Zanthoxylum-specific whole genome duplication and recent activity of transposable elements in the highly repetitive paleotetraploid Z. bungeanum genome.

Zanthoxylum bungeanum is an important spice and medicinal plant that is unique for its accumulation of abundant secondary metabolites, which create a characteristic aroma and tingling sensation in the mouth. Owing to the high proportion of repetitive sequences, high heterozygosity, and increased chromosome number of Z. bungeanum, the assembly of its chromosomal pseudomolecules is extremely challenging. Here, we present a genome sequence for Z. bungeanum, with a dramatically expanded size of 4.23 Gb, assembled into 68 chromosomes. This genome is approximately tenfold larger than that of its close relative Citrus sinensis. After the divergence of Zanthoxylum and Citrus, the lineage-specific whole-genome duplication event η-WGD approximately 26.8 million years ago (MYA) and the recent transposable element (TE) burst ~6.41 MYA account for the substantial genome expansion in Z. bungeanum. The independent Zanthoxylum-specific WGD event was followed by numerous fusion/fission events that shaped the genomic architecture. Integrative genomic and transcriptomic analyses suggested that prominent species-specific gene family expansions and changes in gene expression have shaped the biosynthesis of sanshools, terpenoids, and anthocyanins, which contribute to the special flavor and appearance of Z. bungeanum. In summary, the reference genome provides a valuable model for studying the impact of WGDs with recent TE activity on gene gain and loss and genome reconstruction and provides resources to accelerate Zanthoxylum improvement.

ZbAGL11, a class D MADS-box transcription factor of Zanthoxylum bungeanum, is involved in sporophytic apomixis.

Apomixis is a reproductive model that bypasses sexual reproduction, so it does not require the combination of paternal and maternal gametes but instead results in the production of offspring directly from maternal tissues. This reproductive mode results in the same genetic material in the mother and the offspring and has significant applications in agricultural breeding. Molecular and cytological methods were used to identify the reproductive type of Zanthoxylum bungeanum (ZB). Fluorescence detection of the amplified products of 12 pairs of polymorphic SSR primers showed consistent fluorescence signals for mother and offspring, indicating that no trait separation occurred during reproduction. In addition, the cytological observation results showed differentiation of ZB embryos (2n) from nucellar cells (2n) to form indefinite embryonic primordia and then form adventitious embryos (2n), indicating that the apomictic type of ZB is sporophytic apomixis. The MADS-box transcription factor ZbAGL11 was highly expressed during the critical period of nucellar embryo development in ZB. Unpollinated ZbAGL11-OE Arabidopsis produced fertile offspring and exhibited an apomictic phenotype. The overexpression of ZbAGL11 increased the callus induction rate of ZB tissue. In addition, the results of the yeast two-hybrid experiment showed that ZbAGL11 could interact with the ZbCYP450 and ZbCAD11 proteins. Our results demonstrate that ZbAGL11 can cause developmental disorders of Arabidopsis flower organs and result in apomixis-like phenotypes.

Genomic analysis reveals the genetic diversity, population structure, evolutionary history and relationships of Chinese pepper.

Chinese pepper, mainly including Zanthoxylum bungeanum and Zanthoxylum armatum, is an economically important crop popular in Asian countries due to its unique taste characteristics and potential medical uses. Numerous cultivars of Chinese pepper have been developed in China through long-term domestication. To better understand the population structure, demographic history, and speciation of Chinese pepper, we performed a comprehensive analysis at a genome-wide level by analyzing 38,395 genomic SNPs that were identified in 112 cultivated and wild accessions using a high-throughput genome-wide genotyping-by-sequencing (GBS) approach. Our analysis provides genetic evidence of multiple splitting events occurring between and within species, resulting in at least four clades in Z. bungeanum and two clades in Z. armatum. Despite no evidence of recent admixture between species, we detected substantial gene flow within species. Estimates of demographic dynamics and species distribution modeling suggest that climatic oscillations during the Pleistocene (including the Penultimate Glaciation and the Last Glacial Maximum) and recent domestication events together shaped the demography and evolution of Chinese pepper. Our analyses also suggest that southeastern Gansu province is the most likely origin of Z. bungeanum in China. These findings provide comprehensive insights into genetic diversity, population structure, demography, and adaptation in Zanthoxylum.

Patterns of Drought Response of 38 WRKY Transcription Factors of Zanthoxylum bungeanum Maxim.

The WRKY family of transcription factors (TFs) includes a number of transcription-specific groupings that play important roles in plant growth and development and in plant responses to various stresses. To screen for WRKY transcription factors associated with drought stress in Zanthoxylum bungeanum, a total of 38 ZbWRKY were identified and these were then classified and identified with Arabidopsis WRKY. Using bioinformatics analyses based on the structural characteristics of the conservative domain, 38 WRKY transcription factors were identified and categorized into three groups: Groups I, II, and III. Of these, Group II can be divided into four subgroups: subgroups IIb, IIc, IId, and IIe. No ZbWRKY members of subgroup IIa were found in the sequencing data. In addition, 38 ZbWRKY were identified by real-time PCR to determine the behavior of this family of genes under drought stress. Twelve ZbWRKY transcription factors were found to be significantly upregulated under drought stress and these were identified by relative quantification. As predicted by the STRING website, the results show that the WRKYs are involved in four signaling pathways-the jasmonic acid (JA), the salicylic acid (SA), the mitogen-activated protein kinase (MAPK), and the ethylene signaling pathways. ZbWRKY33 is the most intense transcription factor in response to drought stress. We predict that WRKY33 binds directly to the ethylene synthesis precursor gene ACS6, to promote ethylene synthesis. Ethylene then binds to the ethylene activator release signal to activate a series of downstream genes for cold stress and osmotic responses. The roles of ZbWRKY transcription factors in drought stress rely on a regulatory network center on the JA signaling pathway.

Internal transcribed spacer 2 (ITS2) barcodes: A useful tool for identifying Chinese Zanthoxylum.

PREMISE OF THE STUDY: The genus Zanthoxylum in the Rutaceae family of trees and shrubs has a long history of domestication and cultivation in Asia for both economic and medicinal purposes. However, many Zanthoxylum species are morphologically similar and are easily confused. This often leads to false authentication of source materials and confusion in herbal markets, hindering their safe utilization and genetic resource conservation. DNA barcoding is a promising tool for identifying plant taxa. METHODS: We used three candidate DNA barcoding regions (ITS2, ETS, and trnH-psbA) to identify 69 accessions representing 13 Chinese Zanthoxylum species. The discriminatory capabilities of these regions were evaluated in terms of PCR amplification success, intra- and interspecific divergence, DNA barcoding gaps, and identification efficiency using the BLAST and tree-building methods. RESULTS: ITS2 proved the most useful for discriminating Chinese Zanthoxylum species, with a correct identification rate of 100%, and this region also exhibited significantly higher intra- and interspecific divergence. DISCUSSION: Phylogenetic analysis confirmed that ITS2 has a powerful discriminatory ability both at and below the species level. We confirmed that ITS2 is a powerful barcoding region for identifying Chinese Zanthoxylum species, and will be useful for analyzing and managing Chinese Zanthoxylum germplasm collections.

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.

De novo transcriptome assembly of Zanthoxylum bungeanum using Illumina sequencing for evolutionary analysis and simple sequence repeat marker development.

Zanthoxylum, an ancient economic crop in Asia, has a satisfying aromatic taste and immense medicinal values. A lack of genomic information and genetic markers has limited the evolutionary analysis and genetic improvement of Zanthoxylum species and their close relatives. To better understand the evolution, domestication, and divergence of Zanthoxylum, we present a de novo transcriptome analysis of an elite cultivar of Z. bungeanum using Illumina sequencing; we then developed simple sequence repeat markers for identification of Zanthoxylum. In total, we predicted 45,057 unigenes and 22,212 protein coding sequences, approximately 90% of which showed significant similarities to known proteins in databases. Phylogenetic analysis indicated that Zanthoxylum is relatively recent and estimated to have diverged from Citrus ca. 36.5-37.7 million years ago. We also detected a whole-genome duplication event in Zanthoxylum that occurred 14 million years ago. We found no protein coding sequences that were significantly under positive selection by Ka/Ks. Simple sequence repeat analysis divided 31 Zanthoxylum cultivars and landraces into three major groups. This Zanthoxylum reference transcriptome provides crucial information for the evolutionary study of the Zanthoxylum genus and the Rutaceae family, and facilitates the establishment of more effective Zanthoxylum breeding programs.