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.

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.

Transcriptome Analysis Reveals Candidate Genes Associated with Leaf Etiolation of a Cytoplasmic Male Sterility Line in Chinese Cabbage (Brassica Rapa L. ssp. Pekinensis).

Cytoplasmic male sterility (CMS) is universally utilized in cruciferous vegetables. However, the Chinese cabbage hau CMS lines, obtained by interspecific hybridization and multiple backcrosses of the Brassica juncea (B. juncea) CMS line and Chinese cabbage, show obvious leaf etiolation, and the molecular mechanism of etiolation remains elusive. Here, the ultrastructural and phenotypic features of leaves from the Chinese cabbage CMS line 1409A and maintainer line 1409B are analyzed. The results show that chloroplasts of 1409A exhibit abnormal morphology and distribution. Next, RNA-sequencing (RNA-Seq) is used to identify 485 differentially expressed genes (DEGs) between 1409A and 1409B, and 189 up-regulated genes and 296 down-regulated genes are found. Genes that affect chloroplasts development, such as GLK1 and GLK2, and chlorophyll biosynthesis, such as PORB, are included in the down-regulated DEGs. Quantitative real-time PCR (qRT-PCR) analysis validate that the expression levels of these genes are significantly lower in 1409A than in 1409B. Taken together, these results demonstrate that leaf etiolation is markedly affected by chloroplast development and pigment biosynthesis. This study provides an effective foundation for research on the molecular mechanisms of leaf etiolation of the hau CMS line in Chinese cabbage (Brassica rapa L. ssp. pekinensis).

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.

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.

Yield and size of oyster mushroom grown on rice/wheat straw basal substrate supplemented with cotton seed hull.

Oyster mushroom (Pleurotus ostreatus) was cultivated on rice straw basal substrate, wheat straw basal substrate, cotton seed hull basal substrate, and wheat straw or rice straw supplemented with different proportions (15%, 30%, and 45% in rice straw substrate, 20%, 30%, and 40% in wheat straw substrate) of cotton seed hull to find a cost effective substrate. The effect of autoclaved sterilized and non-sterilized substrate on growth and yield of oyster mushroom was also examined. Results indicated that for both sterilized substrate and non-sterilized substrate, oyster mushroom on rice straw and wheat basal substrate have faster mycelial growth rate, comparatively poor surface mycelial density, shorter total colonization period and days from bag opening to primordia formation, lower yield and biological efficiency, lower mushroom weight, longer stipe length and smaller cap diameter than that on cotton seed hull basal substrate. The addition of cotton seed hull to rice straw and wheat straw substrate slowed spawn running, primordial development and fruit body formation. However, increasing the amount of cotton seed hull can increase the uniformity and white of mycelium, yield and biological efficiency, and increase mushroom weight, enlarge cap diameter and shorten stipe length. Compared to the sterilized substrate, the non-sterilized substrate had comparatively higher mycelial growth rate, shorter total colonization period and days from bag opening to primordia formation. However, the non-sterilized substrate did not gave significantly higher mushroom yield and biological efficiency than the sterilized substrate, but some undesirable characteristics, i.e. smaller mushroom cap diameter and relatively long stipe length.

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.

Genetic characterization of a new cytoplasmic male sterility system (hau) in Brassica juncea and its transfer to B. napus.

A novel cytoplasmic male sterility (CMS) was identified in Brassica juncea, named as hau CMS (00-6-102A). Subsequently, the male sterility was transferred to B. napus by interspecific hybridization. The hau CMS has stable male sterility. Flowers on the A line are absolutely male sterile, and seeds harvested from the line following pollinations with the maintainer gave rise to 100% sterile progeny. The anthers in CMS plants are replaced by thickened petal-like structures and pollen grains were not detected. In contrast, in other CMS systems viz. pol, nap, tour, and ogu, anthers are formed but do not produce viable pollen. The sterility of hau CMS initiates at the stage of stamen primordium polarization, which is much earlier compared with the other four CMS systems. We have successfully transferred hau CMS from B. juncea to B. napus. Restorer lines for pol, ogu, nap, and tour CMS systems were found to be ineffective to restore fertility in hau CMS. Sixteen out of 40 combinations of mitochondrial probe/enzyme used for RFLP analysis distinguished the hau CMS system from the other four systems. Among these sixteen combinations, five ones alone could distinguish the five CMS systems from each other. The evidence from genetic, morphological, cytological and molecular studies confirmed that the hau CMS system is a novel CMS system.