SSR molecular marker developments and genetic diversity analysis of Zanthoxylum nitidum (Roxb.) DC.

Zanthoxylum nitidum (Roxb.) is a commonly used traditional Chinese medicine. However, the collection and protection of wild germplasm resources of Z. nitidum are still insufficient, and there is limited research on its genetic diversity and fingerprint. In the present study, 15 simple sequence repeat (SSR) markers were developed by genotyping based on multiplexed shotgun sequencing. The genetic diversity of 51 populations (142 individuals) of Z. nitidum was evaluated using these 15 SSRs. A total of 245 alleles (Na) were detected, with an average value of 16.333, and the average polymorphism information content was 0.756. The genetic distance among 51 populations was 0.164~1.000, with an average of 0.659. Analysis of molecular variance showed low genetic differentiation (40%) and high genetic differentiation (60%) between populations and individuals, respectively. The genetic differentiation coefficient (Fst) of the population was 0.338, indicating that 66.2% of the genetic variation occurred within the population, and the gene flow (Nm) was 0.636, demonstrating that the gene exchange between populations was low. Clustering analysis revealed that the genetic similarity coefficient was 0.30, dividing the 51 populations into 4 groups of 2, 17, 3, and 29 populations. There was no specific relationship between geographical location differences and genetic distance. The genetic diversity level of Z. nitidum is relatively high, and our results provide a theoretical basis for the rapid identification of Z. nitidum germplasm resources and variety selection.

A NAC transcription factor ZaNAC93 confers floral initiation, fruit development, and prickle formation in Zanthoxylum armatum.

Zanthoxylum armatum is a dioecious prickly plant which developed apomictic reproduction. The increases in male flowers and prickle density in female plants lead to low yield and picking efficiency. However, little is known concerning the mechanisms of floral development and prickle formation. NAC is a well-known transcription factor that participates in multiple aspects of plant growth and development. Herein, we characterize the functions and regulatory mechanisms of candidate NACs controlling both traits in Z. armatum. A total of 159 ZaNACs were identified, and 16 of these were male-biased, represented by the NAP subfamily members ZaNAC93 and ZaNAC34, orthologs of AtNAC025 and AtNARS1/NAC2 respectively. Overexpression of ZaNAC93 in tomato led to modifications in flower and fruit development, including earlier flowering, increased numbers of lateral shoots and flowers, accelerated plant senescence, and reduced size and weight of fruits and seeds. In addition, the trichome density in leaves and inflorescences was dramatically reduced in ZaNAC93-OX lines. Overexpression of ZaNAC93 resulted in the up-/downregulation of genes associated with GA, ABA and JA signaling pathways, such as GAI, PYL and JAZ, as well as several TFs, including bZIP2, AGL11, FBP24 and MYB52. Yeast two-hybrid analysis revealed that ZaNAC93 protein could interact with AP1, GAI, bZIP2 and AGL11 in Z. armatum, which might contribute to floral induction, fruit growth, and trichome initiation. This work provides new insights into the molecular mechanisms of ZaNAC93 in reproductive development and prickle formation in Z. armatum.

Comparative transcriptome analysis reveals hormone, transcriptional and epigenetic regulation involved in prickle formation in Zanthoxylum armatum.

Zanthoxylum armatum is an evergreen plant with high economical and medicinal values. The presence of prickles on stems and leaves is undesirable for them make picking difficult. To date, little is known of prickle formation in Z. armatum. Herein, the morphological and molecular features of prickle initiation in prickless (WC) and three types of prickly Z. armatum were characterized. Compared to WC, the levels of cytokinin and auxin were increased, while GA and JA declined in prickly Z. armatum. Transcriptome analysis identified 6258 differentially expressed genes (DEGs) between prickless and prickly Z. armatum. Among them, several DEGs related to hormone metabolism and signaling, including LOG7, CKX3, AHK1, three DELLAs, six JAZs and TIR1, were candidate genes involved in prickle formation. Transcription factors associated with prickle formation were screened, including MYB6-1/MYB6-2, WER, GL3-2, SPL4/5, SOC1, and SCL32. Of them, MYB6-1 and WER might negatively regulate prickles initiation via interacting with GL3-2. Additionally, the histone acetylation and DNA methylation levels, the transcripts of histone acetyltransferase/deacetylase and DNA methyltransferases showed significant differences between prickless and prickly plants, indicating their involvements in prickle initiation. These findings illustrate the regulation of prickle formation might be mediated by phytohormones (especially cytokinin), transcription factors and epigenetic modifications in Z. armatum.

Genome-Wide Identification and Analysis of the Growth-Regulating Factor Family in Zanthoxylum armatum DC and Functional Analysis of ZaGRF6 in Leaf Size and Longevity Regulation.

Growth-regulating factors (GRFs) are plant-specific transcription factors that play an important role in plant growth and development. In this study, fifteen GRF gene members containing QLQ and WRC domains were identified in Zanthoxylum armatum. Phylogenetic and collinearity analysis showed that ZaGRFs were closely related to CsGRFs and AtGRFs, and distantly related to OsGRFs. There are a large number of cis-acting elements related to hormone response and stress induction in the GRF gene promoter region of Z. armatum. Tissue-specific expression analysis showed that except for ZaGRF7, all the ZaGRFs were highly expressed in young parts with active growth and development, including terminal buds, seeds, and young flowers, suggesting their key roles in Z. armatum growth and development. Eight ZaGRFs were selected to investigate the transcriptional response to auxin, gibberellin and drought treatments. A total of six ZaGRFs in the NAA treatment, four ZaGRFs in the GA3 treatment, and six ZaGRFs in the PEG treatment were induced and significantly up-regulated. Overexpression of ZaGRF6 increased branching and chlorophyll content and delayed senescence of transgenic Nicotiana benthamiana. ZaGRF6 increased the expression of CRF2 and suppressed the expression of ARR4 and CKX1, indicating that ZaGRF6 is involved in cytokinin metabolism and signal transduction. These research results lay a foundation for further analysis of the GRF gene function of Z. armatum and provide candidate genes for growth, development, and stress resistance breeding of Z. armatum.

A methanolic extract of Zanthoxylum bungeanum modulates secondary metabolism regulator genes in Aspergillus flavus and shuts down aflatoxin production.

Aflatoxin B1 (AFB1) is a food-borne toxin produced by Aspergillus flavus and a few similar fungi. Natural anti-aflatoxigenic compounds are used as alternatives to chemical fungicides to prevent AFB1 accumulation. We found that a methanolic extract of the food additive Zanthoxylum bungeanum shuts down AFB1 production in A. flavus. A methanol sub-fraction (M20) showed the highest total phenolic/flavonoid content and the most potent antioxidant activity. Mass spectrometry analyses identified four flavonoids in M20: quercetin, epicatechin, kaempferol-3-O-rhamnoside, and hyperoside. The anti-aflatoxigenic potency of M20 (IC50: 2-4 µg/mL) was significantly higher than its anti-proliferation potency (IC50: 1800-1900 µg/mL). RNA-seq data indicated that M20 triggers significant transcriptional changes in 18 of 56 secondary metabolite pathways in A. flavus, including repression of the AFB1 biosynthesis pathway. Expression of aflR, the specific activator of the AFB1 pathway, was not changed by M20 treatment, suggesting that repression of the pathway is mediated by global regulators. Consistent with this, the Velvet complex, a prominent regulator of secondary metabolism and fungal development, was downregulated. Decreased expression of the conidial development regulators brlA and Medusa, genes that orchestrate redox responses, and GPCR/oxylipin-based signal transduction further suggests a broad cellular response to M20. Z. bungeanum extracts may facilitate the development of safe AFB1 control strategies.

SMRT and Illumina RNA sequencing reveal the complexity of terpenoid biosynthesis in Zanthoxylum armatum.

Sichuan pepper (Zanthoxylum armatum DC) is a popular spice and is often prescribed in traditional Chinese medicine to treat vomiting, diarrhea, ascariasis and eczema, among other conditions. Volatile oils from Z. armatum leaves contain active ingredients, with terpenoids being one of the main components. In the present study, the combination of sequencing data of Z. armatum from PacBio single molecule real time (SMRT) and Illumina RNA sequencing (RNA-Seq) platforms facilitated an understanding of the gene regulatory network of terpenoid biosynthesis in pepper leaves. The leaves of three developmental stages from two Z. armatum cultivars, 'Rongchangwuci' (WC) and 'Zhuye' (ZY), were selected as test materials to construct sequencing libraries. A total of 143,122 predictions of unique coding sequences, 105,465 simple sequence repeats, 20,145 transcription factors and 4719 long non-coding RNAs (lncRNAs) were identified, and 142,829 transcripts were successfully annotated. The occurrence of alternative splicing events was verified by reverse transcription PCR, and quantitative real-time PCR was used to confirm the expression pattern of six randomly selected lncRNAs. A total of 96,931 differentially expressed genes were filtered from different samples. According to functional annotation, a total of 560 candidate genes were involved in terpenoid synthesis, of which 526 were differentially expressed genes (DEGs). To identify the key genes involved in terpenoid biosynthesis, the module genes in different samples, including structural and transcription factors genes, were analyzed using the weighted gene co-expression network method, and the co-expression network of genes was constructed. Thirty-one terpenoids were identified by gas chromatography-mass spectrometry. The correlation between 18 compounds with significantly different contents and genes with high connectivity in the module was jointly analyzed in both cultivars, yielding 12 candidate DEGs presumably involved in the regulation of terpenoid biosynthesis. Our findings showed that full-length transcriptome SMRT and Illumina RNA-Seq can play an important role in studying organisms without reference genomes and elucidating the gene regulation of a biosynthetic pathway.

Transcriptome analysis reveals the molecular mechanisms of response to an emergent yellow-flower disease in green Chinese prickly ash (Zanthoxylum schinifolium).

Chinese prickly ash (Zanthoxylum) is extensively used as spice and traditional medicine in eastern Asian countries. Recently, an emergent yellow-flower disease (YFD) break out in green Chinese prickly ash (Zanthoxylum schinifolium, Qinghuajiao in Chinese) at Chongqing municipality, and then leads to a sharp reduction in the yield of Qinghuajiao, and thus results in great economic losses for farmers. To address the molecular response for the emergent YFD of Qinghuajiao, we analyzed the transcriptome of 12 samples including the leaves and inflorescences of asymptomatic and symptomatic plants from three different towns at Chongqing by high-throughput RNA-Seq technique. A total of 126,550 genes and 229,643 transcripts were obtained, and 21,054 unigenes were expressed in all 12 samples. There were 56 and 164 different expressed genes (DEGs) for the AL_vs_SL (asymptomatic leaf vs symptomatic leaf) and AF_vs_SF (asymptomatic flower vs symptomatic flower) groups, respectively. The results of KEGG analysis showed that the "phenylpropanoid biosynthesis" pathway that related to plant-pathogen interaction were found in AL_vs_SL and AF_vs_SF groups, and the "Plant-pathogen interaction" found in AF_vs_SF group, implying that this Qinghuajiao YFD might cause by plant pathogen. Interestingly, we detected 33 common unigenes for the 2 groups, and almost these unigenes were up-regulated in the symptomatic plants. Moreover, most of which were homologs to virus RNA, the components of viruses, implying that this YFD was related to virus. Our results provided a primary molecular basis for the prevention and treatment of YFD of Qinghuajiao trees.

Genome survey of Zanthoxylum bungeanum and development of genomic-SSR markers in congeneric species.

Zanthoxylum bungeanum, a spice and medicinal plant, is cultivated in many parts of China and some countries in Southeast Asia; however, data on its genome are lacking. In the present study, we performed a whole-genome survey and developed novel genomic-SSR markers of Z. bungeanum. Clean data (∼197.16 Gb) were obtained and assembled into 11185221 scaffolds with an N50 of 183 bp. K-mer analysis revealed that Z. bungeanum has an estimated genome size of 3971.92 Mb, and the GC content, heterozygous rate, and repeat sequence rate are 37.21%, 1.73%, and 86.04%, respectively. These results indicate that the genome of Z. bungeanum is complex. Furthermore, 27153 simple sequence repeat (SSR) loci were identified from 57288 scaffolds with a minimum length > 1 kb. Mononucleotide repeats (19706) were the most abundant type, followed by dinucleotide repeats (5154). The most common motifs were A/T, followed by AT/AT; these SSRs accounted for 71.42% and 11.84% of all repeats, respectively. A total of 21243 non-repeating primer pairs were designed, and 100 were randomly selected and validated by PCR analysis using DNA from 10 Z. bungeanum individuals and 5 Zanthoxylum armatum individuals. Finally, 36 polymorphic SSR markers were developed with polymorphism information content (PIC) values ranging from 0.16 to 0.75. Cluster analysis revealed that Z. bungeanum and Z. armatum could be divided into two major clusters, suggesting that these newly developed SSR markers are useful for genetic diversity and germplasm resource identification in Z. bungeanum and Z. armatum.

Comparative Transcriptome Analysis and Expression of Genes Reveal the Biosynthesis and Accumulation Patterns of Key Flavonoids in Different Varieties of Zanthoxylum bungeanum Leaves.

Zanthoxylum bungeanum (Rutaceae), a popular food flavoring and traditional Chinese medicine ingredient, is an important cash crop. Its leaves are rich in flavonoids with multiple bioactivities. However, the transcriptional sequencing has not been investigated, and the molecular basis for the flavonoid biosynthesis remains unclear in this plant. This paper, the key flavonoids (epicatechin, rutin, hyperoside, trifolin, quercitrin, and afzelin) contents were determined in the leaves of 10 Z. bungeanum varieties from a common garden. Results show the leaves of Z. bungeanum mainly contained hyperoside (11.410-21.721 mg/g) and quercitrin (9.401-18.016 mg/g). The total content of these key components was the highest in Fengxian Dahongpao (66.012 mg/g) and the lowest in Fugu (32.223 mg/g). Three varieties (Hancheng stingless, Fugu, and Fengxian Dahongpao) with significant differences in the total content of key flavonoids were selected for transcriptome analysis to obtain flavonoid biosynthesis-related genes. In total, 83 522 unigenes were obtained, 40 668 (48.69%) unigenes were annotated, and 6656 differentially expressed genes (DEGs) were identified. Comparison of the other two varieties, Fugu had many differentially expressed genes indicating the particularity of its variety. Flavonoid-related DEGs of 22 structural genes, including three PALs, one CYP73A, three 4CLs, six CHSs, one CHI, one F3H, one DFR, two ANSs, one ANR, one FLS, and two CYP75B1s, as well as nine MYBs were obtained. These structural genes had different expression patterns in different Z. bungeanum varieties. It is worth noting that the genes expressing the flavonoid 3'5' hydroxylase are absent in Z. bungeanum. Furthermore, quantitative real-time PCR experiment showed consistent results in transcriptome analysis. The RNA-Seq data set of this study sheds lights on the molecular mechanism of flavonoid biosynthesis in Z. bungeanum, provides valuable information for the metabolic regulation of flavonoids, and may serve as a guide for future breeding programs.

Development of conventional PCR and real-time PCR assays to discriminate the origins of Chinese pepper oil and herbal materials from Zanthoxylum.

BACKGROUND: To ensure the safety, quality and therapeutic efficacy of processed foods and herbal medicines, it is important to identify and discriminate economically motivated adulterants. Zanthoxylum schinifolium is sold at a higher price than other Zanthoxylum species and is frequently adulterated with closely related Zanthoxylum species because of its high demand as a Korean food ingredient and medicinal material in markets. In addition, the pericarps of three Zanthoxylum species (Z. schinifolium, Z. bungeanum and Z. piperitum) are defined as herbal medicine Zanthoxyli Pericarpium in Korean pharmacopoeias, but not Z. piperitum in Chinese pharmacopoeias. Further confusion arises in the morphological similarity between Z. armatum (adulterant) and Z. bungeanum. Therefore, the aim of this study was to develop a sequence characterized amplified region (SCAR) marker for discrimination of four Zanthoxylum species. RESULTS: With the goal of developing rapid and reliable tools for genetic discrimination of authentic Zanthoxyli Pericarpium, we designed species-specific SCAR markers, based on ITS2 sequences, that generate amplicons of less than 200 bp. Using these markers, we established both conventional and real-time PCR assay methods capable of differentiating samples at the species level. We validated the ability of SCAR markers to authenticate edible oil and herbal medicine, and confirmed that some herbal medicines contaminated with Z. armatum are being distributed as Zanthoxyli Pericarpium in Korean and Chinese markets. CONCLUSIONS: The SCAR markers and PCR methods described represent powerful tools for protecting against adulteration and ensuring standardization of processed foods and herbal medicine. © 2018 The Authors. Journal of the Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Genome-wide transcriptome profiling of the medicinal plant Zanthoxylum planispinum using a single-molecule direct RNA sequencing approach.

High-throughput RNA sequencing has revolutionized transcriptome-based studies of candidate genes, key pathways and gene regulation in non-model organisms. We analyzed full-length cDNA sequences in Zanthoxylum planispinum (Z. planispinum), a medicinal herb in major parts of East Asia. The full-length mRNA derived from tissues of leaf, early fruit and maturing fruit stage were sequenced using PacBio RSII platform to identify isoform transcriptome. We obtained 51,402 unigenes, with average 1781 bp per gene in 82.473 Mb gene lengths. Among 51,402, 3963 unigenes showed variety of isoform. By selection of one representative gene among each of the various isoforms, we finalized 46,306 unique gene set for this herb. We identified 76 cytochrome P450 (CYP450) and related isoforms that are of the wide diversity in the molecular function and biological process. These transcriptome data of Z. planispinum will provide a good resource to study metabolic engineering for the production of valuable medicinal drugs and phytochemicals.

Study of Some Zanthoxylum Species by Chemical and DNA Analysis Approaches.

The authentication and traceability of spices is a major concern for industrials and consumers. We focused on species from Zanthoxylum genera which are used for many different applications by local populations and also for trading as spices (dried pericarps or whole fruits). In this case, literature gives contradictory data about botanical names, and commercial labelling is often confusing. We studied commercial fruits pericarps extracts obtained by supercritical CO2 and analyzed them by GC/MS. The very complex volatile and semi volatile fractions composition of each extract is described. The barcoding method including molecular biology and phylogenetic analyses was also developed in order to check the commercial botanical identification of the raw material. This is a robust method to identify species in berries samples. We used one genetic marker to identify two Rutaceae clusters, including several species of Zanthoxylum genus. These results indicate that Fagara and Zanthoxylum groups could be considered as two different genera. Combination of chemical analysis and DNA analysis provides an original approach to increase chemical and botanical Zanthoxylum genus knowledge.

Expression Stabilities of Ten Candidate Reference Genes for RT-qPCR in Zanthoxylum bungeanum Maxim.

Real-time reverse transcription quantitative PCR has become a common method for studying gene expression, however, the optimal selection of stable reference genes is a prerequisite for obtaining accurate quantification of transcript abundance. Suitable reference genes for RT-qPCR have not yet been identified for Chinese prickly ash (Zanthoxylum bungeanum Maxim.). Chinese prickly ash is the source of an important food seasoning in China. In recent years, Chinese prickly ash has also been developed as a medicinal plant. The expression stabilities of ten genes (18S, 28S, EF, UBA, UBQ, TIF, NTB, TUA, RPS, and TIF5A) were evaluated in roots, stems, leaves, flowers and fruits at five developmental stages and also under stress from cold, drought, and salt. To do this we used three different statistical algorithms: geNorm, NormFinder and BestKeeper. Among the genes investigated, UBA and UBQ were found to be most stable for the different cultivars and different tissues examined, UBQ and TIF for fruit developmental stage. Meanwhile, EF and TUA were most stable under cold treatment, EF and UBQ under drought treatment and NTB and RPS under salt treatment. UBA and UBQ for all samples evaluated were most stably expressed, but 18S, TUA and RPS were found to be generally unreliable as reference genes. Our results provide a basis for the future selection of reference genes for biological research with Chinese prickly ash, under a variety of conditions.

[Study on Genetic Diversity of Twelve Natural Zanthoxylum dissitum Populations].

OBJECTIVE: The genetic diversity of twelve natural Zanthoxylum dissitum populations, which is a species of Chinese herbal medicines to four provinces of southwest China, has been investigated. METHODS: By inter-simple sequence repeat markers (ISSR), the eight primers, which could amplify stable, clear and highly polymorphic bands, were screened from 100 candidate primers. RESULTS: 150 total ISSR discernible bands and 147 polymorphic were amplified by the eight checked primers. On one hand, the percentage of polymorphic bands was 98.0%, on the other hand, the population level the percent of polymorphic bands ranged from 26.0% to 62.0%. The Shannon's information index within species (Hsp) was 0.4175, while the values within population (Hpop) were ranged from 0.1328 to 0.3267. Analysis of molecular variance (ANOVA) revealed that the population genetic variation accounted for 47.98% but the intraspecific variation for 52.02%. CONCLUSION: The high level of genetic diversity exists not only in population but also in species. A high degree of genetic differentiation populations is approved to exist in Zanthoxylum dissitum. These results lay a theoretical foundation for genetic diversity analysis of Zanthoxylum dissitum.

[Authentication of Zanthexylum bungeanum Maxin population and adulterants by analysis of rDNA ITS sequences].

AIM: To study the difference of rDNA ITS sequences between Zanthexylum bungeanum populations and their adulterants in main habitants of China so as to provide molecular markers for identifying Zanthexylum bungeanum populations against adulterants. METHODS: rDNA ITS regions (including ITS-1, 5.8S and ITS-2) of 7 populations of Zanthexylum bungeanum which are separate located in Gansu, Shanxi, Sichuan, Hebei provinces, and 3 adulterants were sequenced by PCR products sequencing method or clone sequencing method. RESULTS: The sequences of rDNA ITS region of Zanthexylum bungeanum were reported for the first time, and the sequences of ITS region ranged from 619 to 620 bp, and the length difference amoung Zanthexylum bungeanum and their adulterants is 4 bp. There are 15 variable sites, 12 informative sites and 3 authenticable sites among Zanthexylum bungeanum populations. The difference of rDNA ITS regions amoung Zanthexylum bungeanum and their adulterants is obvious, the number of variable sites is 71. CONCLUSION: The difference of rDNA ITS sequences can be used to authenticate accurately the populations of Zanthexylum bungeanum and their adulterants. These populations of Z. bungeanum which have close relationship always distribute in near geographic areas. The characteristics of rDNA ITS sequence can be used as good markers for authenticating Zanthexylum bungeanum populations form their adulterants.