There were 213 differently expressed transcription factors among them. The MYB and bHLH transcription factors, which may regulate anthocyanin biosynthesis, were up-regulated in the purple leaves. Interestingly, most of the genes involved in plant-pathogen interaction pathway were also up-regulated in the purple leaves. The late biosynthetic genes involved in anthocyanin biosynthesis were highly up-regulated in the purple leaves of B. juncea. The up regulation of BjTT8 and BjMYC2 and anthocyanin biosynthetic genes (BjC4H, BjDFR, and BjANS) may activate the purple leaf formation in B. juncea. This study may help to understand the transcriptional regulation of anthocyanin biosynthesis in B. juncea.

Cytological and iTRAQ-based quantitative proteomic analyses of hau CMS in Brassica napus L.

Hau cytoplasmic male sterility (CMS) is a new type of CMS that was originally identified in Brassica juncea and subsequently transferred to B. napus and B. rapa. To further elucidate the molecular mechanism underlying hau CMS in B. napus, semithin section analysis and iTRAQ-based differential proteomic analysis were performed to compare the hau CMS and its maintainer line. Cytological analysis revealed that abnormal anther development in the hau CMS line was arrested during the differentiation of stamen archesporial cells. qRT-PCR analysis showed that the sterility gene orf288 was expressed at substantially higher levels in CMS anthers than in anthers with restored fertility. In comparison with the maintainer line, a total of 186 differentially abundant proteins were identified in the CMS line, 58 of which exhibited increased accumulation and 128 exhibited decreased accumulation. Bioinformatics analysis showed that proteins involved in carbohydrate and energy metabolism, such as those involved in oxidative phosphorylation, glycolysis/gluconeogenesis and pyruvate metabolism, exhibited decreased accumulation in the hau CMS line, whereas those involved in oxidative stress, antagonism of cell death and protein processing exhibited increased accumulation in the hau CMS line, indicating the potential roles of carbohydrate metabolism and energy supply in the regulation of hau anther abortion. BIOLOGICAL SIGNIFICANCE: Cytoplasmic male sterility (CMS) is one of the most efficient ways to produce hybrid seeds in crops. CMS is mainly caused by mitochondrial mutation and has been an important model for investigation of cytoplasmic and nuclear interactions in various plant species. Hau is a new type of CMS line in Brassica with completely abortive anthers. Although studies have been conducted to identify the key genes associated with CMS, the molecular mechanisms underlying hau CMS remain unclear. In this study, cytological, molecular, and proteomic approaches were used to reveal the mechanism underlying hau CMS in B. napus. Based on a comparison of the protein expression profiles of the hau CMS line and its maintainer line to elucidate the mechanisms underlying hau CMS, a potential protein regulatory network is proposed herein. These results may provide new insights into the molecular basis of hau CMS in B. napus.