Yang Qiu

Transcriptome Analysis of Barbarea vulgaris Infested with Diamondback Moth (Plutella xylostella) Larvae

Background The diamondback moth (DBM, Plutella xylostella) is a crucifer-specific pest that causes significant crop losses worldwide. Barbarea vulgaris (Brassicaceae) can resist DBM and other herbivorous insects by producing feeding-deterrent triterpenoid saponins. Plant breeders have long aimed to transfer this insect resistance to other crops. However, a lack of knowledge on the biosynthetic pathways and regulatory networks of these insecticidal saponins has hindered their practical application. A pyrosequencing-based transcriptome analysis of B. vulgaris during DBM larval feeding was performed to identify genes and gene networks responsible for saponin biosynthesis and its regulation at the genome level. Principal Findings Approximately 1.22, 1.19, 1.16, 1.23, 1.16, 1.20, and 2.39 giga base pairs of clean nucleotides were generated from B. vulgaris transcriptomes sampled 1, 4, 8, 12, 24, and 48 h after onset of P. xylostella feeding and from non-inoculated controls, respectively. De novo assembly using all data of the seven transcriptomes generated 39,531 unigenes. A total of 37,780 (95.57%) unigenes were annotated, 14,399 of which were assigned to one or more gene ontology terms and 19,620 of which were assigned to 126 known pathways. Expression profiles revealed 2,016–4,685 up-regulated and 557–5188 down-regulated transcripts. Secondary metabolic pathways, such as those of terpenoids, glucosinolates, and phenylpropanoids, and its related regulators were elevated. Candidate genes for the triterpene saponin pathway were found in the transcriptome. Orthological analysis of the transcriptome with four other crucifer transcriptomes identified 592 B. vulgaris-specific gene families with a P-value cutoff of 1e−5. Conclusion This study presents the first comprehensive transcriptome analysis of B. vulgaris subjected to a series of DBM feedings. The biosynthetic and regulatory pathways of triterpenoid saponins and other DBM deterrent metabolites in this plant were classified. The results of this study will provide useful data for future investigations on pest-resistance phytochemistry and plant breeding.

Aromatic glucosinolate biosynthesis pathway in Barbarea vulgaris and its response to Plutella xylostella infestation

The inducibility of the glucosinolate resistance mechanism is an energy-saving strategy for plants, but whether induction would still be triggered by glucosinolate-tolerant Plutella xylostella (diamondback moth, DBM) after a plant had evolved a new resistance mechanism (e.g., saponins in Barbara vulgaris) was unknown. In B. vulgaris, aromatic glucosinolates derived from homo-phenylalanine are the dominant glucosinolates, but their biosynthesis pathway was unclear. In this study, we used G-type (pest-resistant) and P-type (pest-susceptible) B. vulgaris to compare glucosinolate levels and the expression profiles of their biosynthesis genes before and after infestation by DBM larvae. Two different stereoisomers of hydroxylated aromatic glucosinolates are dominant in G- and P-type B. vulgaris, respectively, and are induced by DBM. The transcripts of genes in the glucosinolate biosynthesis pathway and their corresponding transcription factors were identified from an Illumina dataset of G- and P-type B. vulgaris. Many genes involved or potentially involved in glucosinolate biosynthesis were induced in both plant types. The expression patterns of six DBM induced genes were validated by quantitative PCR (qPCR), while six long-fragment genes were validated by molecular cloning. The core structure biosynthetic genes showed high sequence similarities between the two genotypes. In contrast, the sequence identity of two apparent side chain modification genes, the SHO gene in the G-type and the RHO in P-type plants, showed only 77.50% identity in coding DNA sequences and 65.48% identity in deduced amino acid sequences. The homology to GS-OH in Arabidopsis, DBM induction of the transcript and a series of qPCR and glucosinolate analyses of G-type, P-type and F1 plants indicated that these genes control the production of S and R isomers of 2-hydroxy-2-phenylethyl glucosinolate. These glucosinolates were significantly induced by P. xylostella larvae in both the susceptiple P-type and the resistant G-type, even though saponins are the main DBM-resistance causing metabolites in G-type plants. Indol-3-ylmethylglucosinolate was induced in the G-type only. These data will aid our understanding of the biosynthesis and induction of aromatic glucosinolates at the molecular level and also increase our knowledge of the complex mechanisms underpinning defense induction in plants.

Interspecific hybridization, polyploidization, and backcross of Brassica oleracea var. alboglabra with B. rapa var. purpurea morphologically recapitulate the evolution of Brassica vegetables.

Brassica oleracea and B. rapa are two important vegetable crops. Both are composed of dozens of subspecies encompassing hundreds of varieties and cultivars. Synthetic B. napus with these two plants has been used extensively as a research model for the investigation of allopolyploid evolution. However, the mechanism underlying the explosive evolution of hundreds of varieties of B. oleracea and B. rapa within a short period is poorly understood. In the present study, interspecific hybridization between B. oleracea var. alboglabra and B. rapa var. purpurea was performed. The backcross progeny displayed extensive morphological variation, including some individuals that phenocopied subspecies other than their progenitors. Numerous interesting novel phenotypes and mutants were identified among the backcross progeny. The chromosomal recombination between the A and C genomes and the chromosomal asymmetric segregation were revealed using Simple Sequence Repeats (SSR) markers. These findings provide direct evidence in support of the hypothesis that interspecific hybridization and backcrossing have played roles in the evolution of the vast variety of vegetables among these species and suggest that combination of interspecific hybridization and backcrossing may facilitate the development of new mutants and novel phenotypes for both basic research and the breeding of new vegetable crops.

Influence of pH, concentration and light on stability of allicin in garlic (Allium sativum L.) aqueous extract as measured by UPLC

BACKGROUND Garlic is one of the most important bulb vegetables and is mainly used as a spice or flavoring agent for foods. It is also cultivated for its medicinal properties, attributable to sulfur compounds, of which allicin is the most important. However, the stability of allicin in garlic extract is not well understood. In this study, using UPLC, the stability of allicin extracted in water from garlic was evaluated in phosphate buffer at different temperatures under light and dark conditions. RESULTS At room temperature, allicin in aqueous extract was most stable at pH 5-6 but degraded quickly at lower or higher pH. It began to degrade within 0.5 h and was not detectable after 2 h when the pH was higher than 11 or lower than 1.5. It degraded quickly when the temperature was higher than 40 °C and especially higher than 70 °C. At room temperature, allicin in water could be stored for 5 days without obvious degradation. Higher concentrations of allicin in solution were somewhat more stable than low concentrations. CONCLUSION Allicin extract was sensitive to pH and temperature of storage but not to light. Higher-concentration allicin solution was more stable.

Comprehensive analysis of expressed sequence tags from cultivated and wild radish (Raphanus spp.)

BackgroundRadish (Raphanus sativus L., 2n = 2× = 18) is an economically important vegetable crop worldwide. A large collection of radish expressed sequence tags (ESTs) has been generated but remains largely uncharacterized.ResultsIn this study, approximately 315,000 ESTs derived from 22 Raphanus cDNA libraries from 18 different genotypes were analyzed, for the purpose of gene and marker discovery and to evaluate large-scale genome duplication and phylogenetic relationships among Raphanus spp. The ESTs were assembled into 85,083 unigenes, of which 90%, 65%, 89% and 89% had homologous sequences in the GenBank nr, SwissProt, TrEMBL and Arabidopsis protein databases, respectively. A total of 66,194 (78%) could be assigned at least one gene ontology (GO) term. Comparative analysis identified 5,595 gene families unique to radish that were significantly enriched with genes related to small molecule metabolism, as well as 12,899 specific to the Brassicaceae that were enriched with genes related to seed oil body biogenesis and responses to phytohormones. The analysis further indicated that the divergence of radish and Brassica rapa occurred approximately 8.9-14.9 million years ago (MYA), following a whole-genome duplication event (12.8-21.4 MYA) in their common ancestor. An additional whole-genome duplication event in radish occurred at 5.1-8.4 MYA, after its divergence from B. rapa. A total of 13,570 simple sequence repeats (SSRs) and 28,758 high-quality single nucleotide polymorphisms (SNPs) were also identified. Using a subset of SNPs, the phylogenetic relationships of eight different accessions of Raphanus was inferred.ConclusionComprehensive analysis of radish ESTs provided new insights into radish genome evolution and the phylogenetic relationships of different radish accessions. Moreover, the radish EST sequences and the associated SSR and SNP markers described in this study represent a valuable resource for radish functional genomics studies and breeding.

Genome-wide identification of microRNAs associated with taproot development in radish (Raphanus sativus L.)

MicroRNAs (miRNAs) are small, endogenous, non-coding RNAs that play vital regulatory roles in plant growth and development. To identify the miRNAs associated with taproot development at the whole genome level, we sequenced five RNA libraries constructed from radish taproots at different developmental stages and generated a total of 148M clean reads. Using an integrative bioinformatics analysis, 494 known miRNAs belonging to 434 families and 220 putative novel miRNAs were identified. Combining the differential expression analysis and target prediction, we found that 77 miRNAs were potentially associated with taproot development. Target transcripts generated significant GO terms relating to cell proliferation, root development and hormone-mediated signaling. The KEGG analyses revealed that plant hormone signal transduction, zeatin biosynthesis, biosynthesis of secondary metabolites, cell cycle, MAPK signaling and p53 signaling were closely associated with taproot development. These findings will provide valuable information for further functional verification of miRNAs and their targets in radish taproot development.

Differential expression of salt tolerance related genes in Brassica campestris L. ssp. chinensis (L.) Makino var. communis Tsen et Lee

We examined salt tolerance responsive genes in Pak-choi under salt stress and analyze their potential function. The mRNA differential display was used to screen the transcript derived fragments (TDFs) related to salinity tolerance in tolerant and moderately tolerant Pak-choi germplasm. Seventy-eight primer combinations generated 101 differential cDNA fragments, which were divided into 10 expression types. Seven cDNA sequences (GenBank accession Nos. DQ006915∼DQ006921) obtained and sequenced were highly homologous to some known expression genes or the genes related to the signaling pathways in plants under different abiotic stress.

Identification of Optimal Reference Genes for Expression Analysis in Radish (Raphanus sativus L.) and Its Relatives Based on Expression Stability

Radish (Raphanus sativus) is an important cruciferous root crop with a close relationship to Chinese cabbage (Brassica rapa). RT-qPCR is used extensively to evaluate the expression levels of target genes, and accurate measurement of target gene expression with this method is determined by the valid reference genes used for data nomalization in different experimental conditions. Screening for appropriate reference genes with stable expression based on RT-qPCR data is important for gene expression and functional analysis research in radish and its relatives. However, many researches have thought that almost no single reference gene is widely suitable for all experimental conditions, and few researchers have paid attention to the validation of reference genes in radish gene expression analysis. In the present study, 12 candidate reference genes were selected for analysis. Their expression in 28 samples, including 20 radish samples from different organs and conditions, four Chinese cabbage organs and four organs of their distant hybrid, was assessed by RT-qPCR and then five software tools—ΔCt, geNorm, NormFinder, BestKeeper and RefFinder—were used to compare their expression stability. The results showed that the most suitable reference genes were different in different organs and conditions. GAPDH, DSS1, and UP2 were optimal reference genes for gene expression analysis in all organs and conditions in radish. UPR, GSNOR1, and ACTIN2/7 were the most stable reference genes in different radish organs. UP2 and GAPDH were suitable reference genes for radish pistil development studies. RPII, UBC9, and GAPDH had the most stable expression in radish under various stresses. DSS1, UP2, and TEF2 were the optimal reference genes for Chinese cabbage organs, whereas TUA was optimal for the distant hybrid. UP2, and TEF2 were appropriate reference genes for all of the samples together. The optimal reference genes we identified, UP2, GAPDH, UPR, and GSNOR1 were verified by normalizing the expression patterns of YAB3, RPL, and FUL. These results will provide important information for selecting target reference genes in different research contexts and improve the accuracy and precision of gene expression analysis for radish, Chinese cabbage and their distant hybrid.

Erratum to: Sprout differentiation and mutation induction of garlic (Allium sativum L.) callus exposed to gamma radiation

Germplasm enhancement and breeding is difficult for garlic (Allium sativum L.) as it can only be vegetatively propagated. Hence, mutation induction is still the most effective way to create new varieties. Calli from two Chinese commercial garlic varieties, Zhoumou (ZM) and Yongnian (YN), were treated with different dosages (1, 3, 5 and 7 Gy) of gamma (γ) radiation. The results showed that the two genotypes differed in their sensitivity to γ-radiation. YN was sensitive to high dosage, while ZM showed better growth at higher dosage. More specifically, the average number of calli producing sprouts, sprout number per callus, total sprouts number, the number of sprouts forming plantlets and plantlet lengths were higher when calli of variety YN were treated with 1 Gy. However, the corresponding parameters in ZM were higher for 7 Gy. Simple sequence repeat analysis with two of 16 novel primers showed genetic variation among the plantlets following γ-radiation treatments of 5 Gy for variety YN and 7 Gy for variety ZM. The methods and mutant materials in this study could be used in future garlic breeding programs.

Insights into the species-specific metabolic engineering of glucosinolates in radish ( Raphanus sativus L.) based on comparative genomic analysis

Glucosinolates (GSLs) and their hydrolysis products present in Brassicales play important roles in plants against herbivores and pathogens as well as in the protection of human health. To elucidate the molecular mechanisms underlying the formation of species-specific GSLs and their hydrolysed products in Raphanus sativus L., we performed a comparative genomics analysis between R. sativus and Arabidopsis thaliana. In total, 144 GSL metabolism genes were identified, and most of these GSL genes have expanded through whole-genome and tandem duplication in R. sativus. Crucially, the differential expression of FMOGS-OX2 in the root and silique correlates with the differential distribution of major aliphatic GSL components in these organs. Moreover, MYB118 expression specifically in the silique suggests that aliphatic GSL accumulation occurs predominantly in seeds. Furthermore, the absence of the expression of a putative non-functional epithiospecifier (ESP) gene in any tissue and the nitrile-specifier (NSP) gene in roots facilitates the accumulation of distinctive beneficial isothiocyanates in R. sativus. Elucidating the evolution of the GSL metabolic pathway in R. sativus is important for fully understanding GSL metabolic engineering and the precise genetic improvement of GSL components and their catabolites in R. sativus and other Brassicaceae crops.