Identification and analysis of major flavor compounds in radish taproots by widely targeted metabolomics.

Radish (Raphanus sativus L.) is an important Brassicaceous vegetable crop that is cultivated worldwide. The taste of radish can be described as pungent, sweet, and crisp. At present, the metabolic characteristics leading to differences in radish taste remain unclear, due to the lack of large-scale detection and identification of radish metabolites. In this study, UPLC-MS/MS-based targeted metabolome analysis was performed on the taproots of eight radish landraces. We identified a total of 938 metabolites, and each landrace exhibited a specific metabolic profile, making it unique in flavor and quality. Our results show that taste differences among the taproots of different radish landraces can be explained by changes in composition and abundance of glucosinolates, polyphenols, carbohydrates, organic acids, amino acids, vitamins, and lipids. This study reveals the potential metabolic causes of variation in the taste and flavor of radish taproots.

Comparative transcriptome analysis reveals key genes associated with pigmentation in radish (Raphanus sativus L.) skin and flesh.

Radish (Raphanus sativus) is an important vegetable worldwide that exhibits different flesh and skin colors. The anthocyanins responsible for the red and purple coloring in radishes possess nutritional value and pharmaceutical potential. To explore the structural and regulatory networks related to anthocyanin biosynthesis and identify key genes, we performed comparative transcriptome analyses of the skin and flesh of six colored radish accessions. The transcript profiles showed that each accession had a species-specific transcript profile. For radish pigmentation accumulation, the expression levels of anthocyanin biosynthetic genes (RsTT4, RsC4H, RsTT7, RsCCOAMT, RsDFR, and RsLDOX) were significantly upregulated in the red- and purple-colored accessions, but were downregulated or absent in the white and black accessions. The correlation test, combined with metabolome (PCC > 0.95), revealed five structural genes (RsTT4, RsDFR, RsCCOAMT, RsF3H, and RsBG8L) and three transcription factors (RsTT8-1, RsTT8-2, and RsPAR1) to be significantly correlated with flavonoids in the skin of the taproot. Four structural genes (RsBG8L, RsDFR, RsCCOAMT, and RsLDOX) and nine transcription factors (RsTT8-1, RsTT8-2, RsMYB24L, RsbHLH57, RsPAR2L, RsbHLH113L, RsOGR3L, RsMYB24, and RsMYB34L) were found to be significantly correlated with metabolites in the flesh of the taproot. This study provides a foundation for future studies on the gene functions and genetic diversity of radish pigmentation and should aid in the cultivation of new valuable radish varieties.

Rapid and Visual Detection of Meloidogyne hapla Using Recombinase Polymerase Amplification Combined with a Lateral Flow Dipstick Assay.

The northern root-knot nematode, Meloidogyne hapla, is a biotrophic parasite that infects many crops and causes severe economic losses worldwide. Rapid and accurate detection of M. hapla is crucial for disease forecasting and control. We developed a recombinase polymerase amplification combined with a lateral flow dipstick (RPA-LFD) assay for rapid detection of M. hapla. The primers and probe were designed based on the effector gene 16D10 sequence and were highly specific to M. hapla. The RPA reaction was performed at a wide range of temperatures from 25 to 45°C within 5 to 25 min, and the amplicon was visualized directly on the LFD within 5 min. The detection limits of the RPA-LFD assay were 10-3 females and 10-2 second-stage juveniles/0.5 g of soil, which was 10 times more sensitive than the conventional PCR assay. In addition, the RPA-LFD assay can detect M. hapla from infested plant roots and soil samples, and the entire detection process can be completed within 1.5 h. These results indicate that the RPA-LFD assay is a simple, rapid, specific, sensitive, and visual method that can be used for rapid detection of M. hapla in the field and in resource-limited conditions.

A Comparative Metabolomics Study of Flavonoids in Radish with Different Skin and Flesh Colors (Raphanus sativus L.).

Radish (Raphanus sativus) is an important worldwide vegetable with a wide variety of colors that affect its appearance and nutritional quality. However, the large-scale detection, identification, and quantification of flavonoids in multicolor radish have rarely been studied. To uncover the diversity and accession-specific flavonoids in radish, liquid chromatography electrospray ionization-tandem mass spectrometry was used to analyze the metabolic profiles in the skin and flesh of six colored radish accessions: light-red Manshenhong, dark-red Touxinhong (TXH), purple Zijinling (ZJL), Xinlimei with red flesh (XLMF) and green skin, white Shizhuangbai (SZB), and black radish. In total, 133 flavonoids, including 16 dihydroflavones, 44 flavones, 14 flavonoids, 9 anthocyanins, and 28 flavonols, were characterized. The flavonoid metabolic profiles differed among the different colored radishes. Red and purple radishes contained similar anthocyanin compounds responsible for color pigmentation, including red cyanidin, callistephin, and pelargonin. Purple ZJL was most enriched with cyanidin o-syringic acid and cyanin, whereas callistephin and pelargonin were more abundant in dark-red TXH. Additionally, the black and white radishes shared similar anthocyanin and flavonoid profiles, suggesting that the color of black radishes was not caused by anthocyanin but by other metabolites. The metabolites in colored radishes that differed from SZB were mainly involved in the biosynthesis of plant secondary metabolites, such as flavonoid, flavone, flavonol, isoflavonoid, and phenylpropanoid biosynthesis. This study provides new insights into the differences in metabolite profiles among radishes with different skin and flesh colors. The results will be useful for aiding the cultivation of valuable new radish varieties.

Antidepressant-like effect of flaxseed in rats exposed to chronic unpredictable stress.

INTRODUCTION: Depression is a serious mental illness. However, a significant proportion of patients with depression fail to achieve remission with antidepressant therapies. This study was conducted to explore the antidepressant-like effect of flaxseed oil and flour in an animal model with depression-like behaviors induced by chronic unpredictable stress (CUS). METHODS: Rats were randomly divided into five groups: normal control (Sham-Sham), CUS plus saline (S-SN), CUS plus escitalopram (S-Esc), CUS plus flaxseeds oil (S-FO), and CUS plus flaxseed flour (S-FF). Behaviors were tested using sucrose preference test and forced swimming test. The serum BDNF concentration, hippocampal BDNF mRNA, and protein expression were measured by enzyme-linked immunosorbent assay, real-time PCR, and Western blot, respectively. RESULTS: The sucrose preference rate was significantly higher in S-FO and S-FF rats than in S-SN and S-Esc rats (p < .01), and lower in S-Esc rats than in Sham-Sham rats (p < .01). The immobility time was significantly shorter in S-FO and S-FF rats than in S-SN rats (p < .01), and shorter in S-Esc rats than in S-SN rats (p < .01). Plasma BDNF concentrations were significantly lower in S-FO, S-FF, and S-Esc rats than in Sham-Sham rats (p < .01); BDNF was lower in S-FO, S-FF, and S-SN rats than in S-Esc rats. The hippocampal BDNF protein expression was significantly higher in S-Esc rats than in S-SN rats (p < .05). The hippocampal BDNF mRNA expression was significantly higher in S-Esc rats than in S-SN rats (p < .01). The BDNF gene expression in plasma and the hippocampus negatively correlated with the immobility time (p < .05), but BDNF gene expression in the hippocampus positively correlated with the sucrose preference rate (p < .05). CONCLUSION: Flaxseed oil and flaxseed flour exert antidepressant-like effect in rats exposed to chronic stress. Flaxseed may have a therapeutic effect on depression.

Antifungal, plant growth-promoting, and mycotoxin detoxication activities of Burkholderia sp. strain XHY-12.

A bacterial strain named XHY-12 was isolated from corn soil samples and identified as Burkholderia sp. based on 16S rDNA sequencing, it displayed high antagonistic activity against 12 fungal pathogens and the common fungal contaminant in grain Aspergillus flavus. Plate experiment showed that XHY-12 fermentation broth reduced the incidence of S. sclerotiorum on detached rape leaves (Brassica campestris L.) by 100%, and a greenhouse experiment showed that it could promote the growth of rape seedlings with significant increases in plant height, root length, and fresh weight. Furthermore, a novel funding was the reduction of aflatoxin B1 and B2 by over 85% in 60 h, and the decomposition enzymes should be extracellular. The results suggest that XHY-12 has a potential for commercial applications as biocontrol, mycotoxin detoxification agent or biofertilizer.

A naturally occurring variation in the BrMAM-3 gene is associated with aliphatic glucosinolate accumulation in Brassica rapa leaves.

Glucosinolate profiles significantly vary among Brassica rapa genotypes. However, the molecular basis of these variations is largely unknown. In this study, we investigated a major quantitative trait locus (QTL) controlling aliphatic glucosinolate accumulation in B. rapa leaves. The QTL, which encompasses three tandem MAM genes and two MYB genes, was detected in two BC2DH populations. Among the five-candidate genes, only the expression level of BrMAM-3 (Bra013007) was significantly correlated with the accumulation of aliphatic glucosinolates in B. rapa leaves. We identified a naturally occurring insertion within exon 1 of BrMAM-3, which is predicted to be a loss-of-function mutation, as confirmed by qRT-PCR. We determined that the loss of function was associated with the low glucosinolate content in B. rapa accessions. Furthermore, overexpressing the BrMAM-3 gene resulted in an increase in total aliphatic glucosinolates in Arabidopsis transgenic lines. Our study provides insights into the molecular mechanism underlying the accumulation of aliphatic glucosinolates in B. rapa leaves, thereby facilitating in the manipulation of total aliphatic glucosinolate content in Brassica crops.

[Genetic diversity of 6 species in Polygonatum by SSR marker].

Polygonatum is a genus of the perennial herb family Liliaceae, with great potential in food, medicine and other field. In this study, genetic diversity and cluster structure analysis of 6 species in Polygonatum were investigated the molecular marker technique of simple sequence repeat (SSR). A total of 49 SSR makers were used to study genetic diversity and population structure within 60 germplasm resources which obtained from 38 counties and cities in 14 provinces of China. A total of 211 alleles were identified and the number of alleles ranged from 2 to 10, with an average of 4.306 1 alleles per SSR. The range of polymorphism information content (PIC) varied from 0.731 8 to 0.031 7, with the average of 0.309 6. The cluster analysis classified 60 germplasm resources into four defined groups at the genetic distance value of 0.26, among which most species with relatives were clustered into the same group. Extraordinarily, there were 6 germplasm resources clustered into other species, indicating that the classification of inter-genus and geographical distribution was crossed in Polygonatum. The genetic diversity index of the 4 geographical populations from high to low was: Western region, Central China, Southern China, Eastern China. The population structure analysis, also indicating divided the entire collection into four groups, which was similar to the assignment pattern of cluster structure analysis. These results suggested that the Polygonatum germplasm resources used in this study is rich in relatively high genetic diversity with large variation range, relatively fuzzy boundaries of species. It appeared the phenomenon that there is a difference decreases between the alternate leaf system and the rotation leaf system. The genetic diversity in the western region was higher than that in other regions, and the western region may be the origin center of the genus Polygonatum.

Transcriptome-referenced association study of clove shape traits in garlic.

Genome-wide association studies are a powerful approach for identifying genes related to complex traits in organisms, but are limited by the requirement for a reference genome sequence of the species under study. To circumvent this problem, we propose a transcriptome-referenced association study (TRAS) that utilizes a transcriptome generated by single-molecule long-read sequencing as a reference sequence to score population variation at both transcript sequence and expression levels. Candidate transcripts are identified when both scores are associated with a trait and their potential interactions are ascertained by expression quantitative trait loci analysis. Applying this method to characterize garlic clove shape traits in 102 landraces, we identified 22 candidate transcripts, most of which showed extensive interactions. Eight transcripts were long non-coding RNAs (lncRNAs), and the others were proteins involved mainly in carbohydrate metabolism, protein degradation, etc. TRAS, as an efficient tool for association study independent of a reference genome, extends the applicability of association studies to a broad range of species.

Comparative Transcriptome Profile of the Cytoplasmic Male Sterile and Fertile Floral Buds of Radish (Raphanus sativus L.).

Radish cytoplasmic male sterility (CMS) has been widely used for breeding in Raphanus and Brassica genera. However, the detailed regulation network of the male sterility remains to be determined. Our previous work has shown that the abnormalities in a CMS radish appeared shortly after the tetrad stage when microspores were malformed and the tapetal cells grew abnormally large. In this work, histological analysis shows that anthers are at the tetrad stage when the radish buds are about 1.5 mm in length. Furthermore, a high throughput RNA sequencing technology was employed to characterize the transcriptome of radish buds with length about 1.5 mm from two CMS lines possessing the CMS-inducing orf138 gene and corresponding near-isogenic maintainer lines. A total of 67,140 unigenes were functionally annotated. Functional terms for these genes are significantly enriched in 55 Gene Ontology (GO) groups and 323 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. The transcriptome detected transcripts for 72 out of a total of 79 protein genes encoded in the chloroplast genome from radish. In contrast, the radish mitochondrial genome contains 34 protein genes, but only 16 protein transcripts were detected from the transcriptome. The transcriptome comparison between CMS and near-isogenic maintainer lines revealed 539 differentially expressed genes (DEGs), indicating that the false positive rate for comparative transcriptome profiling was clearly decreased using two groups of CMS/maintainer lines with different nuclear background. The level of 127 transcripts was increased and 412 transcripts were decreased in the CMS lines. No change in levels of transcripts except CMS-inducing orf138 was identified from the mitochondrial and chloroplast genomes. Some DEGs which would be associated with the CMS, encoding MYB and bHLH transcription factors, pentatricopeptide repeat (PPR) proteins, heat shock transcription factors (HSFs) and heat shock proteins (HSPs), are discussed. The transcriptome dataset and comparative analysis will provide an important resource for further understanding anther development, the CMS mechanism and to improve molecular breeding in radish.

Tracing the transcriptomic changes in synthetic Trigenomic allohexaploids of Brassica using an RNA-Seq approach.

Polyploidization has played an important role in plant evolution and speciation, and newly formed allopolyploids have experienced rapid transcriptomic changes. Here, we compared the transcriptomic differences between a synthetic Brassica allohexaploid and its parents using a high-throughput RNA-Seq method. A total of 35,644,409 sequence reads were generated, and 32,642 genes were aligned from the data. Totals of 29,260, 29,060, and 29,697 genes were identified in Brassicarapa, Brassicacarinata, and Brassica allohexaploid, respectively. We compared 7,397 differentially expressed genes (DEGs) between Brassica hexaploid and its parents, as well as 2,545 nonadditive genes of Brassica hexaploid. We hypothesized that the higher ploidy level as well as secondary polyploidy might have influenced these changes. The majority of the 3,184 DEGs between Brassica hexaploid and its paternal parent, B. rapa, were involved in the biosynthesis of secondary metabolites, plant-pathogen interactions, photosynthesis, and circadian rhythm. Among the 2,233 DEGs between Brassica hexaploid and its maternal parent, B. carinata, several played roles in plant-pathogen interactions, plant hormone signal transduction, ribosomes, limonene and pinene degradation, photosynthesis, and biosynthesis of secondary metabolites. There were more significant differences in gene expression between the allohexaploid and its paternal parent than between it and its maternal parent, possibly partly because of cytoplasmic and maternal effects. Specific functional categories were enriched among the 2,545 nonadditive genes of Brassica hexaploid compared with the additive genes; the categories included response to stimulus, immune system process, cellular process, metabolic process, rhythmic process, and pigmentation. Many transcription factor genes, methyltransferases, and methylation genes showed differential expression between Brassica hexaploid and its parents. Our results demonstrate that the Brassica allohexaploid can generate extensive transcriptomic diversity compared with its parents. These changes may contribute to the normal growth and reproduction of allohexaploids.

A comparative light and electron microscopic analysis of microspore and tapetum development in fertile and cytoplasmic male sterile radish.

To gain further insight into the abortive stages and ultrastructural changes leading to pollen degeneration of a novel cytoplasmic male sterile radish 805A, we compared differences of cellular and subcellular structure of sterile anther with fertile anther by light and electron microscopy analysis. Two types of locule degeneration in sterile anther were detected, of which the time of degeneration occurred and completed was different. In type I, abnormality of pollen mother cells (PMCs) and tapetal cells, including condensation of cytoplasm and large vacuoles within tapetal cells, was shown at PMC stage. In type II, meiosis and early tetrad stage progressed normally except for large vacuoles that appeared in tapetal cells. Ultrastructural alterations of the cellular organization were observed in the type II locules, such as chromatin condensation at the periphery of the nucleus and degeneration of the karyotheca, compared with normal pollen development. The results suggested that the cytoplasmic male sterility anther degeneration was probably caused by dysfunctions of tapetum and vacuolation of tapetum, PMCs, and microspores. Thus, the identical factors, which induced CMS in the same cytoplasmic and nuclear genetic background, might affect development of tapetum and microspore at different stages during the cytoplasmic male sterile 805A anther development.

Cloning and characterization of a novel sulfate transporter gene from radish (Raphanus sativus L.).

A putative sulfate transporter gene, RSultr3.2A, was identified from radish (Raphanus sativus L.). The RSultr3.2A gene encodes a polypeptide of 651 amino acids with a calculated molecular mass of 71701 Da. The deduced amino acid sequence preserves the common motifs of the sulfate transporters and exhibits a high degree of homology to other plant sulfate transporters. RSultr3.2A was not induced by sulfur starvation and was expressed in roots, stems, leaves, flowers and young pods, suggesting a possible need for the nonregulated and constitutive sulfate transport system of the plant. The RSultr3.2A is a single copy gene in the radish genome. A genomic sequence information corresponding to RSultr3.2A cDNA was found by PCR and sequencing. Comparison of cDNA and genomic sequences revealed that the coding sequence of RSultr3.2A consisted of 12 exons. In addition, another cDNA (RSultr3.2B) from radish young pods was found. As compared with RSultr3.2A, RSultr3.2B encodes a 499 amino acid protein with modifications in the carboxy-terminus. Sequence analysis suggested an occasional donor splicing site mutation (GT to TT) in the RSultr3.2A gene from radish young pods leading to the truncated cDNA RSultr3.2B.