Identification and fine mapping of Brmmd1 gene controlling recessive genic male sterility in Brassica rapa L.

Recessive genic male sterility (RGMS) provides an effective approach for the commercial exploitation of heterosis, especially in Brassica crops. Although some artificial RGMS mutants have been reported in B. rapa, no causal genes derived from these natural mutants have been identified so far. In this study, a spontaneous RGMS mutant Bcajh97-01A derived from the 'Aijiaohuang' line traced back to the 1980 s was identified. Genetic analysis revealed that the RGMS trait was controlled by a single locus in the Bcajh97-01A/B system. Bulk segregant analysis (BSA) in combination with linkage analysis was employed to delimit the causal gene to an approximate 129 kb interval on chromosome A02. The integrated information of transcriptional levels and the predicted genes in the target region indicated that the Brmmd1 (BraA02g017420) encoding a PHD-containing nuclear protein was the most likely candidate gene. A 374 bp miniature inverted-repeat transposable element (MITE) was inserted into the first exon to prematurely stop the Brmmd1 gene translation, thus blocking the normal expression of this gene at the tetrad stage in the Bcajh97-01A. Additionally, a co-segregating structure variation (SV) marker was developed to rapidly screen the RGMS progenies from Bcajh97-01A/B system. Our findings reveal that BraA02g017420 is the causal gene responsible for the RGMS trait. This study lays a foundation for marker-assisted selection and further molecular mechanism exploration of pollen development in B. rapa.

Chemical composition analysis and transcriptomics reveal the R2R3-MYB genes and phenol oxidases regulating the melanin formation in black radish.

Melanins are dark-brown to black-colored biomacromolecules which have been thoroughly studied in animals and microorganisms. However, the biochemical and molecular basis of plant melanins are poorly understood. We first characterized melanin from the black radish (Raphanus sativus var. niger) 'HLB' through spectroscopic techniques. p-Coumaric acid was identified as the main precursor of radish melanin. Moreover, a joint analysis of transcriptome and coexpression network was performed for the two radish accessions with black and white cortexes, 'HLB' and '55'. A set of R2R3-type RsMYBs and enzyme-coding genes exhibited a coexpression pattern, and were strongly correlated with melanin formation in radish. Transient overexpression of two phenol oxidases RsLAC7 (laccase 7) or RsPOD22-1 (peroxidase 22-1) resulted in a deeper brown color around the infiltration sites and a significant increase in the total phenol content. Furthermore, co-injection of the transcriptional activator RsMYB48/RsMYB97 with RsLAC7 and/or RsPOD22-1, markedly increased the yield of black extracts. Spectroscopic analyses revealed that these extracts are similar to the melanin found in 'HLB'. Our findings advance the understanding of structural information and the transcriptional regulatory mechanism underlying melanin formation in radish.

Genome-Wide Identification of the CDPK Gene Family and Their Involvement in Taproot Cracking in Radish.

Taproot cracking, a severe and common physiological disorder, markedly reduces radish yield and commercial value. Calcium-dependent protein kinase (CDPK) plays a pivotal role in various plant developmental processes; however, its function in radish taproot cracking remains largely unknown. Here, 37 RsCDPK gene members were identified from the long-read radish genome "QZ-16". Phylogenetic analysis revealed that the CDPK members in radish, tomato, and Arabidopsis were clustered into four groups. Additionally, synteny analysis identified 13 segmental duplication events in the RsCDPK genes. Analysis of paraffin-embedded sections showed that the density and arrangement of fleshy taproot cortex cells are important factors that affect radish cracking. Transcriptome sequencing of the fleshy taproot cortex revealed 5755 differentially expressed genes (DEGs) (3252 upregulated and 2503 downregulated) between non-cracking radish "HongYun" and cracking radish "505". These DEGs were significantly enriched in plant hormone signal transduction, phenylpropanoid biosynthesis, and plant-pathogen interaction KEGG pathways. Furthermore, when comparing the 37 RsCDPK gene family members and RNA-seq DEGs, we identified six RsCDPK genes related to taproot cracking in radish. Soybean hairy root transformation experiments showed that RsCDPK21 significantly and positively regulates root length development. These findings provide valuable insights into the relationship between radish taproot cracking and RsCDPK gene function.

Dynamic profiling of intact glucosinolates in radish by combining UHPLC-HRMS/MS and UHPLC-QqQ-MS/MS.

Glucosinolates (GSLs) and their degradation products in radish confer plant defense, promote human health, and generate pungent flavor. However, the intact GSLs in radish have not been investigated comprehensively yet. Here, an accurate qualitative and quantitative analyses of 15 intact GSLs from radish, including four major GSLs of glucoraphasatin (GRH), glucoerucin (GER), glucoraphenin (GRE), and 4-methoxyglucobrassicin (4MGBS), were conducted using UHPLC-HRMS/MS in combination with UHPLC-QqQ-MS/MS. Simultaneously, three isomers of hexyl GSL, 3-methylpentyl GSL, and 4-methylpentyl GSL were identified in radish. The highest content of GSLs was up to 232.46 µmol/g DW at the 42 DAG stage in the 'SQY' taproot, with an approximately 184.49-fold increase compared to the lowest content in another sample. That the GSLs content in the taproots of two radishes fluctuated in a similar pattern throughout the five vegetative growth stages according to the metabolic profiling, whereas the GSLs content in the '55' leaf steadily decreased over the same period. Additionally, the proposed biosynthetic pathways of radish-specific GSLs were elucidated in this study. Our findings will provide an abundance of qualitative and quantitative data on intact GSLs, as well as a method for detecting GSLs, thus providing direction for the scientific progress and practical utilization of GSLs in radish.

Effects of α-Fe2O3 nanoparticles and biochar on plant growth and fruit quality of muskmelon under cadmium stress.

Cadmium pollution in farmland has become a global environmental problem, threatening ecological security and human health. Biochar is effective in remediation of soil pollution. However, high concentrations of biochar can inhibit plant growth, and low concentrations of biochar have limited mitigation effect on cadmium toxicity. Therefore, the combination of low-concentration biochar and other amendments is a promising approach to alleviate cadmium toxicity in plants and improve the safety of edible parts. In this study, muskmelon was selected as the research object, and different concentrations of α-Fe2O3 nanoparticles were used alone or combined with biochar to explore the effects of different treatments on muskmelon plants in cadmium-contaminated soil. The results showed that the combined application of 250 mg/kg α-Fe2O3 nanoparticles and biochar had a good effect on the repair of cadmium toxicity in muskmelon plants. Compared with cadmium treatment, its application increased plant height by 32.53%, cadmium transport factor from root to stem decreased by 32.95%, chlorophyll content of muskmelon plants increased by 14.27%, and cadmium content in muskmelon flesh decreased by 18.83%. Moreover, after plant harvest, soil available cadmium content in 250 mg/kg α-Fe2O3 nanoparticles and biochar combined treatment decreased by 31.18% compared with cadmium treatment. The results of this study provide an effective reference for the composite application of different exogenous amendments and a feasible idea for soil heavy metal remediation and mitigation of cadmium pollution in farmland.

Genetic Diversity Analysis and Core Germplasm Collection Construction of Radish Cultivars Based on Structure Variation Markers.

Radish is an economically important root vegetable worldwide. In this study, the 217 cultivated radish accessions were collected and genotyped. To detect the genotypes of these accessions, a total of 24 structure variation (SV) markers distributed on nine chromosomes were employed to analyze genetic diversity and construct a core germplasm collection of radish. The results of polymorphism information content (PIC) indicated a good polymorphism of these SV markers. Population structure analysis and principal component analysis (PCA) results showed that the 217 radish accessions fell into three main populations (P1, P2, and P3). Genetic diversity analysis showed that these populations were highly associated with geographical distribution. The values of the fixation index (FST) indicated a high genetic diversity between P2 and P3, and a moderate genetic diversity between P1 and P2, and P1 and P3. Furthermore, the 43 core germplasm were exploited for creating cytoplasmic male sterility (CMS) lines and cultivating new radish varieties. The high genetic diversity of 217 radish germplasms will not only provide valuable resources for future genetic mapping and functional genomic research, but also facilitate core germplasm utilization and the molecular breeding of radish.

Identification of Novel Locus RsCr6 Related to Clubroot Resistance in Radish (Raphanus sativus L.).

Clubroot is a devastating disease that causes substantial yield loss worldwide. However, the inheritance and molecular mechanisms of clubroot resistance during pathogen infection in radish remain largely unclear. In this study, we investigated the inheritance of clubroot resistance in the F2 population derived from crossing clubroot-resistant (CR) and clubroot-susceptible inbred lines "GLX" and "XNQ," respectively. Genetic analysis revealed that a single dominant gene controlled the clubroot resistance of "GLX" with a Mendelian ratio of resistance and susceptibility of nearly 3:1. Bulked segregant analysis combined with whole-genome resequencing (BSA-seq) was performed to detect the target region of RsCr6 on chromosome Rs8. Linkage analysis revealed that the RsCr6 locus was located between two markers, HB321 and HB331, with an interval of approximately 92 kb. Based on the outcomes of transcriptome analysis, in the RsCr6 locus, the R120263140 and R120263070 genes with a possible relation to clubroot resistance were considered candidate genes. In addition, three core breeding materials containing the two reported quantitative trait loci (QTLs) and our novel locus RsCr6 targeting clubroot resistance were obtained using marker-assisted selection (MAS) technology. This study reveals a novel locus responsible for clubroot resistance in radishes. Further analysis of new genes may reveal the molecular mechanisms underlying the clubroot resistance of plants and provide a theoretical basis for radish resistance breeding.

Fine mapping of a candidate gene for cool-temperature-induced albinism in ornamental kale.

BACKGROUND: The symptoms of cool-temperature-induced chlorosis (CTIC) are widely existed in higher plants. Although many studies have shown that the genetic mechanism of CTIC is generally controlled by recessive genes in model plants, the dominant inheritance of albinism has not been reported thus far. Here, two CTIC mutants, Red Kamome and White Kamome, were utilized to analyse the inheritance of the albino trait in ornamental kale. The objective of this investigation is to fine-map the target locus and identify the most likely candidate genes for albinism. RESULTS: Genetic analysis revealed that the albinism in the inner leaves of ornamental kale followed semi-dominant inheritance and was controlled by a single locus in two segregating populations. BSR-seq in combination with linkage analysis was employed to fine-map the causal gene, named AK (Albino Kale), to an approximate 60 kb interval on chromosome C03. Transcriptome data from two extreme pools indicated that the differentially expressed gene of Bol015404, which encodes a cytochrome P450 protein, was the candidate gene. The Bol015404 gene was demonstrated to be upregulated in the albino leaves of ornamental kale by qPCR. Additionally, the critical temperature for the albinism was determined between 10 °C and 16 °C by gradient test. CONCLUSIONS: Using two independent segregating populations, the albino mutants were shown to be controlled by one semi-dominant gene, AK, in ornamental kale. The Bol015404 gene was co-segregated with albinism phenotypes, suggesting this unknown function P450 gene as the most likely candidate gene. The albino trait appeared caused by the low temperatures rather than photoperiod. Our results lay a solid foundation on the genetic control of albinism in ornamental kale.

Identification and characterization of abundant repetitive sequences in Allium cepa.

Species of the genus Allium are well known for their large genomes. Allium cepa is of great economic significance. Among vegetables, it ranks second after tomato in terms of the global production value. However, there is limited genomics information available on A. cepa. In this study, we sequenced the A. cepa genome at low-coverage and annotated repetitive sequences by using a combination of next-generation sequencing (NGS) and bioinformatics tools. Nearly 92% of 16 Gb haploid onion genome were defined as repetitive sequences, organized in 162 clusters of at least 0.01 percent of the genome. Of these, a proportion representing 40.5% of the genome were further analyzed in detail to obtain an overview of representative repetitive elements present in the A. cepa genome. Few representative satellite repeats were studied by fluorescence in situ hybridization (FISH) and southern blotting. These results provided a basis for evolutionary cytogenomics within the Allium genus.

Analysis of bacterial communities in rhizosphere soil of continuously cropped healthy and diseased konjac.

The bacterial community and diversity in healthy and diseased konjac rhizosphere soils with different ages of continuous cropping were investigated using next-generation sequencing. The results demonstrated that the number of years of continuous cropping significantly altered soil bacterial community and diversity. Soil bacterial Shannon diversity index and Chao 1 index decreased with the increasing cropping years of konjac. After 1 year of cropping, the soil exhibited the highest bacterial relative abundance and diversity. Of the 44 bacterial genera (relative abundance ratio of genera greater than 0.3%), 14 were significantly affected by the duration of continuous cropping and plant status. With increasing continuous cropping, Alicyclobacillus decreased, while Achromobacter, Lactobacillus, Kaistobacter, Rhodoplanes increased after 3 years continuous cropping. Continuous cropping altered the structure and composition of the soil bacterial community, which led to the reduction in the beneficial bacteria and multiplication of harmful bacteria. These results will improve our understanding of soil microbial community regulation and soil health maintenance in konjac farm systems.

Histone acetylation and reactive oxygen species are involved in the preprophase arrest induced by sodium butyrate in maize roots.

Histone acetylation plays a critical role in controlling chromatin structure, and reactive oxygen species (ROS) are involved in cell cycle progression. To study the relationship between histone acetylation and cell cycle progression in plants, sodium butyrate (NaB), a histone deacetylase (HDAC) inhibitor that can cause a significant increase in histone acetylation in both mammal and plant genomes, was applied to treat maize seedlings. The results showed that NaB had significant inhibition effects on different root zones at the tissue level and caused cell cycle arrest at preprophase in the root meristem zones. This effect was accompanied by a dramatic increase in the total level of acetylated lysine 9 on histone H3 (H3K9ac) and acetylated lysine 5 on histone H4 (H4K5ac). The exposure of maize roots in NaB led to a continuous rise of intracellular ROS concentration, accompanied by a higher electrolyte leakage ratio and malondialdehyde (MDA) relative value. The NaB-treated group displayed negative results in both TdT-mediated dUTP nick end labelling (TUNEL) and γ-H2AX immunostaining assays. The expression of topoisomerase genes was reduced after treatment with NaB. These results suggested that NaB increased the levels of H3K9ac and H4K5ac and could cause preprophase arrest accompanied with ROS formation leading to the inhibition of DNA topoisomerase.

Histone Modification Is Involved in Okadaic Acid (OA) Induced DNA Damage Response and G2-M Transition Arrest in Maize.

Histone modifications are involved in regulation of chromatin structure. To investigate the relationship between chromatin modification and cell cycle regulation during plant cell proliferation, Okadaic acid (OA), a specific inhibitor of serine/threonine protein phosphatase, was applied in this study. The results showed that OA caused the cell cycle arrest at preprophase, leading to seedling growth inhibition. Western blotting assay revealed that the spatial distribution of phosphorylation of Ser10 histone H3 tails (H3S10ph) signals was altered under OA treatment. Reactive oxygen species (ROS) was found to be at higher levels and TdT-mediated dUTP nick end labeling (TUNEL) assay displayed DNA breaks happened at the chromatin after treatment with OA, companied with an increase in the acetylation of histone H4 at lysine 5 (H4K5ac) level. From these observations, we speculated that the alteration of the spatial distribution of H3S10ph and the level of H4K5ac was involved in the procedure that OA induced DNA breaks and G2-M arrested by the accumulation of ROS, and that the histone H3S10ph and H4K5ac might facilitate DNA repair by their association with the chromatin decondensation.

ABA treatment of germinating maize seeds induces VP1 gene expression and selective promoter-associated histone acetylation.

Seed germination commences from a low metabolic state to a bioactive state and is associated with changes in the pattern of gene expression. Recent studies have revealed that epigenetic processes are involved in abscisic acid (ABA)-regulated seed germination processes. In this study, we showed that the expression of both histone acetyltransferases (HATs) and histone deacetylases (HDACs) was increased gradually during seed germination accompanying an increase in overall acetylation level of histone H3. Application of exogenous ABA repressed the expression of HATs as well as HDACs and delayed histone acetylation. Suppressing HDAC by treatment with an HDAC inhibitor, trichostatin A (TSA), led to an increase in global histone acetylation and inhibited seed germination and growth. However, ABA and TSA both delayed the downregulation of the embryogenesis-related gene viviparous1 (VP1) during seed germination. The further chromatin immunoprecipitation experiments showed that the promoter region of the VP1 gene was deacetylated during seed germination, and this deacetylation event was inhibited by both ABA and TSA. These results suggested that a balance of the two enzymes HATs and HDACs affected the acetylation status of the VP1 gene and ABA selectively activated its transcription by an accumulation of acetylated histone H3 associated with the promoter region during seed germination.